JOURNAL OF VIROLOGY, Nov. 1991, p. 5813-5819 0022-538X/91/115813-07$02.00/0 Copyright C) 1991, American Society for Microbiology

Vol. 65, No. 11

An Attenuated Variant of Coxsackievirus B3 Preferentially Induces Immunoregulatory T Cells In Vivo ROBERT P. LOUDON,1 ALBERT F. MORASKA,2 SALLY A. HUBER,2* PETER SCHWIMMBECK,3 AND PETER SCHULTHEISS3 Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 191071; Department of Pathology, University of Vermont, Burlington, Vermont 05405-00682; and Department

of Internal MedicinelCardiology, University of Duesseldorf, Duesseldorf, Germany3 Received 19 November 1990/Accepted

5

August 1991

BALB/c mice infected with the Woodruff variant of coxsackievirus group B type 3 (CVB3W) develop myocarditis mediated by autoimmune cytolytic T lymphocytes. A variant of CVB3W (designated H3-1OAl) which infects the myocardium but induces minimal mortality or myocarditis compared to the parental virus was selected. Although H3-lOAl infections stimulate normal CTL responses to CVB3-infected myocytes, the autoimmune response to myocardial antigens is absent. Treatment of H3-lOA1-infected mice with 50 mg of cyclophosphamide per kg of body weight, a treatment which preferentially eliminates suppressor cells, allows both the development of the autoimmune cytotoxic T-lymphocyte response and the expression of myocarditis. Similar treatment of CVB3W-infected mice had no effect on the disease. The presence of the immunoregulatory cells was confirmed by adoptive transfer of T lymphocytes from either H3-lOAl or CVB3W-infected donor mice into syngeneic CVB3W-infected recipients. Animals given H3-lOAl-immune cells had minimal myocardial inflammation, while animals given CVB3W-immune lymphocytes developed enhanced cardiac disease. Elimination of the T-lymphocyte population from the donor cells prior to transfer abrogated suppression with the H3-lOAl-immune population, showing that immunoregulation depended upon T lymphocytes. Both H3-lOAl and CVB3W have cross-reactive epitopes between the adenine translocator protein and the virion which are indicative of antigenic mimicry and may be the basis for the autoimmunity to cardiac antigens. These results suggest that immunoregulatory T cells may be primarily responsible for the nonpathogenicity of the H3-lOAl variant. Woodruff (CVB3W) represents a highly pathogenic variant which induces significant cardiac inflammation and potent autoimmune T-cell responses in BALB/c mice (17). A separate isolate, CVB3-Crowell, infects the myocardium but induces only minimal cardiac inflammation (10, 16). Interestingly, no autoimmune T cells occur in CVB3-Crowellinfected mice. Recently, we isolated by plaque assay from CVB3W two virus variants. The first, designated H3, represents a variant which is highly myocarditic. The second, designated H3-1OA1, is an attenuated variant which was selected as an escape mutant from H3 by using the virusneutralizing monoclonal antibody (MAb) lOAl (37). This antibody inhibits myocarditic but not nonmyocarditic CVB3 infections, suggesting that the antibody recognizes differences related to viral pathogenesis (41). Although infection with H3-1OA1 fails to induce autoimmune T cells to myocytes, this variant still shows epitopes cross-reactive (mimicking) with ANT. Reduced pathogenicity in H3-1OA1 infections apparently results from induction of immunoregulatory T cells which override the autoimmune response.

Factors contributing to virus pathogenicity are of significant interest. These factors influence a variety of aspects in the infection cycle, including tropism (15, 18, 25), efficiency of infection and replication (24), cytokine and lymphokine induction (44), alterations in infected cell function and metabolism (19, 29), and other interactions between the virus, infected cell, and immune response. In a number of viral systems, immunity acts to control infections and primarily functions in a beneficial manner (12, 30, 42). In other systems, the immunologic response to infection contributes, in either a major or minor manner, to tissue injury and therefore causes pathogenesis (9, 14, 17, 43). Coxsackievirus B3 (CVB3) infections in mice induce myocarditis in which tissue injury depends predominantly on T-lymphocyte responses (43). Various groups suggest that infection leads to autoimmunity to normally expressed cellular antigens and that this autoimmunity is key to pathogenesis (35, 36). Recently, cross-reactive epitopes have been demonstrated between CVB3 and specific cellular proteins, including the adenosine nucleotide translocator protein (ANT) and cardiac myosin (35, 36). Immunization of animals with either of these proteins produces immune heart disease which in many ways resembles CVB3-induced myocarditis (35). Additionally, antibodies to both ANT and cardiac myosin are associated with either development of myocarditis or cardiac dysfunction (26, 27, 34). Therefore, antigenic mimicry between these proteins and the virus appears to be a prime candidate for explaining CVB3 pathogenicity. Not all CVB3 variants are equally myocarditic. CVB3*

