Altered cytokine genes expression by ConA-activated spleen cells from mice infected by lymphocytic choriomeningitis virus

Immunology Letters, 35 (1993) 247-254

0165 2478 / 93 / $ 6.00 © 1993 Elsevier SciencePublishers B.V. All rights reserved IMLET 01923

Altered cytokine genes expression by ConA-activated spleen cells from mice infected by lymphocytic choriomeningitis virus J e a n - H e r v ~ Colle a, M a r i e - F r a n g o i s e S a r o n b a n d P a o l o T r u f f a - B a c h i a aUnitd d'Immunophysiologie MolOculaire and bLaboratoire de Virologie Expdrimentale Institut Pasteur, Paris, France

(Received 13 January 1993;accepted 19 January 1993)

1.

Summary

The intravenous injection of mice with lymphocytic choriomeningitis virus (LCMV) induces a rapid and long-lasting immunodeficiency. T lymphocytes from 7-day-infected mice do not proliferate in vitro in response to ConA stimulation, do not produce IL-2 but display high affinity IL2 receptors on their membrane. The non-coordinated regulation of these genes suggested that other cytokine-encoding genes may also be affected in their regulation. We have thus analyzed the expression of the genes encoding different cytokines transcribed during spleen cell activation by ConA. The genes encoding T lymphocyte-derived cytokines can be classified in three groups: the genes expressed similarly by normal and LCMVcells (the p55 and the p75 chains of the IL-2 receptor [1]), the genes under expressed in LCMVcells (IL-2, IL-3, IL-4 and IL-5) and the genes over expressed by these cells (GM-CSF and IFN7). These results show that the viral infection has provoked a profound alteration of the overall regulation of the genetic program that follows T lymphocyte activation. Since T cell activation deKey words: Cytokinegene transcription; Immunosuppression;

Lymphocyticchoriomeningitisvirus Correspondence to: Paolo Truffa-Bachi,D~partement d'Immunologie, Unit~ d'ImmunophysiologieMolrculaire, Institut Pasteur, 75724 Paris Cedex 15, France. Abbreviations: LCMV, lymphocytic choriomeningitis virus;

LCMV-cells, spleen cells from 7-day-LCMV-infectedmice.

pends strictly on accessory cell-derived cytokines, we measured the level of transcription of IL-1, IL6 and TNF-e; and our data show that the expression of these genes is equivalent in normal cells and in cells from LCMV-infected mice. 2.

Introduction

T lymphocyte proliferation is characterized by a chronological pattern of gene expression, and many data support the concept that these events are interconnected (reviewed in [2,3]). In particular, the genes encoding the cytokines share analogous targeting sequences for regulatory factors located 5' to their coding sequences [4-6]. However, the regulation of these genes is very complex as optimal transcription requires highly co-operative interactions between different DNA binding proteins ([4,7,8], reviewed in [9]). This complexity is apparent in the dissociation of the co-ordinate expression of IL-2 and IL-2Re genes found when T cell activation is carried out in the presence of the immunosuppressive drug cyclosporin A [10, 11] or when T cell stimulation is ensured by molecules inducing an incomplete proliferative signal [12]. We have previously reported that ConA-stimulated T lymphocytes from LCMV-infected mice do not produce IL-2 while transcribing at a normal level the genes encoding the p55 and p75 chains of the IL-2 receptor [1, 13]. The differential expression of this group of genes suggested that the interconnected regulation of other T-derived cytokine genes could also be affected. We have tested this hypothesis by comparing the le247

vel of expression of the cytokine genes induced during the ConA-induced polyclonal activation of spleen cells from normal or 7-day-LCMV-infected mice. In addition, we have analyzed the capacity of accessory cells to transcribe different cytokines produced during the T-dependent activation of these cells. Our data show that activation of T lymphocytes from LCMV-infected mice results in an increased transcription of IFN-7 and G M - C S F genes, while IL-3, IL-4 and IL-5 encoding genes were expressed at a lower level compared with normal cells. Together with our previous data on IL-2 and 1L-2R gene expression [1, 13], the present results support the hypothesis that infection of mice with LCMV alters the overall regulation of the T-derived cytokine genes without interfering with the capacity of accessory cells to transcribe their relevant cytokines. 3.

3.1.

