Journal of Autoimmunity (1992) 5,5 1 l-525
Effect of Ultraviolet Irradiation on Selected Host Cell Proteins Including Ro/SS-A and Epstein-Barr Virus in Cultured Lymphoblastoid Cell Lines
Marianna M. Newkirk and Christos Tsoukas* Divisions of Rheumatology and *Immunology, Department of Medicine, Montreal General Hospital Research Institute, McGill University, Montreal, Canada (Received 7 November 1991 and accepted16 April 1992) Although systemic lupus erythematosus (SLE) and SjSgren’s syndrome (SS) are distinct collagen vascular illnesses, they share certain features. Both have clinical manifestations involving skin and mucous membranes and characteristically have high titers of circulating autoantibodies to the cellular components Ro/SS-A, calreticulin/Ro, 52 kDa Ro and La/SS-B. Viruses have been postulated to be involved in the pathogenesis of both diseases. Sensitivity to sun is a cardinal feature of SLE, and UV light may be involved in its pathogenesis. Using human B-lymphoblastoid cell lines, the effect of the resident Epstein-Barr virus on the expression of the above cellular components was investigated by flow cytometry. Sublethal irradiation with ultraviolet B light appeared to diminish EBV antigen expression (gp350/220) during the first 48 to 72 hours in culture, whereas there was no change in the expression of MHC class I or immunoglobulin host cell proteins, and an apparent increase in the expression of host cell autoantigens. The virus appeared to be more sensitive to UVB-induced damage yet did appear to be able to undergo repair. No direct correlation could be made between the presence of the virus and the increase in autoantigen expression. La/SS-B and/or 52 kDa Ro antigen(s) were found to be present in the cytoplasm of the B lymphoblastoid cells at a higher base level in EBV-infected cell lines than in the EBV-negative cell lines.
Patients with either subacute cutaneous lupus erythematosus (SCLE) and/or Sjagren’s syndrome (SS) have in common circulating autoantibodies to the protein/ Correspondence to: Marianna M. Newkirk, Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Quebec H3G lA4, Canada. Fax: (514) 937-2796. 511 0896-841 l/92/04051
1 + 15 $03.00/O
0 1992 Academic Press Limited
Marianna M. Newkirk and Christos Tsoukas
RNA complexes Ro/SS-A, calreticulin/Ro, 52 kDa Ro and/or La/SS-B [l-4]. In these individuals, there is a ubiquitous distribution of the antibodies, yet the pathological lesions occur only in specific tissues. The precise nature of these autoantigens, both in their complex formation and in their role in the metabolism of normal or virally infected cells, is not well understood. The La/SS-B protein has been identified as a termination factor of transcription  and the other proteins are thought to be involved in transcription and/or translation events. It has long been suspected that viruses play a role in the etiology of these diseases, but the degree to which they contribute to the pathogenic process has not yet been determined. Patients with autoimmune diseases often have high titers of antiEpstein-Barr virus (EBV) antibodies [6, 71 and, in addition, the viral genome has been identified in the salivary tissue of patients with primary SS [8,9] suggesting a primary role for EBV. These findings, however, have been controversial [ 10-121 and it is likely that a number of factors, including the genetics of the host, may play a role. EBV has been the focus of a substantial body of research. The EBV surface glycoprotein gp350/220 has been identified as the molecule that binds to the CD21 molecule (the complement receptor, CR3) on the B cell [13,14]. Once internalized, EBV can, in fact, cause the transformation of B cells. Moreover, EBV can reside in a latent state in up to 3% of B cells [ 161 and be reactivated periodically during the lifetime of the individual. This virus, which infects B cells, can be found in 70% of the population, including the majority of those with autoimmune diseases [6, 151. Epidemiological studies of the role of this virus in autoimmune disease have been difficult to interpret because of the high infectivity rate. Both SS and SCLE have cyclic courses. In patients with SCLE, lesions often appear after exposure to sunlight [22-241. Since it is well known that both Herpes simplex I and II [25-271 can be induced by exposure to UV rays, we investigated the effect of UVB irradiation on EBV antigen expression in B lymphoblastoid cells in vitro. It has been suggested that the autoantigens Ro/SS-A and La/SS-B can complex with RNA transcribed from EBV and other viruses [ 17-211. The role that these proteins play in the