MATERIALS AND METHODS Mice. BALB/c mice were originally purchased from Cumberland Farms (Clinton, Tenn.). Adult and neonatal animals were derived from colonies of these mice maintained at the University of Vermont. Viruses. CVB3W was originally established by the late Jack Woodruff and has been maintained by passage in either HEP-2 or HeLa cells (American Type Culture Collection [ATCC], Rockville, Md.). H3, a variant of CVB3W, was produced from homogenized heart of an 8-week-old male

Corresponding author. 5813

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J. VIROL.

LOUDON ET AL.

DBA/2 mouse with 104 PFU of CVB3W by plaque isolation as described previously (18). H3-1OA1, a second CVB3 variant, was obtained by infecting HeLa cells with 104 PFU of H3 in the presence of 100 ,ug of MAb 10A, followed by two blind passages of the cell homogenate (37). MAb 1OAl could inhibit 80 to 90% of either CVB3W or H3 infection of HeLa cells but was not inhibitory to H3-1OA1. The D variant of encephalomyocarditis virus (EMCV-D) was obtained from John Craighead (University of Vermont, Burlington) as a heart homogenate. The procedures for the propagation and titration of these viruses have been described in detail earlier (17, 41). Infection of mice. Each animal received 5 x 104 PFU of CVB3 or 30 PFU of EMCV-D virus intraperitoneally (i.p.) in 0.5 ml of phosphate-buffered saline (PBS). Organ titers. Hearts were aseptically removed, homogenized, and centrifuged at 300 x g for 10 min to remove cellular debris. The supernatants were serially diluted in Dulbecco's modified essential medium (DMEM; GIBCO, Grand Island, N.Y.) containing 5% fetal bovine serum (FBS; GIBCO) and supplemented with L-glutamine and antibiotics. Titrations were performed by using the plaque-forming assay on HeLa cells as described previously (17). Preparation of myocytes. The procedure for the preparation of myocytes has been published previously (17). Briefly, hearts were removed aseptically from neonatal mice less than 72 h old, rinsed three times, minced, and subjected to stepwise enzymatic digestion with 0.25% pancreatin (GIBCO) and 0.4% collagenase II (Worthington Biochemical Co., Freehold, N.J.). The isolated cells were washed to remove the enzymes and resuspended in DMEM containing 5% FBS and 10% horse serum. The cells were dispensed into either 1-mm-diameter-well (Belco Glass Co.) or 18-mmdiameter-well (Falcon Plastics, Oxnard, Calif.) tissue culture plates at a concentration of 3.5 x 105 viable cells per ml. The plates were cultured for 3 days at 37°C in a 6% C02-94% air humidified incubator. Preparation of lymphocytes. Spleens or mesenteric lymph nodes were removed from uninfected and CVB3-infected mice and pressed through fine mesh screens to produce a single cell suspension. The cells were washed in DMEM-5% FBS and flotated on Ficoll-Hypaque (Pharmacia Inc., Piscataway, N.J.) to remove erythrocytes (22). Spleen cells were incubated for 30 min at 37°C on nylon wool columns to partially enrich for T lymphocytes. Antibody treatment of cells. Lymphocytes at a concentration of 2 x 107 cells per ml were treated with 0.5 mg of MAb to the pan-T-cell marker (Thy 1.2; clone 30-H12; ATCC), markers for the CD4+ (clone GK 1.5; ATCC) and CD8+ (clone 2.43; ATCC) T-cell subpopulations, and 20% rabbit complement (GIBCO) for 30 min at 37°C. The cells were washed in Hanks' balanced salt solution and resuspended in PBS. Residual viable cells were determined by trypan blue exclusion, and cell suspensions were readjusted to equivalent viable cell numbers. Effectiveness of antibody and complement treatment of cell populations was determined by flow cytometry as described earlier (41). Elimination of greater than 90% of the targeted T-lymphocyte population was required for use of the cells in the study. Adoptive transfer of lymphocytes. Recipient mice were infected with 5 x 104 PFU of CVB3W or 30 PFU of EMCV i.p. and the same day received 2.5 x 106 lymphocytes intravenously (i.v.) in 0.1 ml of PBS through the tail vein. Cell-mediated cytotoxicity assay. Approximately 3.5 x 103 myocytes in 1-mm-diameter-well tissue culture plates were cultured in either medium alone or medium containing 3.5 x