Materials and Methods

Mice, viruses, and cell culture

Eight- to 12-week-old female C3H/HeOu mice from our animal breeding facilities were used. The animals were kept in specific pathogen-free conditions and resisted 1000 rads irradiation, given by a cesium source, with a mean survival time of 10 days. LCMV strain CIVP 76001 was maintained by i.c. passage as previously described [14]. Mice received by intravenous r o u t e 10 3.4 P F U of virus in 0.2 ml Hank's balanced solution supplemented with 2% FCS and were used 7 days after inoculation. Spleen cells (5 x 106 cell/ml) were cultured in RPMI 1640 (Eurobio, France) supplemented with 2 mM L-glutamine, 50 #g/ml streptomycin, 50 U/ml penicillin, 5% heat-inactivated fetal calf serum (Eurobio, France), 5 x 10 -5 M 2-mercaptoethanol and 4 #g/ml concanavalin A (ConA, Miles Yeda, Kankakee, IL) in 12cm Petri dishes in a humidified atmosphere of 5% CO2 in air.

3.2.

absorbency of the samples at 260 nm was used for RNA quantification.

3.3.

The D N A probes used for hybridization were the following: IFN-7, a 910-bp PstI cDNA fragment [17] (a kind gift of Drs. G. Ward and A. Morris); IL-3, a 289-bp BglII-HindIII cDNA fragment [18]; IL-4, a 373-bp RsaI cDNA fragment [19]; GM-CSF, a 216-bp TaqI cDNA fragment [20]; IL-1, a 417-bp PstI-PVUII cDNA fragment [21]; IL-6, a 155-bp BgIII cDNA fragment [22]; TNF-c~, a 298-bp PVUII cDNA fragment [23].

3.4.

Dot hybridization analyses

Four #g of total RNA dissolved in 30/~1 denaturation mixture composed of MOPS 20 mM pH 7, Na acetate 5 mM, E D T A 1 mM, 1.5% formaldehyde, 5% deionized formamide were incubated for 10 min at 65°C and chilled on ice for 2 min. After addition of 30 #1 of 10 x SSC the samples were applied to Hybond-N + membranes (Amersham, Buckinghamshire, UK) using a 96-hole minifold apparatus (BioRad, Richmond, CA) under vacuum. R N A was fixed to filters by baking for 2 h at 80°C. Filters were prehybridized for 4 h at 42°C in a mixture containing 50% formamide, 5 x SSC, 1 x Denhardt's solution, 0.5 M sodium phosphate pH 6.5, 0.1% SDS, 100 ~g/ml salmon sperm DNA and 200/~g/ml of yeast RNA. Hybridization was carried out in the same solution at 42°C for 36 h containing 106 cpm/ml of probe labelled by random priming [24] with ~32p dCTP (dCTP > 800 Ci/mmol, Amersham). The filters were washed for 20 min once with 1 x SSC and once with 0.5 x SSC solution containing 0.1% SDS at room temperature and, finally, with 0.2 x SSC at 55°C for 20 min. Filters were exposed to Kodak XAR-5 film with an intensifying screen at - 7 0 ° C .

RNA preparation 3.5.

Total cellular R N A was extracted from 1.5 × 10v cells cultured as above by homogenization in guanidium isothiocyanate [15]. Total cellular RNA was isolated by phenol extraction [16]. The 248

Probes

RNase protection analysis

RNase protection was used for IL-3 and IL-4 detection. Complementary RNA probes were produced by transcription of linearized plasmid DNA

with T7 or T3 polymerase as described by Pharmacia-LKB (Uppsala, Sweden). The transcription buffer contained unlabeled CTP at a final concentration of 10/~M. Fifty microcuries of c~32p CTP (CTP > 3000 Ci/mmol) were used per 12.5 #1 of reaction. Half to one-fourth/~g of plasmid D N A was used per labelling reaction, and 50-70% of the radioactive nucleotide input was usually incorporated. RNase protection was performed as described by Zinn et al. [25] using an RNA probe (2 5 x 105 cpm) hybridized for 16 h at 45°C with each RNA sample (10 ~tg). After the RNase digestion, the reaction mixtures were denatured for 5 min at 100°C and fractionated on 6% polyacrylamide urea sequencing gels at 42 V/cm [26]. The gels were denatured in 0.5 M NaOH, 1.5 M NaC1, neutralized in 0.5 M Tris-HC1 pH 7.5, 1.5 M NaC1 and transferred to H y b o n d - N + membranes.

