Vγ1⁺ T Cells Suppress and Vγ4⁺ T Cells Promote Susceptibility to Coxsackievirus B3-Induced Myocarditis in Mice¹

Myocarditis represents an inflammation of the heart muscle and usually follows microbial infections (1). Although the pathogenesis of myocardial injury may be complex, with both immune and microbial-induced damage occurring, substantial clinical and experimental evidence indicates that autoimmunity directed at heart Ags such as cardiac isoforms of myosin produces significant disease (2, 3, 4, 5). However, not all individuals infected with a specific pathogen will develop myocarditis. Clearly, the genetic composition of the host is important in determining disease susceptibility (6, 7). At least part of this genetic susceptibility is determined by the type of immune response generated by the host. Studies by Rose and his colleagues (8) demonstrated that exogenous administration of IL-1 and TNF-α to myocarditis-resistant mice infected with a myocarditic Coxsackievirus B3 (CVB3)³ variant restored disease susceptibility. This laboratory has demonstrated that myocarditis susceptibility additionally depends upon activation of T cells expressing the γδ TCR (9, 10, 11, 12).

T cells can be defined by the type of TCR they express. Most Ag-specific T cells have a TCR consisting of an α- and a β-polypeptide chain. In the periphery, a small proportion of T cells express a receptor consisting of a γ and a δ polypeptide chain. Although the role of αβ⁺ T cells in most immunological diseases is clear, the role for γδ⁺ T cells is usually more obscure. γδ⁺ T cells often accumulate at sites of inflammation, whether caused by viral (13, 14, 15), bacterial (16), or parasite (17, 18). In some cases, these responses are subdivided by variable (V) gene expression of the γ- and δ-chains, such that there are distinct patterns of infiltration into inflammation sites depending upon the disease (17, 19, 20, 21, 22). This suggests that only specific γδ⁺ cell populations may participate in disease processes. How γδ⁺ cells participate in disease is also complex, as both beneficial and detrimental roles have been attributed to these cells in various experimental models (23, 24, 25, 26). Previously, we have demonstrated that different subpopulations of γδ⁺ T cells, based on their Vγ/Vδ use, were activated in the resistant (C57BL/6) vs the susceptible (Bl.Tg.Eα) mouse strain following infection with CVB3, with Vγ1 cells dominating in the former and Vγ4 cells dominating in the latter strain. In this paper, we show that the different γδ⁺ cell populations are selectively responsible for both disease resistance and susceptibility. Thus, cells expressing the Vγ1 gene suppress myocarditis, while cells expressing the Vγ4 gene promote disease. This is the first demonstration we could find that in the same disease model different γδ⁺ cell subpopulations had both beneficial and detrimental effects.

C57BL/6 and C57BL/6J^{−Tcrdtm1Mom} (C57BL/6 γδ knockout (ko)) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) as breeder pairs. Breeding pairs of transgenic C57BL/6 mice expressing the MHC class II Eα^k gene from A/J mice (IA⁺IE⁺; Bl.Tg.Eα strain) were initially obtained from Dr. Chella David (Department of Immunology, Mayo Clinic, Rochester, MN). Characterization and description of these mice has been published previously (12, 27). Bl.Tg.Eα mice lacking γδ⁺ T cells were produced by mating Bl.Tg.Eα male to C57BL/6 γδ ko females, backcrossing the F₁ progeny to the C57BL/6δ ko parental strain for seven generations, and screening for IE^k+ progeny using cytometric analysis of PBLs. IE^k+ male and female mice were inbred for a further six generations. All progeny are screened for IE^k expression (Bl.Tg.Eαγδ ko strain).

Animals were infected by i.p. injection of 0.5 ml PBS containing 10⁴ PFUs of CVB3 (H3 variant), derived from Cos cells transfected with the infectious cDNA of this virus (28). For virus titration, hearts were homogenized in 0.9 ml RPMI 1640 containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 5% FBS. Cellular debris was removed by centrifugation at 1045 × g for 10 min. The supernatant was titered by the plaque-forming assay on HeLa cell monolayers as described previously (29).

