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. 2000 Aug;74(16):7496-507.
doi: 10.1128/jvi.74.16.7496-7507.2000.

Murine model of interstitial cytomegalovirus pneumonia in syngeneic bone marrow transplantation: persistence of protective pulmonary CD8-T-cell infiltrates after clearance of acute infection

J Podlech  1 , R Holtappels, M F Pahl-Seibert, H P Steffens, M J Reddehase
Affiliations

Murine model of interstitial cytomegalovirus pneumonia in syngeneic bone marrow transplantation: persistence of protective pulmonary CD8-T-cell infiltrates after clearance of acute infection

J Podlech et al. J Virol. 2000 Aug.

Abstract

Interstitial pneumonia (IP) is a severe organ manifestation of cytomegalovirus (CMV) disease in the immunocompromised host, in particular in recipients of bone marrow transplantation (BMT). Diagnostic criteria for the definition of CMV-IP include clinical evidence of pneumonia together with CMV detected in bronchoalveolar lavage or lung biopsy. We have used the model of syngeneic BMT and simultaneous infection of BALB/c mice with murine CMV for studying the pathogenesis of CMV-IP by controlled longitudinal analysis. A disseminated cytopathic infection of the lungs with fatal outcome was observed only when reconstituting CD8 T cells were depleted. Neither CD8 nor CD4 T cells mediated an immunopathogenesis of acute CMV-IP. By contrast, after efficient hematolymphopoietic reconstitution, viral replication in the lungs was moderate and focal. The histopathological picture was dominated by preferential infiltration of CD8 T cells confining viral replication to inflammatory foci. Notably, after clearance of acute infection, CD62L(lo) and CD62L(hi) subsets of CD44(+) memory CD8 T cells were found to persist in lung tissue. One can thus operationally distinguish an early CMV-positive IP (phase 1) and a late CMV-negative IP (phase 2). According to the definition, phase 2 histopathology would not be diagnosed as a CMV-IP and could instead be misinterpreted as a CMV-induced immunopathology. We document here that phase 1 as well as phase 2 pulmonary CD8 T cells are capable of exerting effector functions and are effectual in protecting against productive infection. We propose that antiviral "stand-by" memory-effector T cells persist in the lungs to prevent virus recurrence from latency.