105 PFU of CVB3 for 2 h at 37°C and subsequently labeled with 1 ,uCi of 51Cr (Na51CrO4; ICN Radiochemicals, Irvine, Calif.) for 2 h. The monolayers were washed three times with medium and overlaid with 3.5 x 105 viable lymphocytes in DMEM-10% FBS (for a 100:1 effector/target cell ratio) or with other lymphocyte concentrations to give appropriate effector/target cell ratios as indicated in the text. The cultures were incubated for 18 h at 37°C. Radioactivity in the supernatant and cell pellet was determined by using an Intertechnique CG4000 gamma counter. Percent 51Cr release represents [(cpm in supernatant)/(cpm in supernatant + cpm in cell pellet)] x 100. Percent lysis was determined as [(percent 51Cr release in test sample) - (mean percent 51Cr release in medium control group)/(mean percent 51Cr release in freeze-thaw group - mean percent 51Cr release in medium control group)] x 100. All groups contained a minimum of five replicate cultures. CY treatment. Cyclophosphamide (CY; Sigma) was dissolved in sterile PBS immediately before use. Animals were given 50 mg of CY/kg of body weight i.p. on the same day as virus infection. Enzyme-linked immunoadsorption assay (ELISA). Approximately 107 PFU of CVB3 per ml was suspended in carbonate buffer (pH 9.6) and dispensed in 0.05-ml aliquots into 96-well plates (Dynatek Immunolon). After incubation overnight at 4°C, the plates were washed and incubated in saline containing 1% bovine serum albumin (Sigma) to block unbound attachment sites. Sera, diluted 1:50 in PBS containing 0.05% Tween 20, were added to the wells, which were incubated at 37°C for 30 min. The plates were washed and subsequently incubated with a 1:1,000 dilution of ureaseconjugated goat anti-mouse immunoglobulin G and antimouse immunoglobulin M combined for 30 min at 37°C. The secondary antibody was removed by washing with PBSTween and then with distilled water. The wells were incubated with 50 RI of urease substrate (0.008 g of bromcresol purple, 0.1 g of urea, and 0.074 g of EDTA in 100 ml of distilled water, pH 4.8) for 60 min at 37°C. Changes in optical density were determined at 599 nm, using a multiwell mi-

croplate spectrophotometer (Bio-Tek Instruments, Inc.). Histology. The hearts were removed, fixed in 10% buffered formalin, and sectioned laterally approximately midway between the apex and atria, resulting in cross sections of both ventricles. The sections were stained with hematoxylin and eosin and scored blindly by S. A. Huber for inflammation, using a 0-to-4 scale. A score of 0 represents no inflammation; scores of 1 (1 to 10 inflammatory foci), 2 (11 to 20 foci), 3 (21 to 40 foci), and 4 (widespread and confluent lesions) represent increasing severity of cardiac disease. Statistical analysis. The Wilcoxon ranked score test was used to evaluate histology, organ titers, and serology. The Student t test was used for cytotoxicity assays. RESULTS

Generation of cytolytic activity to infected and uninfected myocardial cells in mice infected with CVB3W, H3, and H3-lOAl. BALB/c mice were injected i.p. with 5 x 104 PFU of either CVB3W, H3, or H3-1OA1 and sacrificed 7 days

later. T lymphocytes derived from these animals were assayed for cytotoxicity to either uninfected myocardial cells or targets infected at 100 PFU per cell with one of the three viruses (Table 1). Controls consisted of target cells cultured with nonimmune lymphocytes. Generally, there was no significant difference between 51Cr release from myocyte targets in the presence of normal lymphocytes and release