Ab class control (isotype control) and Ag-specific Abs were obtained from PharMingen (San Diego, CA). These included the following: purified rat anti-mouse CD16/CD32 (Fc Block; clone 2.4G2); Cy-Chrome, FITC, and PE-rat IgG1 (clone R3-34); biotinylated and PE-rat anti-mouse IFN-γ (clone XMG 1.2); biotinylated and PE-rat anti-mouse IL-4 (clone BVD4-1D11); Cy-Chrome-rat-anti-mouse CD4 (clone GK 1.5), purified mouse-anti-IA^b (clone AF6-120.1); PE-mouse-anti-IE^k (clone 14-4-4S); purified hamster anti-TCRβ (clone H57-597); purified mouse anti-NK1.1 (clone PK136); FITC and PE-hamster IgG; FITC and PE-hamster-anti-γδ TCR (clone GL3); and purified mouse anti-hamster IgG (clones G70-204/G94-56) Abs. Additional purified FITC- and biotin-conjugated Abs to Vγ1 (clone 2.11) and Vγ4 (clone UC3) were prepared and tested in the laboratory of Dr. Rebecca O’Brien (National Jewish Medical and Research Center, Denver, CO). Cy-Chrome, FITC, and PE-conjugated streptavidin were purchased from PharMingen.

For isolation of γδ⁺ T cells, mice were euthanized by injecting 120 mg/kg sodium pentobarbital in PBS i.p. The spleens were removed, disrupted to produce single cell suspensions, and washed in RPMI 1640 medium containing 5% FBS and antibiotics. After removing tissue debris by sedimentation, the cells were centrifuged at 225 × g for 10 min at 5°C. The cell pellet was resuspended in medium, layered over Histopaque-1077 (Sigma, St. Louis, MO), and centrifuged at 1048 × g for 15 min, and the lymphoid cells at the interface were retrieved. After washing the cells in medium with 5% FBS, they were incubated for 30 min on nylon wool columns containing 0.5 g nylon wool (Polysciences, Warrington, PA) at 37°C. The nonadherent cells were retrieved by washing the column with 30× void volume of medium. The cells were resuspended in medium and counted by trypan blue exclusion. For every 10⁶ lymphocytes in 100 μl medium with 5% FBS, 1 μl each of FcBlock, anti-αβ TCR, anti-IA^b, and anti-NK1.1 were added to the cell suspension. The cells were incubated for 20 min at 4°C on a rocker platform, washed once, and resuspended in medium 1:100 dilution of mouse anti-hamster IgG for 20 min at 4°C. The cells were then washed once more and incubated with magnetic particles conjugated with anti-mouse IgG (PerSeptive Biosystems, Framingham, MA) for 30 min at 4°C. The cell suspension was passed twice over a magnet (PerSeptive Biosystems) to remove Ab-bound cells. This produced a cell population containing ∼50% γδ⁺ T cells as determined by staining with PE-anti-γδ TCR Ab. To improve the purity of the cell population further, and isolate the specific Vγ4 subpopulation, the cells were stained with PE-anti-γδ TCR and biotinylated hamster anti-Vγ4 mAb for 20 min, washed, and incubated with FITC-streptavidin for 20 min at a 1:50 dilution. After washing, the cells were resuspended in PBS/2% BSA and sorted in a Coulter (Palo Alto, CA) Epics Elite flow cytometer into RPMI 1640 containing 20% FBS as described below. Flow diagrams of the initial (Fig. 1,A), positively selected (Fig. 1,B), and residual (Fig. 1 C) populations are given for γδ⁺ cells stained for the Vγ4 receptor. For adoptive transfer, a total of 2.1 × 10⁶ Vγ4⁺ cells were isolated from 35 pooled spleens (initial starting population after nylon wool of 6.9 × 10⁸ lymphocytes).

For preparation of splenocytes, spleens were removed, pressed through fine mesh screens, washed twice, and centrifuged on Histopaque.

For preparation of PBLs, blood was obtained by cardiac puncture at the time of euthanization, using a 26 gauge needle and a 1-cc syringe containing 50 μl of a 0.15% EDTA (Sigma) solution. The blood was diluted 1:10 with medium, layered on Histopaque, and centrifuged at 1048 × g for 15 min. The lymphoid cells at the interface were removed, washed once, and counted by trypan blue exclusion.

For cell surface marker staining, 1 × 10⁵ lymphocytes were washed in PBS containing 1% BSA and 0.1% sodium azide (PBS-BSA) and resuspended in 0.1 ml PBS-BSA containing a 1:100 dilution of fluorochrome-labeled Ab and a 1:100 dilution of Fc-Block. After incubation for 30 min on ice, the cells were washed twice in PBS-BSA, and fixed in 2% formaldehyde for flow analysis.

