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Role of CD1d in Coxsackievirus B3-Induced Myocarditis ¹

Sally Huber^2,*, Danielle Sartini^* and Mark Exley

^* Department of Pathology, University of Vermont, Burlington, VT 05446; and Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215

	Abstract

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The myocarditic (H3) variant of Coxsackievirus B3 (CVB3) causes severe myocarditis in BALB/c mice and BALB/c mice lacking the invariant J281 gene, but minimal disease in BALB/c CD1d^-/- animals. This indicates that CD1d expression is important in this disease but does not involve the invariant NKT cell often associated with CD1d-restricted immunity. The H3 variant of the virus increases CD1d expression in vitro in neonatal cardiac myocytes whereas a nonmyocarditic (H310A1) variant does not. V4⁺ T cells show increased activation in both H3-infected BALB/c and J281^-/- mice compared with CD1d^-/- animals. The activated BALB/c V4⁺ T cells from H3-infected mice kill H3-infected BALB/c myocytes and cytotoxicity is blocked with anti-CD1d but not with anti-MHC class I (K^d/D^d) or class II (IA/IE) mAbs. In contrast, H3 virus-infected CD1d^-/- myocytes are not killed. These studies demonstrate that CD1d expression is essential for pathogenicity of CVB3-induced myocarditis, that CD1d expression is increased early after infection in vivo in CD1d⁺ mice infected with the myocarditic but not with the nonmyocarditic CVB3 variant, and that V4⁺ T cells, which are known to promote myocarditis susceptibility, appear to recognize CD1d expressed by CVB3-infected myocytes.

	Introduction

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Myocarditis is an inflammation of the myocardium, which often follows microbial infections (1). Two variants of Coxsackievirus B3 (CVB3)³ have been used to study myocarditis in BALB/c mice. One variant, designated H3, causes severe cardiac inflammation and high animal mortality (2). The second variant, designated H310A1, differs from the H3 virus by a single amino acid mutation in the VP2 capsid protein and induces minimal myocarditis and mortality (3, 4). Why this single amino acid difference between H3 and H310A1 viruses should have such a strong biological effect on disease susceptibility is not clearly understood, but may partially depend upon differences in activation of innate immunity represented by proinflammatory cytokines and ⁺ T cells. H3 infection of cultured monocytes induces strong TNF- secretion which is not observed with H310A1 (5). TNF- is necessary for CVB3 pathogenicity (6, 7, 8). A second difference between H3 and H310A1 virus infections is that only the former virus activates V4⁺ T cells, which promote myocarditis susceptibility (9). ⁺ cells comprise up to 50% of the T cell infiltrate in the hearts of H3-infected animals (8, 10). As with TCR⁺ cells, ⁺ T cells can be distinguished by their variable (V) gene usage. V1⁺ T cells induce myocarditis resistance while V4⁺ T cells induce susceptibility (9, 11). In the present studies, we show that H3 virus infections up-regulate expression of CD1d (a nonpolymorphic MHC I-like molecule often associated with innate immunity and control of infectious agents (12, 13, 14, 15, 16, 17, 18) both in vitro on infected myocytes and in vivo in the heart. V4⁺ cells isolated from H3 virus-infected BALB/c mice kill H3 virus-infected myocytes by recognition of CD1d in vitro. Since the classical invariant NKT (J281⁺) cells are not involved in CVB3-induced myocarditis and since V4⁺ cells have already been shown to determine myocarditis susceptibility, we hypothesize that the requirement for CD1d in this disease is probably functioning at the level of the V4⁺ cells.

	Materials and Methods

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Mice

Male BALB/cJ, BALB/c-CD1d KO (CD1d^-/-), and BALB/c J281^-/- mice, 6–8 wk of age, were used in these experiments. BALB/cJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Breeding pairs of BALB/c CD1d^-/- mice were kindly supplied by Dr. M. Grusby (Harvard School of Public Health, Boston, MA); breeding pairs of BALB/c J281^-/- mice were kindly supplied by Dr. M. Taniguchi (Chiba University, Chiba, Japan, and currently RIKEN, Japan).

Virus, virus infection, and virus titration

Animals were infected by i.p. injection of 0.2 ml of PBS containing 10⁴ PFU of CVB3 H3 or H310A1 variants as described previously (9). Virus titers in tissues or in in vitro cell populations were determined by the plaque-forming assay as described earlier. Infectious center assay was used to determine the percentage of myocytes infected (19).