3.6.

Polymerase chain reaction (PCR) analysis

1.5 /~g of total RNA was reverse transcribed using a 15-17 oligo (dT) primer (Pharmacia, Uppsala, Sweden) and Superscript M-MLV RNaseH RT (Gibco BRL) in a 20 /A reaction mixture as specified by the manufacturer. 2 ~tl ot a 1/10 dilution of the reverse transcript reaction were used for each PCR. Conditions were as follows: 25 pmol of each primer (see below), 125/~M each of dGTP, dATP, dCTP and dTTP (Pharmacia, Uppsala, Sweden), 50 mM KC1, 10 mM TrisHCI, pH 8.3, 1.5 mM MgC12, 1 mg/ml gelatine, 100 #g/ml non-acetylated BSA and 1 U of recombinant Taq D N A polymerase (Perkin Elmer Cetus, Norwalk, ME) in 50/A. The primers were the following:

natured in 0.5 M NaOH, 1.5 M NaC1, neutralized in 0.5 M Tris-HCl pH 7.5, 1.5 M NaC1 and transferred to H y b o n d - N + membranes. Hybridization and revealing of bands were carried out as described above.

4. Results Cells from normal or 7-day-LCMV-infected (LCMV-cells) C3H/HeOu mice were activated with ConA (4 #g/ml). Cells were collected at different times, the RNAs prepared and the level of transcripts encoding different cytokine genes was determined.

4.1.

Low level of IL-3, IL-4 and IL-5 transcripts in T lymphocytes from LCMV-infected mice upon ConA-activation

The scarcity of IL-3, IL-4 and IL-5 mRNAs in ConA-activated cells precluded the dot blot analysis, and the more sensitive RNase protection technique was therefore used. The RNA prepara-

Time (h)

0 3 [6 12 24 48

IL-3 LCMV

IL-4

IL-5 sense (5'-CTGCACTTGAGTGTTCTGAC-3') IL-5 anti-sense (5'-GTACTCATCACACCAAGGAA-Y) IL-6 sense (5'-TGGAGTCACAGAAGGAGTGGCTAAG-3') IL-6 anti-sense (5'-TCTGACCACAGTGAGGAATGTCCAC-3')

The IL-5 and IL-6 primers allow for the generation of a fragment of 353 bp and 156 bp, respectively. Amplifications were performed in a Pharmacia-LKB Gene ATAQ Controller for 20 cycles (denaturation: 60 s at 94°C; annealing: 60 s at 55°C; extension: 60 s at 72°C). 10 /A of each sample were loaded on an agarose gel (1.5%) in TBE buffer. For the other genes, the gels were de-

IL-5

LcMvN°rmal

Fig. 1. Low level of IL-3, IL-4 and IL-5 transcripts in T lymphocytes from LCMV-infected mice upon ConA-activation. Spleen cells (5 x 106 cell/ml) from normal or LCMV-infected mice were stimulated with ConA (4 #g/ml). Total RNA was extracted at the indicated times, and the presence of IL-3, IL-4 and |L-5 m R N A was revealed by RNase protection as described in Materials and Methods. Exposure time: 72 h.

249

GM-CSF

IFNo'~

Time (h)

0

3

6

I

112

24 48

0

3

6

12 24

48

I Normal

~:~~'~

"

~,

LCMV iiii~iii!!iii~lil]iiii!i!i!i!iiii~iiii!ili!li~l[! ]

Fig. 2. Up regulation of interferon-~ and GM-CSF genes in LCMV-cells upon ConA activation. Spleen cells (5 × 106 cell/ml) from normal or LCMV-infected mice were stimulated with ConA (4 pg/ml). Total RNA was extracted at the indicated times, and the presence of IFN- 7 and GM-CSF specific m R N A was revealed by dot blot analysis using the appropriate radiolabeled probes as described in Materials and Methods. Exposure time: 72 h.

tions were incubated with IL-3, IL-4 or IL-5 radiolabelled probes. After digestion with RNase H, the samples were submitted to electrophoresis, as specified under Materials and Methods. The autoradiograms presented in Fig. 1 show that IL-3, IL-4 and IL-5 specific mRNAs were extremely scarce in cells from LCMV-infected mice compared to normal cells. It should be pointed out that the expression of IL-4 in C3H/HeOu cells peaked at around 3 h, a very early transcription time compared with the 12 h found in other mice strains [27].