For intracellular cytokine staining, a modification of the method of Picker et al.(30) was used to evaluate intracellular cytokines in splenocytes. Briefly, 1 × 10⁶ spleen cells were cultured in medium containing 10 μg/ml brefeldin A, 50 ng/ml PMA, and 500 ng/ml ionomycin (Sigma) for 4 h at 37°C in 5% CO₂. The cells were subsequently resuspended in medium containing 50 μg/ml rat polyclonal IgG (Zymed, San Francisco, CA) and brefeldin A, incubated for 10 min at 5°C, washed, and resuspended in medium containing Fc-Block (PharMingen) and either fluorochrome-labeled surface marker Abs or appropriate Ig isotype controls. After incubation on ice for 30 min, the cells were washed in PBS-BSA-brefeldin A, and fixed for 10 min in 2% paraformaldehyde. The cells were then washed once in PBS-BSA buffer, incubated for 10 min in PBS-BSA buffer containing 0.5% saponin, and stained for intracellular cytokines using either FITC-anti-IFN-γ and PE-anti-IL-4. As an Ab class control for the intracellular staining, we used PE-rat IgG. All staining was performed in buffer containing Fc-Block and 50 μg/ml polyclonal rat IgG to block nonspecific Ab binding. After incubation for 30 min, the cells were washed twice in PBS-BSA-saponin and once in saponin-free PBS to close the membrane, then resuspended in PBS/azide containing 2% paraformaldehyde. Positive controls for cytokine staining were 2.5 × 10⁵ of either MIC-1- (IFN-γ) and MIC-2 (IL-4)-fixed cells obtained from PharMingen. Control cell populations were permeabilized and stained as described above.

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), and Cy-Chrome (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. Generally, the results were expressed as the percentage of gated cells which stained positively for each marker, or as the percentage of positive cells after gating using an additional marker, after subtracting the percent positive cells in the Ab class control. For example, the percentage of CD4⁺ cells in peripheral blood staining for IL-4 represents the following: (CD4⁺/IL-4⁺ cells)/(CD4⁺/IL-4⁻ and CD4⁺/IL-4⁺ cells) × 100. In some cases (Fig. 3 and Table II), values may represent the percent of the total cell population staining for specific markers. Each study was repeated at least two times, and the data from a representative experiment are presented.

Hearts were removed, fixed in 10% buffered formalin, paraffin embedded, sectioned, and stained with von Kossa. Stained sections were used for image analysis in transmitted light mode with an Olympus (New Hyde Park, NY) BX50 compound light microscope (×4 objective lens; numerical aperture, 0.13). True color digital images (640 by 480 pixels) were captured with a Sony (Tokyo, Japan) DXC-960 MD/LLP video camera connected via an RS170 cable to a video frame grabber on a Sun SPARCstation 5 (Mountain View, CA). Image processing and analysis were accomplished with IMIX software (Princeton Gamma Tech, Princeton, NJ). Final percent cardiac injury was calculated by dividing the area of injury by the total area of the heart.

Statistical evaluation was performed using the Wilcoxon ranked score method.

C57BL/6, C57BL/6 γδ ko, Bl.Tg.Eα, and Bl.Tg.Eα γδ ko mice were infected with CVB3 and euthanized 7 days later. Hearts were evaluated for myocardial inflammation (Table I and Fig. 2) and cardiac virus titers (Table I). PBLs were evaluated for CD4⁺ cells, IL-4⁺, and IFN-γ⁺ cells using intracellular cytokine staining and flow analysis. For this evaluation, PBLs were surface stained for CD4 (Cy-Chrome) and intracellularly stained for IFN-γ (FITC) and IL-4 (PE). Fig. 3 provides a flow diagram for one animal (highlighted in bold in Table I) from each group. In Fig. 3, the numbers in the upper right corner represent the total percent of all cells in each quadrant. Table I provides the individual animal values for myocarditis, cardiac virus titers, percent of CD4⁺ cells in the peripheral blood staining intracellularly for either IFN-γ or IL-4, and the calculated ratio of CD4⁺ Th1/Th2 cells. As can be seen in Fig. 3, while CD4⁺ cells clearly stain for either IFN-γ or IL-4, non-CD4⁺ cells are also cytokine positive in most animals. The identity of non-CD4⁺ cells staining for cytokine has not been determined.