Antibodies

Ab class control (isotype control) and Ag-specific Abs were obtained from BD PharMingen (San Diego, CA). These included: PE-conjugated anti-CD3 (clone 17A2); purified rat anti-mouse CD16/CD32 (Fc Block; clone 2.4G2); CyChrome, FITC, and PE-rat IgG1 (clone R3-34); FITC- and PE-conjugated anti-mouse IFN- (clone XMG 1.2); PE-rat anti-mouse IL-4 (clone BVD4-1D11); PE-hamster anti-mouse CD69 (clone H1.2F3); CyChrome-rat anti-mouse CD4 (clone GK 1.5); purified, FITC, and PE-hamster IgG; purified and FITC anti-mouse CD1d (clone 1B1); purified anti-mouse H-2D^d (clone 34-2-12); purified anti-mouse H-2K^d (clone SF1-1.1); purified anti-mouse IA/IE (clone 2G9); and FITC-anti- TCR (clone GL3). FITC-conjugated mAbs to V1 (clone 2.11) and V4 (clone UC3) were prepared and tested in the laboratory of Dr. R. O’Brien (National Jewish Medical and Research Center, Denver CO). PE-streptavidin was purchased from BD PharMingen.

Isolation of splenic lymphocytes

Isolation of cell populations has been described previously (9). Briefly, spleens were pressed through fine mesh screens, centrifuged on Histopaque-1077 (Sigma-Aldrich, St. Louis, MO), to remove RBC, and incubated on nylon wool for 30 min at 37°C. The nonadherent cells were retrieved. To isolate enriched V1⁺ or V4⁺ cells, splenocytes after nylon wool incubation were depleted of CD4⁺ and MHC class II (IA/IE⁺) cells using anti-CD4 and anti-IA/IE Ab treatment of the cells followed by incubation with anti-rat IgG-conjugated magnetic particles. The remaining cells were divided into three different tubes and incubated in 500 µl of PBS-1% BSA containing a 1/50 dilution of either FITC anti-V4 and PE anti-CD3 (tube 1) or anti-V1 and PE-anti-CD3 (tube 2) for 20 min on ice, washed, and resuspended in RPMI 1640 containing 10% FBS (Life Technologies, Grand Island, NY) and sterile sorted.

Isolation of neonatal myocytes

Isolation of neonatal mouse myocytes has been described previously (20). Briefly, hearts are obtained from mice within 72 h of birth, minced finely, and subjected to sequential digestion with 0.25% pancreatin (Life Technologies) and 0.4% collagenase (Worthington Biochemical, Freehold, NJ). The single-cell suspension was washed and depleted of endothelial cells and fibroblasts by two sequential 1-h adsorptions to plastic. The nonadherent population was retrieved and plated into 1-mm tissue culture wells (Falcon Plastics, Oxnard, CA) at 1 x 10 ³ cells/well. After 48 h at 37°C in a humidified 5% CO₂ incubator, the wells were used for cytotoxicity assays.

Confocal microscopy

BALB/c and CD1d^-/- mice were uninfected or were infected i.p. with H3 or H310A1 virus and then killed 3 days later. Hearts were perfused with PBS, snap frozen in Tissue Freezing Medium (TBS; Triangle Biological Sciences, Durham, NC) in liquid nitrogen, then sectioned using a TBS cryostat into 10-µm sections and placed on glass slides (Fisher Superfrost Plus; Fischer Scientific, Pittsburgh, PA). Tissue was fixed for 10 min in -20°C acetone, rehydrated in PBS containing 1% BSA for 20 min, incubated for 30 min in PBS-BSA containing 1% normal goat serum, and then incubated for 1 h in PBS-BSA containing a mixture of 1/100 dilutions of mouse anti-CVB3 (IgM, clone 8A6; Ref.21), anti-CD1d (rat IgG, clone 1B1; BD PharMingen) and anti-muscle -actin (mouse IgG, clone 5F8; InnoGenex, San Ramon, CA). The tissues were washed and incubated with PBS-BSA containing a mixture of 1/100 dilutions of Cy5-anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA), Alexa 568 anti-mouse IgG and Alexa 488 anti-rat IgG (Molecular Probes, Eugene OR) for 1 h, washed three times, and evaluated using a Bio-Rad MRC 1000 confocal scanning laser system (Hercules, CA) mounted to an Olympus BX 50 microscope (Melville, NY) using an iris setting of 2.0.