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Figures

FIG. 1
FIG. 1
Role of T-cell subsets in the control of CMV disease during hematopoietic reconstitution. (A) Effect of selective T-cell subset depletion on survival rate. BMT and mCMV infection were performed on day 0, and T-cell subsets were depleted by two consecutive (on days 7 and 14) i.v. infusions of MAbs directed against CD4 or CD8. Shown are Kaplan-Meyer plots for 10 recipients per group. Solid and open symbols indicate the presence and absence of CD8 T cells, respectively. (B) Effect of selective T-cell subset depletion on mCMV replication in the lungs. With additional recipients in each of the four experimental groups, the number of infected lung tissue cells was determined by quantitative viral DNA ISH on day 19, that is, at a stage when CMV disease was prefinal in CD8-depleted recipients. Symbols correspond to those in panel A and represent data from individual recipients. The median value is marked by a horizontal bar. The left-hand scale shows the absolute numbers of ISH-positive lung cells present in representative 100-mm2 areas compiled from lung tissue sections. The right-hand scale relates these numbers to the number of nucleated stromal and parenchymal cells detected by hematoxylin staining. The total number of lung cells is ca. 6 × 107.
FIG. 2
FIG. 2
Viral histopathology in the lungs after T-cell subset depletion. A two-color IHC analysis of lung tissue was performed on day 19 after BMT and mCMV infection (corresponding to Fig. 1) for recipients depleted either selectively of CD4 T cells (A1, overview; A2, details) or depleted of both T-cell subsets (B1, overview; B2, details). Infected cells are visualized by red staining of intranuclear viral IE1 protein, and infiltrating T cells are visualized by black staining of membrane CD3ɛ. Counterstaining was performed with hematoxylin. The arrows in panel A2 point to uninfected alveolar macrophages recruited to the inflammatory focus. Bars, 25 μm.
FIG. 3
FIG. 3
Longitudinal analysis of pulmonary infection and T-cell infiltration after BMT. Histopathology in the lungs was studied by quantitative two-color IHC, with infected cells being visualized by red staining of intranuclear viral IE1 protein (red dots) and infiltrating T cells by black staining of membrane CD3ɛ (black dots). (A) Time course of infection and T-cell infiltration. Each symbol represents data for individual recipients. The lines connect median values. Negative counts are depicted only on the first occasion in the kinetics and remained negative throughout. The left-hand scale and the right-hand scale show the numbers of infected lung cells and of lung-infiltrating T cells, respectively, for representative 100-mm2 areas compiled from several lung tissue sections. In a control group receiving BMT but not infection, the tissue architecture was not pathologically altered (19) (not shown), and in phase 2, the average number of T cells per 100 mm2 was 880 (arrowhead), in comparison to ca. 200 in lungs of normal mice. (B) Examples of lung histology representative of phase 1 (B1, CMV-positive IP) and of phase 2 (B2, CMV-negative IP). (B1) Perivascular inflammatory focus seen 3 weeks after BMT and infection in tissue that connects two neighboring vessels. T cells located within the inflammatory focus are activated lymphoblasts, as shown by halo-like membrane staining (see also Fig. 2A2). (B2) Random distribution of residual interstitial T cells after clearance of productive infection, as observed after 3 months. Note that most of the T cells (some of which are marked by an arrow) are considerably smaller than those in phase 1 and apparently represent resting cells. Counterstaining was performed with hematoxylin. Bars, 25 μm.
FIG. 4
FIG. 4
Phenotypes of phase 1 and phase 2 pulmonary infiltrate T cells. Pulmonary infiltrate cells were isolated at 4 weeks (phase 1) and at 10 weeks (early in phase 2) after BMT and mCMV infection. (A and B) Three-color cytofluorometric analysis was performed for the marker combinations FITC (FL-1)-CD8, PE (FL-2)-TCRα/β, and PE-Cy5 (FL-3)-CD4. A gate was set on lymphocytes, and the analysis was restricted to ca. 20,000 α/β T cells by a second gate set on positive FL-2. (C and D) Three-color cytofluorometric analysis was performed for the marker combinations FITC (FL-1)-CD44, PE (FL-2)-CD8, and RED613 (FL-3)-CD62L. A gate was set on lymphocytes, and the analysis was restricted to ca. 10,000 CD8 T cells by a second gate set on positive FL-2. FL-3 (ordinate) versus FL-1 (abscissa) log fluorescence intensities are shown for gated cells as contour plots in a 70% log-density mode (threshold, 2%; smoothing factor, 5). Percentages of relevant T-cell subsets and the ratios of CD8 to CD4 T cells (CD8/CD4) are indicated.
FIG. 5
FIG. 5
Cytolytic activity of pulmonary infiltrate CD8 T cells after polyclonal CD3ɛ signaling. Immunomagnetically purified CD8 T cells (phase 1, 4 weeks; phase 2, 16 weeks) were tested for cytolytic activity by the assay of CD3ɛ-redirected lysis with effector-to-target cell ratios (E/T) as indicated. Note that T cells isolated from lungs after BMT with no infection did not exert detectable cytolytic activity at an E/T ratio of 40 (data not shown here; see reference 19).
FIG. 6
FIG. 6
Production of IFN-γ after polyclonal CD3ɛ signaling. (A) BMT and mCMV infection. (B) BMT with no infection. Pulmonary infiltrate cells (phase 1, 4 weeks; phase 2, 16 weeks) were stimulated for 5 h with MAb anti-CD3ɛ in the presence of brefeldin A. Control groups were treated accordingly except for polyclonal CD3ɛ stimulation. Three-color cytofluorometric analysis was performed for the marker combination FITC (FL-1)-CD62L, PE (FL-2)-IFN-γ, and PE-Cy5 (FL-3)-CD8. A gate was set on lymphocytes, and the analysis was restricted to ca. 25,000 CD8 T cells by a second gate set on positive FL-3. FL-1 (ordinate) versus FL-2 (abscissa) log fluorescence intensities are shown for gated cells as dot plots, with 10,000 dots displayed. Percentages are indicated for CD62Lhi CD8 T cells (upper left quadrants) and for CD62Llo CD8 T cells with intracellular accumulation of IFN-γ (lower right quadrants). The isotype controls (PE-conjugated rat IgG1) are shown for cells stimulated with anti-CD3ɛ.
FIG. 7
FIG. 7
Comparison of the in vivo antiviral function of phase 1 and phase 2 CD8 T cells. Graded numbers of immunomagnetically purified pulmonary infiltrate CD8 T cells (phase 1, 4 weeks; phase 2, 12 weeks) were transferred by i.v. infusion into immunocompromised and infected indicator recipients. Infectious virus in lungs and spleen of the recipients was measured on day 12 after infection and cell transfer by a virus plaque assay. PFU*, PFU determined under conditions of centrifugal enhancement of infectivity. Dots represent individual transfer recipients. The median values are marked by a horizontal bar. The dotted line indicates the detection limit (DL) of the assay. Ø, positive control of virus replication in the absence of cell transfer.
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