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VOL. 65, 1991

TABLE 1. Characteristics of CVB3 infectiona Animals infected with:

No virus

CVB3W

Myocarditis

score

(mean + SEM)b

0.2

1.8

0.3

0.4

H3

1.4

0.4

H3-1OA1

0.6

0.2e

Effector/target

%

cell ratio

No virus

150:1

0 ± OC 0 ± 0C 0±0

100:1 50:1 25:1 150:1 100:1 50:1 25:1 150:1 100:1 50:1 25:1 150:1 100:1 50:1 25:1

Lysis (mean

0±0 36 ± 4

18 ± 3 5±4 3±3 49 ± 2 29 ± 5 8±3 0±0 1 ± Vc 0 ± 0C 0±0 0±0

±

SEM) in myocytes infected withd: H3

H3-1OA1

8 ± 4c

5 ± 2c

0 ± 0C

10 ± 7c 12 ± 0 0±0 41 ± 4 41 ± 4 23±10 4±2 52 ± 4 27 ± lc 15 ± 6 13±8 36 ± 5 24 ± 6c 5±3 5±3

12 ± 6c 3±3 1± 1 58 ± 3 42 ± 3 14±0 7±4 60 ± 1 48 ± 5 32 ± 9 11±2 36 ± 10 23 ± 6c 8±2 7±0

1 ±1c 3±3 0±0

CVB3W

24 ± 6 25 ± 7

3±1 0±0 44 ± 1 25 ± 3 13 ± 4

13±4 49 52 7 0

5 26 3 0

a Infected mice (five to eight per group) received 5 x 104 PFU of virus i.p. in 0.5 ml of PBS. All animals were sacrificed 7 days after infection. b Scale of 0 to 4. c Significantly less than for CVB3W-immune lymphocytes at P - 0.01. d A cell-mediated cytotoxicity assay was performed by using mesenteric lymph node cells from infected and uninfected animals cultured at different effector/target cell ratios on 51Cr-labeled myocyte targets. Results represent mean ± SEM percent lysis of six replicate cultures for each point. e Myocarditis in H3-lOA1-infected mice was significantly lower than in other infected groups (P - 0.05).

from targets incubated in medium alone. Infection of mice with any of the three CVB3 variants induced significant cytolytic activity to all CVB3-infected targets. However, only animals inoculated with the pathogenic virus variants (CVB3W and H3) produced reactivity to uninfected targets. Demonstration of immunoregulatory T lymphocytes in H3lOAl-infected animals which prevent myocarditis and autoreactive T-celi responses. Previous studies from this laboratory have demonstrated that myocarditis during CVB3 infection correlates to the induction of cytolytic T lymphocytes (CTL) to uninfected myocyte targets (17). Failure of H3-lOA1infected mice to induce autoreactive CTL may explain the attenuation of this virus variant. Why autoreactive CTL are absent during H3-1OA1 infections is another question. At present, antigenic mimicry between viruses and self-antigens is considered a major factor in virus-induced autoimmunity (28). Mimicry between ANT of the heart and CVB3 has been described and associated with cardiac pathogenesis (3436). Use of anti-ANT antibodies against sucrose-purified

CVB3W, H3, and H3-1OA1 in an ELISA indicates that all three virus variants contain at least one mimicking epitope between the viruses and ANT (results not shown). We do not know whether identical ANT-mimicking epitopes are detected in all virus variants, however, since the anti-ANT represented a polyclonal antiserum. A second explanation for the absence of autoimmune effectors may reflect the preferential induction of immunoregulatory cells in H3lOAl-infected animals. Low doses of CY have been used successfully in BALB/c mice to selectively alleviate T-cellmediated suppression without inhibiting either humoral or cell-mediated immune responses (3, 13, 21). Therefore, BALB/c mice were inoculated with 5 x 104 PFU of CVB3W or H3-1OA1 and treated with CY at 50 mg/kg (Table 2). CVB3W-infected mice developed significant levels of myocarditis 7 days after infection and cytolytic lymphocytes to both infected and uninfected myocyte targets. CY treatment only slightly enhanced either parameter in these animals. In H3-1OA1-infected mice, however, myocarditis increased