Cell-mediated cytotoxicity assay

The cell-mediated cytotoxicity assay has been described in detail previously (20). Infected targets were incubated with 100 multiplicity of infection virus for 1 h, washed, and incubated with fresh medium for 12 h. Targets were labeled with 100 µCi of ⁵¹Cr (Na₂⁵¹CrO₄; ICN Pharmaceuticals, Irvine, CA) for 2 h at 37°C, washed four times, and cultured for 12 h at 37°C with 1 x 10⁴ lymphocytes (E:T ratio, 10:1). Supernatants were removed and counted in a Packard Gamma Counter (Packard Instrument; Downers Grove, IL). Pellets were lysed using 6 N HCl and the pellets were removed and counted. Percent ⁵¹Cr release was calculated as: cpm in supernatant/(cpm in supernatant +cpm in pellet) x 100 for each well. Percent specific lysis represents: (percent ⁵¹Cr release in experimental wells) - (percent ⁵¹Cr in wells without lymphocytes; medium control wells). In some experiments, soluble recombinant Fas-Fc (10 µg/ml; R&D Systems, Minneapolis, MN) or concanamycin A (CMA, 100 nM; Sigma-Aldrich) was added to the assay wells.

Flow cytometry

Myocytes were labeled with 1/100 dilutions of FITC anti-V1 or FITC-anti-V4 Ab for 20 min at 4°C. The cells were washed in PBS-1% BSA and fixed for 10 min in 2% paraformaldehyde, then incubated with a 1/100 dilution mouse anti--actin, followed by incubation with a 1/100 dilution of PE-anti-mouse IgG. For labeling of cell surface markers (CD3, CD4, V1, V4, CD69), cells were incubated in PBS-1% BSA containing a 1/100 dilution of the relevant fluorochrome-labeled Abs and a 1/100 dilution of Fc-Block for 20 min at 4°C, washed twice with PBS-BSA, and resuspended in 2% paraformaldehyde. For intracellular cytokine staining, cells were cultured in medium containing 10 µg/ml brefeldin A, 50 ng/ml PMA, and 500 ng/ml ionomycin (Sigma-Aldrich) for 4 h at 37°C in 5% CO₂. The cells were washed once in PBS-BSA containing brefeldin A and resuspended in PBS-BSA-brefeldin A containing a 1/100 dilution of CD4 for 20 min at 4°C, washed, and fixed in 2% paraformaldehyde for 10 min. The cells were washed once in PBS-BSA buffer containing 0.05% saponin and resuspended in PBS-BSA-saponin containing 1/100 dilutions of Fc Block, FITC-anti-IFN-, and PE-anti-IL-4 Abs and 50 µg/ml rat polyclonal IgG (Zymed Laboratories, San Francisco, CA). After incubation for 30 min, the cells were washed twice in PBS-BSA-saponin, then once in saponin-free PBS-BSA to close the membrane, and resuspended in PBS containing 2% paraformaldehyde. Positive controls for cytokine staining were 2.5 x 10⁵ of either MIC-1 (IFN-) and MIC-2 (IL-4) fixed cells obtained from BD PharMingen. Negative controls were fluorochrome-conjugated species- and isotype-matched Ig combinations as used in specific Ab-staining samples. Stained cell populations were analyzed using a Coulter Epics Elite instrument with a single excitation wavelength (488 nm) and band filters for PE (575 nm), FITC (525 nm), Red613 (613 nm), and CyChrome (670 nm). Each sample population was classified for cell size (forward scatter) and complexity (side scatter), then gated on a population of interest. At least 10,000 cells were evaluated for each sample. Criteria for positive staining were established using isotype controls. The results are expressed as the percentage of total splenocytes staining for a particular marker minus the isotype control.

Histology

Hearts were removed, fixed in 10% buffered Formalin, paraffin embedded, sectioned, and stained with H&E. Stained sections were evaluated for myocarditis as described previously (9).

Statistics

Statistical evaluation was performed using the Wilcoxon ranked score method. Each experiment was repeated at least two times.