C57BL/6 mice are myocarditis resistant despite high levels of virus in the heart. As shown previously (12), Bl.Tg.Eα mice, transgenic C57BL/6 mice that express MHC class II IE Ag, in contrast, are myocarditis susceptible with approximately six times more cardiac inflammation than wild-type animals. As can be seen in Table I, removing γδ⁺ T cells reversed this pattern (i.e., C57BL/6 γδ ko mice showed higher levels of myocarditis, whereas B1.Tg.Eα γδ ko mice had lower levels). This suggests that γδ⁺ T cells are involved in both protection and promotion of cardiac inflammation. C57BL/6 γδ ko mice had fewer Th2 (CD4⁺/IL-4⁺) but more Th1 (CD4⁺/IFN-γ⁺) cells than C57BL/6 mice (p < 0.05). In contrast, B1.Tg.Eα γδ ko mice showed significantly more Th2 cells than B1.Tg.Eα animals, but fewer Th1 cells. This suggests that γδ⁺ cells might be directly or indirectly modulating Th cell phenotype.

Previous studies showed that Vγ1⁺ cells dominate among γδ⁺ T cells in the hearts of CVB3-infected C57BL/6 mice while Vγ4⁺ cells dominate in the hearts of infected Bl.Tg.Eα animals. To evaluate whether Vγ populations might differentially affect myocarditis susceptibility, C57BL/6 and Bl.Tg.Eα mice were depleted of each subset by injecting either 100, 200, or 400 μg anti-Vγ1 or anti-Vγ4 Abs i.v. through the tail vein. Three days later, the mice were injected i.p. with CVB3. Seven days after infection, mice were killed. PBLs were evaluated by intracellular cytokine staining for Th1 and Th2 cells (Table II, Fig. 3). Splenocytes were evaluated first for the percentage of total splenocytes which are γδ⁺ and either Vγ1⁺ or Vγ4⁺ (Table II). Secondly, flow was gated on the γδ⁺ population and evaluated for Vγ1⁺ or Vγ4⁺ cells (Fig. 4). γδ⁺ constitute a small subpopulation of total splenocytes. Gating specifically on the γδ⁺ cell population can better demonstrate the effectiveness of Ab depletion. The heart was processed for histology (Table II, Fig. 2). Ab depletion was effective, especially at the 400-μg dose, in reducing the number of Vγ1⁺ or Vγ4⁺ cells invivo. Anti-Vγ4 treatment of Bl.Tg.Eα mice resulted in reduction of myocarditis (Table II) and an increase in CD4⁺ Th2 cells in peripheral blood. Anti-Vγ1 Ab treatment produced the opposite result, a modest increase in myocarditis and decrease in CD4⁺Th2 cells.

To confirm the roles of Vγ4⁺ cells in CVB3 myocarditis, C57BL/6 and Bl.Tg.Eα mice lacking γδ⁺ cells were reconstituted with Vγ4⁺ cells purified from day 7 CVB3-infected Bl.Tg.Eα mice by sterile sorting. Lymphocytes were retrieved from the spleens and enriched for γδ⁺ cells as described in Materials and Methods. Fig. 1 shows flow cytometric analysis of the Vγ4⁺ cell population before and after purification. C57BL/6 γδ ko and Bl.Tg.Eα γδ ko mice were infected with CVB3 and injected i.v. with either 10², 10⁴, or 2.5 × 10⁵ of these cells 1 day later. All animals were killed 7 days after infection. Table III shows the effect of the γδ⁺ cell transfer on myocarditis. Giving Vγ4⁺ cells to either Bl.Tg.Eα γδ ko or C57BL/6 γδ ko mice resulted in enhanced myocarditis. Aliquots of the Vγ4⁺ cells were stimulated with PMA and ionomycin for 4 h in the presence of brefeldin A, then intracellularly stained with either Cy-Chrome-streptavidin and biotinylated anti-IFN-γ or anti-IL-4 to evaluate cytokine production (Fig. 5). Vγ4⁺ cells stained for IFN-γ but not for IL-4.