TABLE 2. Effect of CY on CVB3W and H3-1OA1 infectionsa Group

CY treatment

1 (uninfected) +

2 (CVB3W infected)

+

3 (H3-1OA1 infected)

+

Heart virus titer (loglo PFU)

NAC NA 6.00 ± 5.26 7.18 ± 6.91 4.93 ± 4.51 6.29 ± 5.81

% Lysis (mean + SEM)

score Myocarditis (mean + SEM)b

Uninfected myocytes

0.58 ± 0.12 0.14 ± 0.06

0.49 0.31 2.8 ± 0.093

1.27 ± 1.51 ±0.18 0.24 ± 0.08 1.37 ± 0.

CVB3W-infected

myocytes

3.0 + 0.88 5.9 ± 2.5 24.1 0095 40.0 ± 5 35.6 ± 4.8f 36.3 ± 4.5e 33.9 ± 4.6f 2.6 ± 1.1 +

37.6 ± 5.2e

37.2 ± 5.3f

a Infected mice received 5 x 104 PFU of virus i.p. in 0.5 ml of PBS. Where indicated, mice received a single low-dose injection of CY (50 mg/kg of body weight; 0.5 ml in PBS) i.p. on the day of virus injection. Groups consisted of 7 to 11 mice each. b Scale of 0 to 4. c ND, not determined. d Significantly greater than for groups 1 and 3 (P - 0.01). Mesenteric lymph node cells were inoculated on uninfected or CVB3W-infected myocytes at an effector/target cell ratio of 100:1). e Significantly greater than for groups 1 and 3 (P s 0.001). f Significantly greater than for group, 1 (P s 0.001).

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LOUDON ET AL. TABLE 3. Adoptive transfer of spleen cells into CVB3W- and EMCV-D-infected recipients'

Recipients

Spleen cells infected transferred with: with:

No virus CVB3W

None None Normal

Anti-Thy 1.2 + complement treatment treatment

Heart virus titer (log1o) (mean t SEM)

Myocarditis score

NA NA -

ND 7.03 ± 0.08 6.89 ± 0.08 ND 7.17 ± 0.10 7.05 ± 0.08 7.06 ± 0.14 6.99 ± 0.07 7.01 ± 0.12 7.05 ± 0.09 ND ND ND ND ND

ND 1.45 ± 0.14 1.55 ± 0.14

(mean

t

SEM)'