	Results

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Myocarditis in BALB/c, BALB/c CD1d^-/-, and BALB/cJ281^-/- mice

Male mice were infected with H3 virus and surviving animals were euthanized 7 days later. Cumulative mortality was 67% (10/15) for wild-type, 50% (5/10) for J281^-/-, and 25% (2/8) for CD1d^-/- mice. No significant difference was observed between the strains in cardiac virus titers (Table I); however, myocarditis was significantly reduced in CD1d^-/- animals (0.5% of the myocardium inflamed compared with 9.3% for BALB/c mice). CD1d^-/- mice also had decreased numbers of CD4⁺ IFN-⁺, but greater numbers of CD4⁺IL-4⁺ cells in the spleen (Table I). Total numbers of splenic CD4⁺ cells were not significantly different between wild-type (38.9%) and CD1d^-/- (42.1%) mice, indicating that reduced CD4⁺ IFN-⁺ numbers do not reflect selective loss of CD4⁺ cells in the CD1d^-/- mouse. We have previously shown that V4⁺ cells promote myocarditis susceptibility (9). Wild-type BALB/c and J281^-/- mice showed increased numbers of V4⁺ cells compared with infected CD1d^-/- mice, and most of the V4⁺ cells are positive for the early activation marker CD69. Uninfected BALB/c, J281^-/-, and CD1d^-/- mice had <0.01% CD69⁺ cells, indicating that infection is necessary for activation marker induction. The data shown in Table I represents one of three experiments performed. Results obtained in replicate experiments was similar. These studies demonstrate that H3 virus fails to activate V4⁺ cells or induce myocarditis in mice lacking CD1d, whereas mice lacking the classical J281⁺ NKT cell as well as wild-type mice are susceptible to myocarditis.

Since H3 only induces myocarditis in CD1d⁺ mice, the next studies investigated whether CD1d expression alters in vivo and in vitro during H3 and H310A1 infection. In initial studies, neonatal cardiac myocytes were isolated, infected with 100 PFU of virus/cell for 18 h, then labeled with PE-anti--actin and FITC anti-CD1d. After gating on the -actin-positive population, the percentage of myocytes positive for CD1d was determined (Fig. 1). Approximately 37.2% of H3 virus-infected BALB/c myocytes expressed CD1d compared with <2% of either uninfected or H310A1 virus-infected cells. To confirm specificity of the CD1d labeling, myocytes were isolated from CD1d^-/- mice and infected with H3 virus. None of these myocytes were labeled with anti-CD1d Ab above the isotype control level.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. CD1d expression in neonatal myocytes. Myocytes were cultured from 3-day-old BALB/c (A–D) and CD1-/- (E) neonatal mice. Some cells were uninfected (B) while the remaining cells were infected with 100 multiplicity of infection of H3 virus (A, C, and E) or H310A1 virus (D) for 18 h. Cells were labeled with FITC-anti-CD1d, fixed with paraformaldehyde, and then resuspended in buffer with saponin and mouse anti--actin and PE-anti-mouse IgG. Flow analysis was gated on the -actin-positive cell population which represented >80% of the total cells. Graphs indicate CD1d staining of these -actin-positive cells. The isotype control represents infected myocytes incubated with FITC-rat IgG and was set so that between 1 and 2% of cells fall in the positive range. Numbers in B–E indicate the percentage of CD1d+ cells above isotype control.

Next, BALB/c and CD1d^-/- mice were infected with either H3 or H310A1 virus. On day 3 after infection, the hearts were perfused with PBS, snap frozen, and sectioned for confocal microscopy (Fig. 2). The myocardium was visualized using anti--actin and Cy3-conjugated anti-mouse IgG Ab (red, stains myocardial cells). Virus-infected cells were identified using monoclonal anti-CVB3 Ab (mouse IgM) and Cy5-anti-mouse IgM (µ-chain specific; blue). CD1d expression was determined using anti-CD1d (rat IgG) and Alexa 488 anti-rat IgG Ab (green). Uninfected BALB/c mice showed actin staining but neither virus nor CD1d. Both H3-infected CD1d^-/- and H310A1-infected BALB/c hearts showed infected myocytes (purple due to colocalization of blue and red), but no significant CD1d expression. In contrast, hearts from H3-infected BALB/c mice showed substantial numbers of CD1d⁺ cells. Although CD1d is associated with virus-positive cells, other CD1d⁺ cells are not myocytes and do not appear to be infected. These may represent CD1d⁺ inflammatory cells rapidly infiltrating the heart during H3 virus infection. Double-color analysis of CD1d (green) and virus (blue) provides better visualization of virus-positive and virus-negative CD1d⁺ cells without the complication of -actin staining (Fig. 3). Analysis of hearts at a later time (day 6 after infection) showed more widespread CD1d⁺ cells in H3-infected BALB/c mice but few virus-positive cells. Again, few CD1d⁺ cells were seen in H310A1-infected BALB/c hearts at this time (data not shown).