life cycle of the virus is not known. We speculated that UVB might alter virus and/or host proteins such as Ro/SS-A (52 kDa, 60 kDa), calreticulin/Ro or La/SS-B. These proteins, which are probably involved in hostcell transcription or translation, could possibly be utilized in viral replication. Set in an appropriate genetic environment 1281, these host proteins could become antigenie, leading to the production of autoantibodies. The distribution of viruses within an individual is generally not ubiquitous and is dependent on the location of virus receptors and/or the site of latency (the receptor can be lost). Upon further viral reactivation events, periodic expression of the antigens could occur in regions where the virus is resident. This hypothesis would then account for the cyclic nature of the diseases and, more importantly, could explain the tissue-specific manifestation of the disease which may be linked to the resident virus. Materials
Human B-lymphoblastoid cell lines utilized included P3HR-1 (gift of Dr Qualtiere), BJAB (gift of Dr Jolicoeur), RAJI (NIGMS, Camden, NJ, USA), B95-8 and
UVB irradiation of EBV lymphoblasts Table
1. Presence of viralgenomes
Hum-BL latent NP 50
Hum-BL producer NT 300-1000
Marmoset producer T 63
#EBV genome copies EBV Proteins EBNA- 1 EBNA-2 Detection by PCR: EBV CMV HHV-6 EL = Burkitt’s
in human and marmoset B-lymphoblastoid
NE’ = non-producer;
NT = non-transforming;
T = transforming.
RAMOS (American Type Culture Collection, Rockville, MD, USA). The viral status of the cell lines is listed in Table 1. The presence of the DNA genomes for EBV, cytomegalovirus (CMV) and human herpes virus-6 (HHV-6) was determined using established polymerase chain amplification methods [9,29,30].
Surface expression of EBV gp350/220 was detected using mouse monoclonal antigp350/220 F34-5H7-DlO  (gift of Dr L. Qualtiere). The EBV nuclear antigen was detected using mouse monoclonal anti-nuclear antigen (Chemicon, St Laurent, Quebec, Canada). Lymphoblastoid cells were incubated with the appropriate mouse monoclonal antibodies for 30 min at 4°C. After washing, the cells were incubated with fluorescein isothiocyante (FITC)-conjugated goat anti-mouse IgG (Tago, Markham, Ontario, Canada) for a further 30min. Staining was analysed by flow cytometry using a FACS-SCAN (Becton Dickinson, San Jose, CA, USA). For cytoplasmic staining, the cells were fixed in - 20°C ethanol for 30 min prior to staining. Comparative analysis of the FACS data was carried out using the mode values. This value gives a better representation of the staining, taking into consideration the percentage of cells staining as well as the intensity of staining.
Host cell proteins
Ro/SS-A and calreticulin/Ro were detected using F(ab’), fragments of rabbit antipeptide antibodies. The peptides utilized for the induction of the antisera were: Ro/SS-A [32, 331, peptide A, amino acids 220-231 [KLKWRPDEEILK(C)], peptide B amino acids 367-379 [RKKMDIPAKLIVC] and calreticulin/Ro  amino acids 7-24 [KEQFLDGDWTSRWIESK(C)]. F(ab’), fragments of IgG from
Marianna M. Newkirk and Christos Tsoukas
a patient with SLE, specific by western blot analysis for both La/SS-B (48 K) and the 52 kDa Ro/SS-A antigen, were also used and will be referred to as anti-La/52K Ro. Identity of La was confirmed using the CDC standard reagent. In immunoprecipitation experiments using this sera, no appreciable binding to the 60 kDa Ro proteins was observed (not shown). Class I MHC antigens were monitored using a mouse monoclonal specific for HLA-ABC (Chemicon, IngramandBell, St Laurent, Quebec, Canada). Immunoglobulin expression was measured using FITC-conjugated antitotal Ig (Sigma, St Louis, MO, USA). Staining for cell surface or cytoplasmic expression was as above, however, using the appropriate fluorescein isothiocyante (FITC)-conjugated anti-rabbit Fab (Cappel, Organon Teknika, Scarborough, Ontario, Canada) or anti-human IgG antisera (Sigma). Ultraviolet
Lymphoblastoid cells were irradiated for O-45 seconds with UVB (302 nm, 2.0 mJ/ cm’, Transilluminator, Spectroline, Fisher, St Laurent, Quebec, Canada) at a density of 2 x lo5 cells/ml in 100 x 20 mm plastic petri dishes, (10 ml/dish, depth of liquid < 2 mm). According to the manufacturer, there is no UVA component in the irradiation. After irradiation thecells were centrifuged and the medium replaced. Cells were cultured at a density of 5 x lo5 cells/ml in 5% CO, at 37°C for up to 72 h. Statistical
two tailed t-tests were calculated using InStat
Results Presence of viral genomes in lymphoblastoid