% Lysis (mean t SEM)' Uninfected infected ~~~~~~~~ ~ CVB3Wmyocytes

~~~~~~~~~~~~myocytes

10.2 2.3d 29.0 ± 2.8 37.0 ± 2.2 38.8 ± 3.6 47.8 ± 4.8 35.5 ± 3.2 32.3 ± 4.9 61.8 ± 0.9 11.0 2.7d 45.5 ± 7.0 ND ND ND ND ND

16.0 ± 2.2 23.2 + 2.1 26.4 ± 3.0e + ND 3.0 ± 2.6e 2.29 ± 0.19 CVB3W-immune 35.5 ± 3.8e + 1.71 ± 0.46 34.3 t 3.7e H3-immune 1.58 ± 0.07 26.3 ± 4.2e + 1.84 ± 0.43 26.2 ± 0.2e 0.96 O.O9f 36.0 ± 5.6e H3-1OAl-immune + 1.63 ± 0.19 30.3 ± 2.4e 2.67 ± 0.24 NA ND EMCV-D None Normal 2.0 0.32 ND 2.1 ± 0.23 CVB3W-immune ND 2.3 ± 0.27 H3-immune ND 2.7 ± 0.25 H3-lOA1-immune ND a BALB/c CUM mice injected i.p. with 5 x 104 PFU of CVB3W or 25 PFU of EMCV-D simultaneously received 3.0 x 106 donor spleen cells either untreated or T-cell depleted, using anti-Thy 1.2 antibody plus complement injected i.v. (0.1 ml; Hanks' balanced salt solution). Number of animals per group ranged from 8 to 18. NA, not applicable; ND, not determined. b

Scale of 0 to 4. c Cell-mediated cytotoxicity, determined by using mesenteric lymph nodes on uninfected and CVB3W-infected myocytes at an effector/target cell ratio of 100:1. Significantly less than for other CVB3W-infected recipients within the uninfected-myocyte group (P - 0.01). e Significantly greater than for untreated mice (P c 0.05). f Significantly lower than for other groups (P s 0.02). d

from background levels in the absence of CY treatment to levels observed in pathogenic CVB3W-infected animals. Similarly, cytolytic activity to uninfected myocytes by cells derived from H3-1OA1-infected mice was minimal (2.6%) but rose to 37.6% in animals given both H3-1OA1 and CY. CY treatment had minimal effects on either cardiac virus titers or cytolytic activity to CVB3-infected myocytes in animals infected with either virus variant. To confirm the presence of immunoregulatory cells which prevent myocarditis in H3-1OA1-infected mice, adoptive transfer studies were performed. Donor BALB/c mice were inoculated with 5 x 104 PFU of either CVB3W, H3, or H3-1OA1 and sacrificed 7 days later. Spleen cells were retrieved from normal (uninfected) and the various infected mice. Syngeneic recipient mice were infected i.p. with either 5 x 104 PFU of CVB3W or 30 PFU of EMCV-D. Later on the same day, these recipient mice were given 2.5 x 106 donor lymphocytes i.v. In some groups, donor lymphocytes were treated with anti-Thy 1.2 antibody and rabbit complement to eliminate T lymphocytes prior to adoptive transfer into recipient animals. Additional control mice were infected with either CVB3W or EMCV-D but not given spleen cells (Table 3). All recipient animals were sacrificed 7 days after infection. CVB3W infection of recipient mice given either no or normal lymphocytes resulted in nearly equivalent amounts of cardiac inflammation and virus titers. Also, lymphocytes derived from these animals showed cytolytic activity to both infected and uninfected myocyte targets. Adoptive transfer of H3-immune cells into CVB3W-infected recipient animals had little significant effect on any of these parameters, but transfer of CVB3W-immune splenocytes actually enhanced myocarditis. In contrast, adoptive transfer of H3-1OA1-immune lymphocytes inhibited myocarditis in CVB3W-infected recipients while having no effect on virus concentrations in the heart. Interestingly, these latter animals also showed reduced cytolytic activity to uninfected myocytes although cytotoxicity to infected targets was sim-

ilar to that in other CVB3W-infected mice. T-cell depletion of the donor H3-1OA1-immune lymphocytes prior to transfer abrogated the inhibition of both myocarditis and uninfected myocyte cytotoxicity. Similar treatment of H3-immune lymphocytes enhanced cytotoxicity but had little effect on myocarditis. Presumably, pathogenicity correlates to either the presence or absence of autoimmunity in the animals. Once autoimmunity is induced, however, the severity of the myocardial injury may not be directly proportional to the level of autoimmune CTL activity in peripheral lymphoid organs. Factors such as emigration and homing of the immune lymphocytes from distal sites to target tissue may influence how much myocarditis actually occurs. While H3-1OA1-immune lymphocytes reduced myocarditis due to CVB3W infection, these cells had no effect on myocarditis induced by EMCV-D, suggesting virus specificity to this immunoregulatory activity. Finally, studies were performed to identify the T-cell subpopulation responsible for immunoregulation. Donor mice were either not infected or infected with 5 x 104 PFU of H3-1OA1 and sacrificed 7 days later. The lymphocytes were retrieved and treated with either rabbit complement alone or