View larger version (63K): [in this window] [in a new window]   FIGURE 2. Confocal microscopy of infected hearts. Mice (BALB/c or CD1d-/-) were either uninfected or infected 3 days previously with H3 (myocarditic) or H310A1 (nonmyocarditic) variants of CVB3. Hearts were perfused, snap frozen, and sectioned. All sections (uninfected BALB/c, H3-infected BALB/c, H3-infected CD1-/-, and H310A1-infected BALB/c) were incubated with buffer containing 1/100 dilutions of mouse IgG anti--actin (red), mouse IgM anti-CVB3 (blue), and rat IgG anti-CD1d (green). The sections were washed and then incubated with buffer containing 1/100 dilutions of Cy5-goat-anti-mouse IgM (µ-chain specific), Cy3-goat-anti-mouse IgG and Alexa 488 goat-anti-rat IgG. Secondary control represents H3 virus-infected BALB/c myocardium incubated with the three fluorochrome-labeled secondary Abs only.

View larger version (84K): [in this window] [in a new window]   FIGURE 3. Confocal analysis of H3-infected BALB/c mice from Fig. 2 for virus (blue) and CD1d (green) only. Cells assumed to represent infiltrating CD1d+ inflammatory cells are marked (CD1d+).

Lysis of H3-infected myocytes by V4⁺ cells

The following studies investigated whether V1⁺ or V4⁺ effector cell populations could directly kill infected myocyte targets in vivo. V1⁺ and V4⁺ effector cell populations were isolated from uninfected BALB/c mice or from BALB/c or CD1d^-/- mice infected 7 days earlier with either H3 or H310A1 virus. Fig. 4 shows representative purity of the sorted effector cell populations. Effectors were assayed on ⁵¹Cr-labeled H3 virus-infected myocyte targets. Fig. 5A shows that only V4⁺ cells from H3-infected BALB/c mice showed cytotoxicity to the infected targets. Neither V1⁺ cells isolated from H3-infected BALB/c mice nor V4⁺ cells from uninfected BALB/c mice were cytolytic. This indicates that not all ⁺ cell subpopulations from H3 virus-infected mice kill cardiac myocytes and that virus infection of V4⁺ cell donors is required to activate cytotoxic potential. In a second set of experiments, V4⁺ cells were isolated from H3-infected BALB/c donors and assayed for cytotoxicity against BALB/c (uninfected, H3, or H310A1 infected) and CD1d^-/- (H3 infected) myocyte targets (Fig. 5B). The V4⁺ effectors were cytolytic to the H3-infected BALB/c (CD1d⁺) but not to CD1d^-/- myocytes. Also, no cytotoxicity was observed to either uninfected or H310A1 virus-infected BALB/c myocytes. One possibility is that V4⁺ cells from H3-infected mice do not kill H310A1 virus-infected targets due to viral specificity of the V4⁺ cell populations. In Fig. 5C, V4⁺ cells were isolated from H310A1-infected BALB/c mice and assayed for cytotoxicity to both H3 and H310A1 virus-infected myocytes. The V4⁺ cells from H310A1 virus-infected donors were not significantly lytic to either infected target. Thus, not only do the effectors need to come from H3-infected donors, but these cells can distinguish between uninfected and H3-infected targets.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Post-sort of CD3+V1+ and CD3+V4+ lymphocytes from H3 virus-infected BALB/c mice. Number indicates percentage of lymphocytes that are positive for both CD3 and indicated V TCR.