We confirmed the presence of the EBV genome in the Raji, P3HR-1 and B95-8 cell lines by polymerase chain amplification techniques (Table 1). In order to determine whether viruses other than EBV in the EBV-negative transformed cells might contribute to the changes in protein expression, we tested the cell lines for the presence of two other Herpes viruses which are lymphotrophic. We determined that both B JAB and RAMOS, which are negative-for EBV, do carry the genome for HHV-6 (Table l), while none of the EBV-infected cells do. No cell line in this study was found to carry the genome for CMV. Impact of UVB
on cell viability andgeneration
Irradiation of the lymphoblastoid cells for up to 45 s did not appear to affect the ability of the cells to exclude trypan blue when assessed immediately after irradiation. Viability at the end of each incubation period (up to 72 h) was significantly lower only in the UVB-irradiated cells which were EBV-infected (mean viability, no UVB, 86.5 + 2.14; with UVB, 79.55 + 2.19, P= 0.03). The population doubling time was significantly increased during the first 3 days post-irradiation in all cell lines regardless of the presence of the EBV genome (Figure 1; Table 2). Five seconds of irradi-
UVB irradiation of EBV lymphoblasts
c 1,000 - ” P z 5 800 - .... -. .. “” 4 2
1. Doubling potential of lymphoblastoid daily for 5 days following irradiation. -
of UVB irradiation
cell lines irradiated with 2 mJ/cm’ BJAB;-+ -- P3HR-1.
UVB for 0 to 45 s
Table 2. Effect of 5 s UVB irradiation on the mean doubling time of human B-lymphoblastoid cell lines as measuredfor up to 72 h following irradiation
Doubling time (h) Cell line
Net UVB induced increase
EBV-negative RAMOS BJAB
EBV-infected RAJI P3HR- 1 P95-8
140 46 96
164 60 L 200
1.17 1.30 2.08
Mean _t SEM
ation was chosen for all subsequent replicating, albeit with an increased E#ect of UVB
experiments, since the cells were still capable doubling time (mean increase = 1.66 fold).
expression in B lymphoblastoid
During the 72-h incubation period following irradiation of the cell lines, UVB was found to have little effect on the cell surface or cytoplasmic expression of the
Marianna M. Newkirk and Christos Tsoukas
Time in culture
Figure 2. Cytoplasmic binding (immunofluorescence intensity, mode) of antibody specific for EBVgp350/220 in lymphoblastoid ceil lines irradiated with UVB and then cultured for up to 72 h. Background staining with a mouse monoclonal isotype control antibody was subtracted. W RAMOS; q RAJI; q P3HR-1.
EBV-encoded gp350/220. Only in the P3HR-1 cell line was there recovery of the expression of the EBV-encoded antigen in the cytoplasm after the initial loss induced by UVB (Figure 2). Although the RAMOS lymphoblasts had..been determined to be EBV genome negative (Table l), significant cytoplasmic binding of the monoclonal antibody to gp350/220 was observed, which may reflect molecular mimicry of a host cell protein. To investigate the possibility of a UVB-induced redistribution of gp350/220, the distribution of the total antigen expression between the cytoplasm and the cell surface was calculated. As shown in Table 3, for both P3HR-1 and B95-8 cell lines there appeared to be a small increase in the amount of gp350/220 expressed on the surface compared with that expressed in the cytoplasm following UVB irradiation. No gp350/220 was detected on the surface of the majority of RAJI cells regardless of whether they had undergone irradiation. In < 1 o/0of RAJI cells there did appear to be redistribution of the EBV-encoded protein (the change was two-fold) in UVB irradiated cells. We investigated whether this decrease after UVB irradiation occurred only in the gp350/220 glycoprotein or was more general for EBV. We found that the expression of the EBV nuclear antigen similarly decreased by 25 y0 in the first 16 h after UVB irradiation in the P3HR-1 cell line (data not shown). Eflect of UVB Class I MHC,
on host cell encoded protein expression
The quantification of the expression of host cell class I MHC antigens and immunoglobulin (immunofluorescence intensity, mode) 24 h after UVB irradiation from two representative cell lines is illustrated in Figure 3. UVB irradiation did not significantly
UVB irradiation of EBV lymphoblasts Table
3. Surface antigen expression as a percentage of cytoplasmic antigen detected in UVB-irradiated B lymphoblastoid cell line after 24 h in culture o/0 Surface/cytoplastnic UVB
0 2 (41)* 0(25) 0 (22) 0 0 0 2.2
2 6 (68) 0 0 0 1 3.2 7.5
La/52 kDa Ro
28 25 0.4 (6) 0.1(4) 0 0.2 6 6
0 0 0 0 1.3 1.0 0.4 0.6
0 0 0 (43) 0 (86) 0 5.2 5.3 7.5
*( ) represents subpopulation of < 5% of the total cells. Unless designated, this subpopulation was not detected.