antibody to pan-T-cell (Thy 1.2) or CD4+ (GK 1.5), or CD8+ (2.43) T-cell populations. Recipient BALB/c mice were infected with 5 x 104 PFU of H3 and adoptively transferred i.v. with either normal lymphocytes (from uninfected syngeneic mice), H3-1OA1-immune lymphocytes, or the H3-1OA1-immune lymphocytes treated as indicated (Table 4). All animals were sacrificed 7 days later and evaluated for cardiac virus titers, myocarditis, and CTL activity to uninfected myocyte targets. Selective elimination of CD4+ T cells from the H3-lOA1-immune lymphocyte population completely reversed immunoregulation. Although often suppressor T cells belong to the CD8+ T-cell population, suppressor cells can also belong to the CD4+ lymphocyte subset (7). Presumably, the immunoregulation observed in

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5817

TABLE 4. Evidence that the immunoregulator T lymphocyte belongs to the CD4+ cell populationa

Recipients Spleen cells transferred with: with: No virus H3

Antibody

Heart virus titer

Myocarditis score

treatment

(log1o)

(mean + SEM)b

None None

None None

0 ± od 5.75 ± 0.08

0 ± od 1.33 ± 0.29

Normal lymphocytes

None None

6.29 ± 0.19 5.77 ± 0.14

1.24 + 0.43

C' only Anti-Thy 1.2 + C' Anti-CD4 + C' Anti-CD8 + C'

6.31 6.06 5.78 5.54

H3-lOA1-immune lymphocytes

± 0.33 + 0.21 ± 0.17 ± 0.20

% Lysis, uninfected

myocytes ~~~~~~~~~~~~~~~~~~~~~~~~ ±SEM)C 4.8 ± 5.8d 39.8 ± 7.6

55.4 ± 7.5

0.5

0.35d

9.3

0.61 1.11 1.24 0.57

± 0.49d ± 0.55 ± 0.25

5.6 56.5 59.9 0

± 0.38d

15.6d ± 6.5d ± 7.8 ± 5.2

± od

a BALB/c CUM mice were injected i.p. with 5 x 104 PFU of H3 and simultaneously received 2.5 x 106 donor spleen cells from either uninfected (normal lymphocytes) or H3-1OA1-infected (H3-lOA1-immune lymphocytes) syngeneic donor mice. The H3-1OA1-infected donor mice had received 5 x 104 PFU of H3-1OA1 i.p. 7 days before sacrifice. Donor lymphocytes were treated with 100 Fg of MAb to Thy 1.2 (pan-T-cell marker), CD4 or CD8 T-cell markers, and 20% rabbit complement (C') to remove selected T-cell subpopulations prior to adoptive transfer of the cells. Control groups received H3-1OA1-immune lymphocytes which were untreated or treated with complement only but no antibody. Results are for group of three to nine animals each. b Scale of 0 to 4. c Effector/target cell ratio of 100:1. d Significantly lower than for recipients given H3 but with no spleen cells transferred at P s 0.05.

our model is mediated through these CD4+ suppressorinducer cells.

DISCUSSION Preparations of many viruses contain variants differing widely in pathogenicity (4, 44). Often the pathogenicity of a particular variant will depend on the virus's ability to infect specific cells or tissues (tropism) (15, 18, 25), resemble self-epitopes leading to induction of autoimmunity (antigenic mimicry) (28, 31, 38), or alter cytokine or lymphokine induction in either infected or neighboring tissue cells (44) and in the cells of the inflammatory response. Previously, we described the isolation of an attenuated variant of CVB3 by using a MAb that distinguishes between myocarditic and nonmyocarditic CVB3 variants (37). This attenuated variant (H3-lOA1) differed from the pathogenic parent (H3) in several ways. First, H3-1OA1 is less effective in interfering with the cellular metabolism of infected cells. Second, less virus is produced in infected myocyte cultures, suggesting that the efficiency of replication might be lower. Third, H3-1OA1infected mice show dramatically reduced animal mortality and develop less myocarditis than do H3-infected animals. The former two characteristics of the attenuated virus might certainly explain the third observation. A virus which is inefficient in either causing direct cellular dysfunction or producing high concentrations of progeny virus to spread the infection into greater numbers of cells might be more easily eliminated and cause a restricted amount of damage in the interim. However, in the present case, this does not appear to be the explanation for attenuation of H3-1OA1. Rather, the escape mutant virus triggers an immunoregulatory T-cell response which is either absent or weaker in the H3-infected

animals. Numerous studies document the induction of autoimmunity resulting from infections with various viruses (1, 5, 8, 17), and several different mechanisms for this phenomenon have been implicated. Inadvertent induction of class I or class II major histocompatibility complex antigens on peripheral infected tissues may result from lymphokines released during viral immune responses (2). This allows the constituents of these tissues to act as antigen-presenting cells not only for virus antigens (thus aiding in virus immunity and clearance of the infectious agents) but also for