4.2.

Up regulation of interferon-7 and GM-CSF genes in LCMV-cells upon ConA activation

IFN-7 and G M - C S F were detected by dot blot hybridization. As shown in Fig. 2, the kinetics was very similar in both groups of cells. However, accumulation of mRNAs was more elevated in LCMV-cells than in controls: 3 h after activation, the amount of IFN-7 m R N A was already similar to that accumulated by normal cells at the peak of the expression (12 h). At the peak of expression in LCMV-cells the IFN-7 transcripts were 10-fold higher compared with the control group.

4.3.

Similar expression of the macrophage-derived cytokines upon ConA activation of spleen cells from normal or LCMV-infected mice

The ConA-induced T-dependent expression of 250

IL-1, IL-6 and T N F - e genes by accessory cells was analyzed at different times during the activation process of normal and LCMV-cells. Total R N A preparations (4 #g) were blotted on Hybond + membranes as described under Materials and Methods and analyzed using IL-1, IL-6 or TNF-c~ specific radiolabelled probes. The autoradiograms presented in Fig. 3 show the kinetics of IL-1, IL-6 and TNF-~ gene expression. Accumulation of these transcripts occurred with similar kinetics in both experimental groups suggesting that the viral infection had not modified the capacity of accessory cells to produce these cytokines.

5.

Discussion

We have previously reported that ConA-triggered splenic T lymphocytes from 7-day-LCMVinfected mice initiate the genetic program characterizing cell activation but do not proliferate [1, 13]. The abortive activation process is exemplified by the differential transcription of IL-2 and p55 IL-2R genes, the former remaining silent and the latter being normally induced [1]. We report here that the expression of other cytokine encoding genes is also profoundly modified. Resting T lymphocytes are transcriptionally silent for the genes encoding the cytokines. These genes are actively transcribed after antigenic or mitogenic stimulation in a chronological order suggesting that the different events controlling their expression are interconnected (reviewed in [2, 3]). Several c/s-acting regulatory D N A sequen-

T i m e (h)

Normal LCMV

Time (h) Normal LCMV

Time (h) Normal LCMV Fig. 3. Similar expression of the macrophage-derivedcytokines upon ConA activation of spleen cells from normal or LCMVinfected mice. Spleen cells (5 x l 0 6 cell/ml) from normal or LCMV-infected mice were stimulated with ConA (4 /~g/ml). Total RNA was extracted at the indicated times, and the presence of IL-I, IL-6 and TNF-~ mRNA was measured. IL-1 and TNF-ct transcripts were detected by direct dot blot hybridization. IL-6 was detected after reverse transcription and gene amplification as described in Materials and Methods. Exposure time: 48 h. ces have been identified and characterized in the cytokine genes. The coordinate expression of the cytokine genes may rely on the fact that some of these genes share homologous targeting sequences. For example, N K x B binding sites have been characterized in the 5'-flanking region of IL-2 and p55 IL-2R genes [5, 28-30], while SP1 binding sites were found in the regulatory regions of

IL-3 and G M - C S F ([31, 32], reviewed in [33]). Therefore, some trans-acting factors interact with various cytokine genes, while others are specific for a given gene. As exemplified by the regulation of the IL-2 and p55 IL-2R gene expression, optimal transcription also requires co-operative interactions between different D N A binding proteins ([4, 7, 30], reviewed in [9]). Although there is a consensus that the cytokine genes are co-ordinately regulated, little is known about the global regulation of their expression once the activation signal(s) are transmitted to the cell nucleus. It is precisely this co-ordinate wave of expression that is altered in ConA-triggered T lymphocytes from LCMV-infected mice. The cytokine genes tested can be classified into three groups: the genes which are transcribed as in normal T lymphocytes (IL-2R~ and IL-2R/~ genes, [1]), the genes which are poorly expressed (IL-2, IL-3, IL-4 and IL-5) and the genes for which R N A accumulation is increased (IFN-7 and GM-CSF). The altered expression of certain cytokine genes cannot be ascribed to the existence of ConA-sensitive and ConA-refractory T cell subsets but is a characteristic of the entire T cell population from L C M V infected mice. This conclusion takes into account our previous reports that all T lymphocytes from LCMV-infected mice display the IL-2R~ chain upon ConA activation [13] and that the vast majority ( > 7 0 % ) of these cells also transcribe the interferon-~' gene (Colle et al., International Immunology, 1993, in press). The finding that a set of cytokine genes remained silent whereas another set was transcribed at high levels raises the question of their differential regulation. Accumulation of cytokine m R N A s in activated T cells is synchronized, protein synthesis-dependent and primarily regulated at the level of gene transcription (reviewed in [2, 3]). The hypothesis of a direct influence of the virus on the differential gene expression implies that the vast majority of the lymphocytes carry the virus. This is not the case in L C M V infections as the frequency of spleen or blood cells infected is of the order of 1% [34, 35]. In addition, infectious viral particles which could interfere with the cell physiology [36-39] were not detected either at the onset of the culture or in 48 h ConA supernatants (results not shown) making the pos251