RAMOS + UVB Lyrnphoblastoid
Figure 3. Expression of Class I MHC and immunoglobulin in RAMOS and RAJI lymphoblasts 24 h post-irradiation with UVB. W Class I (s); t&rclass I (c); q Ig (s); q Ig (C). s = Surface expression; c = cytoplasmic expression.
alter the expression of either ofthese proteins inmost cell lines tested. The cytoplasmic expression of class I MHC was found to be two-fold higher after UVB irradiation in the RAJI cell-line between 48 and 72 h post-irradiation (data not shown). Autoantigens:
of the autoantigens tested could be detected in the majority of cells for all of the cell lines (Figure 4). UVB irradiation did not appear to induce surface expression, except in small subpopulations of cells. For example, in In general,
M. Newkirk and Christos Tsoukas
. ,._ ................................. :: ................................... ................................... . . .. .. .
..I .. -I
:::I ,... .
.1. 1 ..
. .:. ::
:: .. ..
. u ‘:
Figure 4. Expression of calreticulin/Ro, Ro/SS-A and La/52 kDa Ro in lymphoblast cell lines 24 (RAMOS, RAJI, P3HR-1) or 48 h (El958) post-UVB irradiation. n Calreticulin/Ro (s); q calreticulin/Ro (c); q Ro/SS-A (s); q Ro/SS-A (c); q La/52K Ro (s)/; q La/52K Ro (C). s = Surface expression; c = cytoplasmic expression.
3.5% and 2.2% of RAMOS and RAJI cells, respectively, significant expression of Ro/SS-A (modes of 26 and 29 respectively) was detected during the first 24 h following UVB irradiation. Similarly, as with Ro/SS-A, 2.8% of the RAMOS cells appeared to express calreticulin/Ro on the surface, 24 h after irradiation. This small population was not detected in either the RAJI or PSHR-1 cell lines. It is possible that this ‘surface’ expression is restricted to cells that are lethally damaged by the UVB. Interestingly, with the RAJI cells, significant surface expression of La/52 kDa
UVB irradiation of EBV lymphoblasts
Ro was detected at 48 h after UVB irradiation (12.8% of the cells having an immunofluorescence mode of 41.3). This subsequently diminished to the value of the unirradiated control by 72 h (data not shown). Cytoplasmic expression of all autoantigens was readily detected in all cell lines tested (Figure 4). The impact of UVB irradiation was found to vary for each autoantigen and each particular cell line. The cell line that showed an irradiationinduced cytoplasmic expression of all of the autoantigens (1.2-fold for La/52 kDa Ro, four-fold for calreticulin/Ro and 2%fold for Ro/SS-A) was PSHR-1 which is an EBV-producer cell line. In the RAJI cell line (EBV latent), however, no substantial induction (greater than 1.4 fold) of any of the autoantigens in the cytoplasm could be detected. The UVB-induced changes which occurred for La/52 kDa Ro showed an interesting pattern. The base amount of La/52 kDa Ro in the cytoplasm of unstimulated EBV negative cell lines (B JAB, RAMOS) was found to be 189 f 41 and in the human EBV-positive cell lines 571& 164 (P=O.O8). Cytoplasmic expression of these proteins was induced in three of the four cell lines regardless of the EBV status of the line, in the initial 24 h after UVB irradiation (Figure 3). The greatest induction (3.8 fold increase) occurred in the RAMOS, EBV negative cell line. Only in the PSHR-1 cell line was this increase maintained, similar to the unirradiated control. In all other cell lines there appeared to be a subsequent loss of antigen expression (both for unirradiated and irradiated cells) with time in culture. In order to determine whether there was UVB-induced redistribution of the proteins, calculations of the percentage of their surface expression, relative to the amount detected in the cytoplasm, were made in both irradiated and non-irradiated cells (Table 3). No marked differences in the expression of the proteins (surface to cytoplasm) was found for the majority of cell lines. Both the RAMOS and B95-8 cells showed slight increases in surface compared with cytoplasmic expression of the Ro/SS-A and calreticulin/Ro proteins following irradiation.