self-peptides (resulting in autoimmunity) (11). Additionally, when infectious agents share conserved peptide sequences with cellular molecules, immunity to the microbe will often result in autoreactivity as well (28, 31, 38). In a third mechanism, some viruses not only infect lymphoid cells but differentiate between different cell subpopulations. EpsteinBarr virus, for example, recognizes receptors on CD8+ lymphocytes, a subpopulation usually associated with both cytotoxic and suppressor T-cell functions (33). Presumably, selective infection of an immunoregulatory cell population might disrupt normal mechanisms of self-tolerance, resulting in either nonspecific immunosuppression (if the virus induces polyclonal stimulation of suppressor cells) or autoimmunity (if the infection induces suppressor cell dysfunction). A fourth mechanism may involve aberrations in cytokine and lymphokine production. Certain of these agents potently influence immunoregulation. For example, beta interferon selectively blocks suppressor cell responses (32). Similarly, known T-lymphocyte responses require two signals to trigger proliferation (39, 40). One signal is the antigenic peptide associated with the appropriate major histocompatibility complex molecule. The second signal is presumably a cytokine such as interleukin-1. Several studies indicate that antigenic presentation in the absence of the secondary signal results in tolerization of the antigen-specific T lymphocyte (20, 23). Thus, should any virus infection alter production of either interleukin-1 or other signal cytokines, immunoreactivity could be detrimentally affected. Variants of the picornavirus EMCV have distinct pathogenic activities and have already been shown to differ in cytokine induction (44). EMCV-D, which is a highly diabetogenic variant, is a poor inducer of interferon. EMCV-B, which is a nondiabetogenic variant, is a high inducer of interferon. Similarly, Palmenberg and colleagues (29a) report that pathogenic and nonpathogenic variants of EMCV may have differential effects on the infection and biological function of macrophage, specifically upon the ability of infected macrophage to produce cytokines during immunological responses. In the CVB3 myocarditis model, infection with pathogenic variants induces an autoimmune CTL response which is directly responsible for cardiac injury in BALB/c mice (17). Two nonpathogenic CVB3 variants have been evaluated,

5818

LOUDON ET AL.

and in each case, infection with the variant fails to elicit the autoimmune response (6). Interestingly, both nonpathogenic variants, CVB3C and H3-1OA1, contain appropriate signals for autoimmunity induction, but the autoimmune signal is overridden by a signal for immunoregulation. This is shown by CY treatment of H3-1OA1-infected mice, which relieves the immunosuppression and allows both development of the autoimmune CTL and expression of myocarditis. Similar treatment of the H3-infected animals which lack immunoreg-

ulatory cells has little effect on either CTL or cardiac injury. This finding suggests that the enhanced responses in H3lOAl-infected mice do not result from nonspecific mechanisms of immunoenhancement. The nature of the autoimmune signal is not conclusively known. However, since both pathogenic and nonpathogenic CVB3 variants show equivalent cross-reactivity with ANT-specific antibodies, and since immunization with ANT is capable of triggering autoimmune heart disease, presumably autoimmune CTL could arise due to the antigenic mimicry between the virions and selfmolecules. The question remains whether H3 infections actively deactivate immunoregulatory cells (which are designed to prevent autoimmunity to cardiac antigens and are normally present in the uninfected host) or whether H3-1OA1 infections actively induce such immunoregulatory responses de novo. In the former instance, one might hypothesize that H3 virus either infects the immunoregulatory cell population while H3-1OA1 virus does not (difference in cellular tropism), or both virus variants infect the immunoregulatory cells but the greater efficiency of H3 in inhibiting cellular metabolism results in only the pathogenic variant deactivating these cells. Similarly, should H3 induce higher beta interferon responses, this cytokine might act to eliminate suppressor cell function. Experiments are presently under way to investigate these possible explanations. ACKNOWLEDGMENT We thank Laurie Sabens for help in the preparation of the

manuscript.

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