sibility of a direct effect of the virus very unlikely. The ConA-induced activation of T lymphocytes depends on the co-stimulation of accessory cells which produce different cytokines and particularly ILl, which has been identified as the main T-cell co-stimulator factor. We found that IL-1 expression was similar in normal cells and in cells from infected mice, a result in agreement with the biological titration of this cytokine previously reported [13]. In addition, no differences between these two groups of ConA-stimulated cells could be shown for the transcription of two other macrophage-derived cytokines, IL-6 and TNF-~. These analyses confirm our previous assumption that these cells are not adversely affected by the viral infection [13]. This conclusion is not incompatible with that of Odermatt et al. [40] and Althage et al. [41], who reported that the general immunosuppression could arise from the destruction of the marginal zone macrophages and the dendritic follicular cells by anti-LCMV cytotoxic T lymphocytes [40, 41]. Our present experiments do not take into account the viral-induced modifications of the organization of the follicular centre since we have analyzed the in vitro activation by ConA of the total splenic population. The finding that the genetic program leading to T cell activation and proliferation is not completed even though the accessory cells appear normally activated implies that additional events other than the destruction of the follicular organization of the spleen are involved in the viral-induced immunosuppression. As previously reported, these additional events are not related to active suppression or suppressive factors [13]. Besides its relevance to the general problem of all viral-induced immunodeficiencies, the particular activation pattern of the cytokine genes induced by ConA in T cells from LCMV-infected mice appears as a particularly effective approach for the delineation of the mechanisms participating in the global regulation of cell activation and proliferation.

Acknowledgements The authors would like to thank Drs. Genevi6ve Milon and Anne Galelli for many discussions and kindly reviewing the manuscript. We acknowledge the technical help of Ms. Francine 252

Guillon and Mr. Raymond Perret. This work was supported in part by grants from the CNRS (LA 359) and ARC (6244).

References [1] Saron, M.F., Colle, J.-H., Dautry-Varsat, A. and TruffaBachi, P. (1991) J. lmmunol. 147, 4333. [2] Crabtree, G.R. (1989) Science 243, 355. [3] Ullman, K.S., Northrop, J.P., Verweij, C.L. and Crabtree, G.R. (1990) Ann. Rev. Immunol. 8, 421. [4] Durand, D.B., Shaw, J.P., Bush, M.R., Replogle, R.E., Belagaje, R. and Crabtree, G.R. (1988) Mol. Cell. Biol. 8, 1715. [5] Hoyos, B., Ballard, D.W., B6hnlein, E., Siekevitz, M. and Greene, W.C. (1989) Science 244, 457. [6] Shibuya, H., Yoneyama, M., Nakamura, Y., Harada, H., Hatakeyama, M., Minamoto, S., Kono, T., Doi, T., White, R. and Taniguchi, T. (1990) Nucleic Acids Res. 18, 3697. [7] Serfling, E., Barthelm/is, R., Pfeuffer, l., Schenk, B., Zarius, S., Swoboda, R., Mercurio, F. and Karin, M. (1989) EMBO J. 8, 465. [8] Shibuya, H. and Taniguchi, T. (1989) Nucleic Acids Res. 17, 9173. [9] Muegge, K. and Durum, S.K. (1989) New Biol. 1,239. [10] Dos Reis, G.A. and Shevach, E.M. (1982) J. Immunol. 129, 2360. [11] Kr6nke, M., Leonard, W.J., Depper, J.M., Arya, S.K,, Wong-Staal, F., Gallo, R.C., Waldmann, T.A. and Greene, W.C. (1984) Proc. Natl. Acad. Sci. USA 81, 5214. [12] Proust, J.J., Shaper, N.L., Buchholz, M.A. and Nordin, A.A. (1991) Eur. J. Immunol. 21,335. [13] Saron, M.F., Shidani, B., Nahori, M.A., Guillon, J.C. and Truffa-Bachi, P. (1990) J. Virol. 64, 4076. [14] Saron, M.F., Launay, F. and Guillon, J.C. (1980) Ann. Virol. (Inst. Pasteur) 131E, 407. [15] Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294. [16] Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156. [17] Gray, P.W. and Goeddel, D.V. (1983) Proc. Natl. Acad. Sci. USA 80, 5842. [18] Kindler, V., Thorens, B., de Korsodo, S., Allet, B., Eliason, J.F., Thatcher, D., Faber, N. and Vassali. P. (1986) Proc. Natl. Acad. Sci. USA 83, 1001. [19] Noma, Y., Sideras, P., Naito, T., Bergstedt-Lindqvist, S., Azuma, C., Severinson, E., Tanabe, T., Kinashi, T., Matsuda, F., Yaoita, Y., et al. (1986) Nature 319, 640. [20] Gough, N.M., Gough, J., Metcalf, D., Kelso, A., Grail, D., Nicola, N.A., Burgess, A.W. and Dunn, A.R. (1984) Nature 309, 763. [21] Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Girl, J.G., Pan, Y.-C.E., Collier, K., Semionow, R., Chua, A.O. and Mizel, S.B. (1984) Nature 312, 458. [22] Van Snick, J., Cayphas, S., Szikora, J.P., Renauld, J.C.,