To date, there is little information available concerning the effect of UVB on viral replication or antigen expression. Our intention was to examine whether UVB irradiation could affect the expression of viral and/or host protein expression. In particular we were interested in determining the effect of irradiation on the proteins that are the target of the autoimmune response in patients with sun-induced SLE. In our study of the effect of UVB irradiation on EBV antigen expression in B lymphoblastoid cells in vitro, we did not find a marked viral reactivation of EBV measurable in the first 72 h after UVB irradiation. Instead, we saw a clear loss in the expression of the EBV surface glycoprotein and the nuclear antigen during the initial phase. Existing evidence reveals that doses of one to four minimum erythema doses (MED) of UVB were required to reactivate Herpes Simplex I or II in vivo [26,27]. One MED is defined as the amount of UVB that will turn skin pink and varies with the skin type but in general is about 15-75 J/cm2 for Caucasians. The skin lesions in SLE have been shown to be induced by doses of UVB irradiation in a range of l-4 MED . Since lymphoblasts in vitro are not protected by keratin or layers of
Marianna M. Newkirk and Christos Tsoukas
squamous cells, we chose a dose that was not lethal to host cells, less than 1 MED (2mJ/cm2), but one that is thought to equate approximately 20 mJ/cm’ in viva . This amount of UVB irradiation allowed the cells to continue metabolizing while prolonging their doubling time. The primary damage induced by UVB is the formation of pyrimidine dimers and cyclobutane, and is dependent on both the sequence and structure of the DNA [42-44]. Based on in vitro studies of keratinocytes , the single break lesions in the DNA would be expected to occur (at a wavelength of 310) roughly at a frequency of 1.3 per 1013 Da, using the dose of radiation administered in the current study. For EBV, this amount of UVB should induce a lesion in one of every two virions. Our data would suggest that EBV appears to be exquisitely sensitive to UVB damage since there was a dramatic loss in the expression of the EBV-specific gp350/220 suggesting that little or no new transcription or translation of this protein was occurring. Only in the P3HR- 1 cells was there a slight recovery in the expression of this protein by 72 h. The half life of the gp350/220 protein is not known but is clearly induced in infected cells by treatment with phorbol over a 3-day period . Enzymatic repair of the pyrimidine dimers begins in vivo approximately 5 h following UVB irradiation . Full recovery of the DNA to the pre-irradiation state is dependent on both the number and position of the lesions. The precise mechanism for this enzymatic recovery has not been fully characterized to date. The recovery in the expression of the gp350/220 in P3HR- 1 cells suggests that this repair mechanism may function for EBV. The host cell proteins monitored in this study appeared to be more resistant to UVB induced damage than the EBV-encoded molecules. Both class I MHC antigens and immunoglobulin appeared to be unaffected, except in the RAJI cells, where there appeared to be a modest increase in cytoplasmic expression of class I glycoproteins between 48 and 72 h following irradiation. UVB has previously been found to induce the expression of a number of different proteins, few of which have been fully characterized [48-5 11. The size of the specific protein pool within the cell and the half-life of individual proteins may obscure UVB-induced events. However, in the RAMOS cell line, where the baseline expression of class I MHC was found to be much lower than for RAJI cells, no net loss or induction could be seen. Previous studies have shown that class II MHC expression is inhibited by UVB irradiation. However, when doses of UVB radiation similar to those administered to cells in the current study are used, the initial loss can be recovered by synthesis and re-expression of the glycoproteins . The host cell proteins (Ro/SS-A; calreticulin/Ro, La/SS-B and/or 52 kDa Ro) that are the target of autoantibodies in patients with SLE appeared to be induced by UVB irradiation, primarily in the cytoplasm of some cell lines. Each individual cell line showed a slightly different pattern with respect to the specific proteins that were induced and the degree of induction. Interestingly, the proteins La/SS-B and/or 52 kDa Ro were markedly induced by UVB irradiation in the majority of cell lines. The base line level of the protein(s) was higher initially in all of the EBV infected cells. Previous studies have shown that viral infection or cell transformation (spontaneous or virally induced) may induce an increase in La/SS-B expression in epithelial cells, mouse lymphoblasts or fibroblasts [2 1,531. The most dramatic induction was found in our study to occur in the EBV-