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Cytotoxicity of uninfected and virus-infected neonatal cardiac myocytes by purified lymphocyte subpopulations: A,V1+ and V4+ cell populations were isolated from uninfected, H3-, or H310A1-infected BALB/c mice and from H3-infected CD1-/- mice 7 days after infection as described in Materials and Methods. Neonatal cardiac myocytes were isolated from 3-day-old BALB/c mice infected with H3 virus at 100 PFU/cell for 12 h, then incubated with the effector cells at an E:T ratio of 10:1 and incubation time of 6 h. B, Neonatal cardiac myocytes were isolated from 3-day-old BALB/c or CD1-/- mice. Cells were either uninfected or infected with 100 PFU/cell H3 or H310A1 virus for 12 h, then incubated with V4+ cells obtained from H3-infected BALB/c mice 7 days after infection. C, Neonatal BALB/c cardiac myocytes were uninfected or infected with either H3 or H310A1 virus, then incubated with V4+ cells isolated from H310A1 virus-infected BALB/c mice 7 days after infection. Results represent the mean percent lysis of four or more mice per group ± SEM in one of two replicate experiments.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Blocking of cell-mediated cytotoxicity using mAbs. Anti-CD1d, anti-H-2Dd, anti-H-2Kd, anti-CD1d, and anti-IA/IE mAbs were added at 1 µg/ml to wells containing purified CD3+V4+ cells from H3 virus-infected BALB/c mice and BALB/c neonatal myocytes infected with H3 virus for 12 h in a cell-mediated cytotoxicity assay. The E:T ratio was 10:1 and the incubation time (lymphocytes and myocyte targets) was 6 h. Results represent the mean percent lysis in four wells per group ± SEM in one of two replicate experiments. *, Different from groups containing effectors without Ab at p < 0.05.

View larger version (13K): [in this window] [in a new window]   FIGURE 7. Blocking of cytotoxicity with CMA and Fas-Fc. Purified CD3+V4+ cells were isolated from H3 virus-infected BALB/c mice and assayed at a 30:1 E:T ratio on H3-infected (CD3+V4+ effectors only) with either 100 nM CMA or 10 µg/ml Fas-Fc. Results represent the mean cytotoxicity ± SEM of four replicate cultures per group. *, Data are significantly different from cultures without CMA or Fas-Fc at p 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

MHC class I-like CD1 molecules are involved in innate immunity to many infectious agents (12, 13, 14, 15, 22, 23, 24, 25). Most often, CD1d-restricted cells belong to the invariant NKT subpopulation (12, 13, 14, 15, 22, 24, 26), although T cells expressing noninvariant TCR have also been reported to be CD1 restricted (26, 27, 28). Finding a CD1d-restricted murine CTL belonging to the ⁺ T cell population extends current knowledge regarding these diverse types of CD1d-restricted immune responses in the mouse (29, 30). CD1d-restricted T cells can make either Th1 (IFN-) or Th2 (IL-5, IL-4) cytokines, depending upon the model system used (14, 26, 30, 31). Although cytokine bias of the CD1d-restricted effector might reflect an inherent characteristic of the T cell, CD1d is expressed on dendritic and other APCs, and the APC may be the determining factor in T cell cytokine bias (23, 32, 33). Our studies in CVB3-induced myocarditis resemble those in hepatitis C virus-infected humans (30), in which CD1-restricted intrahepatic lymphocytes were increased in patients with chronic hepatitis C infections, but cells expressing the invariant TCR were rare. The CD1d-restricted intrahepatic lymphocytes were strongly Th1 biased and cytolytic. Despite TCR utilization by NKT cells for CD1d recognition, it is not certain whether CD1d recognition by V4⁺ T cells is through the TCR. This point will need further investigation.

Generally, CD1 molecules bind hydrophobic lipids or peptides (17). In bacterial infections, the lipid Ags can derive from the bacterial membrane. This cannot be true of CVB3 because these are nonenveloped viruses. However, CVB3-infected cells might release glycolipid Ags, which could be exogenously taken up by target CD1d⁺ cells, or glycolipids endogenous to the CD1d⁺ cell might provide the relevant Ag. A hydrophobic virus peptide is also a possible candidate for the CD1d-restricted Ag. A large number of CD1d⁺ cells rapidly appear in the hearts of H3-infected BALB/c mice. The nature of these cells in not known, but presumably represent various lymphoid cells infiltrating the heart and may even be the ⁺ cells themselves (16, 34).