Van Roost, E., Boon, T. and Simpson, R.J. (1988) Eur. J. Immunol. 18, 193. [23] Pennica, D., Hayflick, J.S., Bringman, T.S., Palladino, M.A. and Goeddel, D.V. (1985) Proc. Natl. Acad. Sci. USA 82, 6060. [24] Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6. [25] Zinn, K., DiMaio, D. and Maniatis, T. (1983) Cell 34, 865. [26] Maxam, A.M. and Gilbert, W. (1980) Methods Enzymol. 65, 499. [27] Le Moal, M., Colle, J.H., Galelli, A. and Truffa-Bachi, P. (1992b) Res. Immunol. 143, 701. [28] Fujita, T., Shibuya, H., Ohashi, T., Yamanishi, K. and Taniguchi, T. (1986) Cell 46, 401. [29] Durand, D.B., Bush, M.R., Morgan, J.G., Weiss, A. and Crabtree, G.R. (1987) J. Exp. Med. 165, 395. [30] Shibuya, H., Yoneyama, M. and Taniguchi, T. (1989) Int. Immunol. I, 43. [31] Miyatake, S., Yokota, T., Lee, F. and Arai, K. (1985) Proc. Natl. Acad. Sci. USA 82, 316. [32] Miyatake, S., Otsuka, T., Yokota, T., Lee, F. and Arai, K.

(1985) EMBO J. 4, 2561. [33] Arai, K., Lee, F., Miyajima, A., Miyatake, S., Arai, N. and Yokota, T. (1990) Ann. Rev. Biochem. 59, 783. [34] Ahmed, R., King, C.C. and Oldstone, M.B.A. (1987) J. Virol. 61, 1571. [35] Tishon, A., Southern, P.J. and Oldstone, M.B.A. (1988) J. Immunol. 140, 1280. [36] Doyle, M.V. and Oldstone, M.B.A. (1978) J. Immunol. 121, 1262. [37] Oldstone, M.B.A., Holmstoen, J. and Welsh Jr., R.M. (1976) J. Cell. Physiol. 91,459. [38] Rivi6re, Y., Ahmed, R., Southern, P.J., Buchmeier, M.J., Dutko, F.J. and Oldstone, M.B.A. (1985) J. Virol. 53, 966. [39] Klavinskis, L.S. and Oldstone, M.B.A. (1987) J. Gen. Virol. 68, 1867. [40] Odermatt, B., Eppler, M., Leist, T.P., Hengaertner, H. and Zinkernagel, R.M. (1991) Proc. Natl. Acad. Sci. USA 88, 8252. [41] AIthage, A., Odermatt, B., Moskophidis, D., Kundig, T., Hoffmanrohrer, U., Hengartner, H. and Zinkernagel, R.M. (1992) Eur. J. Immunol 22, 1803.

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