UVB irradiation oEEBV lymphoblasts
negative cell line @AMOS) which brought the amount of this protein or proteins almost to the base line level of the EBV-carrying cell lines. Further studies are in progress to identify more precisely the induced protein(s) recognized by antibodies to La/SS-B and 52 kDa Ro. It is interesting to speculate about the role of the protein(s) that is/are induced. There may be a UVB-induced conformational change in an already existing protein or a movement from the nucleus to the cytoplasm, which could account for its increased detection after irradiation. The latter has been demonstrated for the 48 kDa RLa/SS-B protein in keratinocytes . Alternatively, the inducible protein may reflect net translation increases and may play a role in the repair process either directly or indirectly (aiding in the translation of the repair enzyme). Our data would argue that this induction is not EBV-related; however, we cannot rule out an effect of HHV-6 in the transformed RAMOS or B JAB cell lines. The question of surface expression of all of the autoantigens is of interest clinically. Because these proteins, as well as the autoantibodies, have a ubiquitous distribution throughout the body, for these antigens to be involved in the tissue specific injury, the most appealing hypothesis is one of surface expression of these proteins. The use of F(ab’), antibody fragments in our labelling studies ensured that we were not measuring Fc related absorption. Since FACS analysis allows one to choose a homogeneous population and eliminate the non-viable cells from analysis, we are confident that we are not measuring cytoplasmic events in our surface staining protocols. It was of interest, therefore, that there appeared to be little or no surface expression of these proteins. In some instances, only a small population of the cells (less than 5oj, of the total) expressed significant amounts of the Ro/SS-A proteins (RAMOS with UVB, RAJI both with and without UVB) or calreticulin/Ro (RAMOS with UVB). Since this small population was induced in the RAMOS cell line, we can conclude that it is not related to the presence of EBV. In the case where the whole population appeared to express the proteins, although at low levels, again the presence of EBV did not appear to play a role since this occurred in both the EBVpositive and -negative cell lines. Our data suggest that UVB alone can induce the surface expression of these proteins but only in some cell lines (not RAJI or P3HR1). Previous studies have demonstrated the presence of the Ro/SS-A and La/SS-B antigen on the surface of UVB-irradiated keratinocytes [41, 551. We could not demonstrate UVB induced surface expression of La and/or 52 kDa Ro on the majority of the cell lines in our study. This difference may be due to the nature of the cells studied and/or the fact that in the previous studies the UVB cells were treated with trypsin prior to FACS analysis [41,55], or to the use of whole human antibodies which may have been surface bound through Fc receptors . The results of our study demonstrate that at sublethal doses of UVB radiation, the host cell appears to be more resistant to UVB-induced damage than does the resident virus. However, it appears that the virus does have some capacity for DNA repair, possibly utilizing host cell repair enzymes. The presence of the EBV does not apparently correlate with either UVB-induced changes or lack of change in the host cell proteins which may or may not be targets of the autoantibodies in patients with SLE. There was, however, a higher base line level of the proteins La/SS-B and/or 52 kDa Ro in the EBV-infected cell lines which may predispose such cells to tissue injury in the event of membrane disruption.
522 Marianna M. Newkirkand Christos Tsoukas Acknowledgements The authors are grateful to Drs Karen Duffy, Tianying Long, Jianhui Zhu, MS Monidapta Dasgupta, Mr Bernard N6el and MS Louise Gilbert for technical assistance and to Dr Carolynn Pietrangeli for helpful discussions. This research was supported by an operating grant from the Arthritis Society of Canada. Dr Newkirk is a recipient of a Basic Science Associateship from the Arthritis Society of Canada.
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UVB irradiation of EBV lymphoblasts
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