V4⁺ effectors could directly induce cardiac injury since these cells are cytolytic to infected myocytes in vitro. It is not clear how much of the total myocyte destruction results from the V4⁺ effector. Certainly, ⁺ cells comprise a large proportion of the inflammatory cells in myocarditis (8). We have shown that up to 50% of the lymphoid cells recoverable at the time of peak myocarditis are ⁺ TCR⁺ and that these effectors are strongly positive for Fas ligand expression. However, maximum Coxsackievirus replication is observed between 3 and 4 days after infection and the infectious virus is rapidly cleared by day 10 (35). Since V4⁺ cells kill infected but not uninfected myocytes in vitro, presumably damage mediated by this effector should be limited to periods of virus infection. If persistent virus infections are established, as has been reported by some investigators in CVB3-induced heart disease (36), the amount of cytotoxicity may be significant. V4⁺ cells kill CD1d⁺ H3-infected neonatal myocytes in vitro. It is not known whether they can also kill adult myocytes. Unfortunately, isolation of viable adult myocytes is extremely difficult, if not impossible. Although we infer that V4⁺ cells also kill adult cells of the same type, this may not be true. An alternative hypothesis is that V4⁺ cells affect myocarditis susceptibility in more than one way. As reported earlier and as shown in Table I, myocarditis susceptibility correlates to preferential activation of CD4⁺ Th1 (IFN⁺) cell responses (9). Both NKT and ⁺ cells are known to produce pro- and anti-inflammatory cytokines as part of the innate immune response and influence developing adaptive immunity (14, 30, 37, 38). The V4⁺ cells in H3 virus-infected BALB/c mice are strongly IFN-⁺ and production of this cytokine is important for their ability to bias CD4⁺ T cell responses toward a Th1 phenotype (9). It is presently not known whether V4⁺ cells induced in CD1d⁺ mice and apparently recognizing CD1d Ag are primarily pathogenic through their direct ability to kill infected cells or through their ability to modulate adaptive immunity through production of IFN- early in the infection.

The cytolytic V4⁺ cell population is primarily (>80%) CD4^-CD8⁺. The V1⁺ cell population is primarily CD4^-CD8^- (>95%). However, in both populations, a minority of other phenotype cells are present (20% CD4^-CD8^- V4⁺ cells and 5% CD4^-CD8⁺V1⁺ cells). No CD4 expression was noted in either sorted cell population. It is not known what proportion of the total V4⁺ cells isolated from H3-infected BALB/c mice actually modulate myocarditis. This is likely to be a heterogeneous population with regard to V gene usage and only a specific V4⁺ V population might prove pathogenic. Positive selection of NKT cells during ontogeny involves CD1d (39, 40), like conventional T cell MHC restriction (41, 42). Although V4⁺ cells are found in CD1^-/- mice, it is possible that the specific CD1d-reactive V4⁺ subpopulation may be absent rather than not activated.

A remaining question is why the single amino acid mutation between the H3 and H310A1 viruses had such a dramatic effect on CD1d expression in vitro and in vivo in the heart. Some reports find that multiple cytokines can up regulate CD1d expression on various tumor cell lines including IFN-, IL-4, IL-10, and GM-CSF (31). Other investigators find constitutive expression of CD1d without marked enhancement by these cytokines (34, 43, 44), although activation of T cells can up-regulate CD1d expression through unknown mechanisms (45). Since infections with H3 virus induce high expression of at least certain cytokines (TNF-; Ref.5), it is possible that one or more of these factors could increase CD1d expression.

	Acknowledgments

 
We thank Dr. Steven P. Balk for advice and discussions on this work, Dr. Michael Grusby for the BALB/c CD1d^-/- mice, and Dr. Masuru Taniguchi for the J281^-/- mice. We also thank Drs. O’Brien and Born for the mAbs to V1 and V4 TCR. We thank Colette Charland for expert technical assistance with flow analysis and Kevin Kolinich for help in preparation of this manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants HL 58583 (to S.A.H.), 1P01 AI45666-01, and CA89567 (to M.A.E.).

² Address correspondence and reprint requests to Dr. Sally A. Huber, Department of Pathology, University of Vermont, 208 South Park Drive, Suite 2, Colchester, VT 05446. E-mail address: Sally.Huber{at}uvm.edu

³ Abbreviations used in this paper: CVB3, Cocksackievirus B3; CMA, concanamycin A.

Received for publication July 13, 2002. Accepted for publication January 3, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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