These studies show that in CVB3-induced myocarditis, Vγ1⁺ T cells suppress myocardial inflammation whereas Vγ4⁺ T cells promote disease. Thus the type of γδ⁺ cell dominating, or responding in an animal will determine its disease susceptibility or resistance. We believe that this is the first study to demonstrate distinct roles for different γδ⁺ T cell subpopulations within the same disease. The mechanism of γδ⁺ cell regulation seems to be mediated through effects on Th subset response because Th2 cell responses, for the most part, correlate with Vγ1⁺ cell dominance and Th1 cell responses correlate with dominant Vγ4⁺ cells. Th1/Th2 dichotomy in disease susceptibility and resistance is well-recognized (31, 32, 33, 34, 35, 36). In diseases dependent on cellular inflammation and pro-inflammatory cytokines such as IL-1 and TNFα, it is reasonable that Th1 cell responses should be pathogenic while Th2 cell responses should be protective. This is especially true in CVB3-induced myocarditis, because T cell-dependent responses are not required for virus clearance (37) and, therefore, are not “beneficial” in terms of the infection itself. How γδ⁺ cells modulate Th1/Th2 responses has not been thoroughly addressed in this communication, although we show that Vγ4⁺ cells which promote Th1 cell responses do produce IFN-γ. Th cell modulation by γδ⁺ cells has been reported previously (38, 39), and presumably occurs through cytokines released by these effectors. IL-4 would promote Th2 cell differentiation if present early in the Ag-specific T cell response, while IFN-γ directly or indirectly would promote Th1 cell responses (31, 40). Other possible mechanisms for γδ⁺ cell-induced immunomodulation are possible. One such mechanism could be direct killing of specific CD4⁺ Th cells by γδ⁺ lymphocytes. Evidence supporting selective γδ⁺ cell lysis of CD4⁺ lymphocytes comes from studies in Lyme arthritis (26) and from CVB3-induced myocarditis (12). In the latter studies, γδ⁺ cells directly killed differentiated Th2 cell lines through Fas-dependent mechanisms. What structural differences between Th1 and Th2 cells allow γδ⁺ cell recognition and/or killing of the Th2 but not the Th1 cells is not known. It is equally possible that γδ⁺ cells recognize both Th1 and Th2 cells, but the Th1 cells are rescued from death while the Th2 cells are not. Certainly, T cell apoptosis can be abrogated with appropriate positive signals (31). An additional possibility is that γδ⁺ cells influence other cell types, such as macrophage, which subsequently alter Th development (41).

Experiments were performed with both direct Ab-induced depletion of γδ⁺ cells in vivo and by adoptive transfer of positively sorted γδ⁺ cell subpopulations into genetically γδ⁺ cell-deficient recipients. Both sets of experiments indicate a role for specific γδ⁺ subpopulations in regulating both Th cell phenotype and myocarditis susceptibility. Because Ab binding to the TCR might activate cells to release cytokines or immunomodulate αβ⁺ T cell responses before their elimination, the Ab depletion experiments might indicate either that the lack of or activation of specific subpopulations resulted in the observed effects. However, specific Vγ4 Abs were used to isolate purified subpopulations for adoptive transfer. Although these Abs might also have activated the respective cell populations, Vγ4⁺ cells continued to promote myocarditis susceptibility and Th1 responsiveness.

An important question is why dominant Vγ populations differ between genetic strains of mice. Various studies have shown that class II MHC molecules influence γδ⁺ cell subpopulation composition (12, 42, 43, 44). C57BL/6 mice inherently lack MHC class II IE Ag expression due to a naturally occurring mutation in their Eα gene, while Bl.Tg.Eα mice express IE (27). Flow cytometric analysis of γδ⁺ cell populations in the spleens of uninfected C57BL/6 mice show ∼50% of these cells express Vγ1 and 20% express Vγ4 receptors, whereas in uninfected Bl.Tg.Eα mice, ∼40% of γδ⁺ cells express Vγ4 and only 35% express Vγ1 receptors. Thus, the uninfected mice from each strain have the same propensity for differential Vγ subset expression as infected mice. This suggests that predominance of distinct Vγ subpopulations in myocarditis in C57BL/6 and Bl.Tg.Eα mice reflects the inherent influence of MHC class II Ag on clonal selection of Vγ subpopulations during thymic development.

The importance of our study is that it shows the complexity of γδ⁺ cell interactions in immunological disease. As with T cells expressing the αβ⁺ TCR, where different subpopulations might interact negatively or positively with each other, γδ⁺ cell subpopulations may also interact between themselves and also with the αβ⁺ cells. The γδ⁺ cell plays an intricate and critical role in the pathogenesis of at least some diseases. How universally important they might be in other forms of disease will require further investigation.

We thank Debbie Perrotte for the expert secretarial assistance and Colette Charland for the expert flow cytometric analyses.