Preemptive CD8 T-cell immunotherapy of acute cytomegalovirus infection prevents lethal disease, limits the burden of latent viral genomes, and reduces the risk of virus recurrence

Abstract

In the immunocompetent host, primary cytomegalovirus (CMV) infection is resolved by the immune response without causing overt disease. The viral genome, however, is not cleared but is maintained in a latent state that entails a risk of virus recurrence and consequent organ disease. By using murine CMV as a model, we have shown previously that multiple organs harbor latent CMV and that reactivation occurs with an incidence that is determined by the viral DNA load in the respective organ (M. J. Reddehase, M. Balthesen, M. Rapp, S. Jonjic, I. Pavic, and U. H. Koszinowski. J. Exp. Med. 179:185-193, 1994). This predicts that a therapeutic intervention capable of limiting the load of latent viral genome should also reduce the risk of virus recurrence. Here we demonstrate the benefits and the limits of a preemptive CD8 T-cell immunotherapy of CMV infection in the immunocompromised bone marrow transplantation recipient. Antiviral CD8 T cells prevented CMV disease and accelerated the resolution of productive infection. The therapy also resulted in a lower load of latent CMV DNA in organs and consequently reduced the incidence of recurrence. The data thus provide a further supporting argument for clinical trials of preemptive cytoimmunotherapy of human CMV disease with CD8 T cells. However, CD8 T cells failed to clear the viral DNA. The therapy-susceptible portion of the DNA load differed between organs and was highest in the lungs. The existence of an invariant, therapy-resistant load suggests a role for immune system evasion mechanisms in the establishment of CMV latency.

FIG. 1

CD8 T cells are essential for the control of CMV infection after BMT. Shown are Kaplan-Meier survival plots for groups of 20 mice, giving the survival rates (ordinate) as a function of time (abscissa) after syngeneic BMT, performed with the indicated doses of BMC, followed by intraplantar infection with murine CMV. Note that in the absence of infection, survival rates after hematoablative γ-ray treatment with 6 Gy ranged from 40 to 60% in groups without BMT and were 100% after reconstitution with any of the indicated doses of BMC (not shown). The dashed line indicates the control group given hematoablative γ-ray treatment with 6 Gy followed by infection but no BMT. In all other experimental groups, the recipients received BMC and were infected. The presence and absence of CD8 T cells are indicated by solid and open symbols, respectively. Solid squares indicate groups of recipients reconstituted by BMT with the indicated doses of BMC. Solid circles indicate immunotherapy by adoptive transfer of 10⁶ CD8 T cells. Open circles indicate adoptive transfer of 10⁶ CD4 T cells. Solid diamonds indicate in vivo depletion of CD4 T cells. Open diamonds indicate in vivo depletion of CD8 T cells.

FIG. 2

CD8 T-cell immunotherapy modulates the course of primary CMV infection. Throughout, BMT was performed with 10⁷ BMC and was followed by intraplantar infection (corresponding to Fig. 1, bottom). The group that did not receive immunotherapy served as a reference showing the normal course of infection during reconstitution after BMT (top). In the remaining three groups, immunotherapy was performed by adoptive transfer of the indicated doses of CD8 T cells. Virus titers in organs (ordinate) were monitored as a function of time after BMT and infection (abscissa). The dashed line gives the detection limit of the infectivity assay, which was 100 PFU* per organ. Individual titers are depicted for three mice per time point. The median value is marked by a short horizontal bar. Cases in which virus titers were uniformly negative for an organ are depicted only on the first occasion, but the titers then remained negative throughout the kinetics. Symbols: open circles, salivary glands; solid circles, lungs; solid squares, adrenal glands.

FIG. 3

Verification of CMV latency in the lungs. The analysis is shown for the group with no CD8 T-cell therapy. (Top right) Scheme of the lobular anatomy of the lungs in ventral view is depicted at the upper right. For each individual mouse included in a latency analysis performed 12 months after infection, the left lung (LL) and postcaval lobe (PCL) were used to verify the absence of infectious virus by the RT-PCR-based focus expansion assay (FEA) whereas the superior lobe (SL), middle lobe (ML), and inferior lobe (IL) were used to detect the presence of latent viral DNA. (Bottom right) Nine 2-ml aliquots of the homogenate of the LL and PCL were tested for the presence of infectivity in cultures of permissive cells by the RT-PCR-based focus expansion assay. As a positive control, aliquot 1 was supplemented with 0.05 PFU of purified murine CMV. Poly(A)⁺ RNA derived from this culture was serially diluted as indicated, whereas for each of the remaining cultures, 100 ng of poly(A)⁺ RNA was subjected to ie1 exon 3/4-specific RT-PCR, leading to an amplification product of 188 bp. The autoradiograph was obtained after hybridization with a γ-³²P-end-labeled oligonucleotide probe directed against the splice junction. For culture 9, the presence of RNA was verified by an RT-PCR specific for the HPRT housekeeping gene transcript. (Left) DNA isolated from the SL, ML, and IL was subjected to an ie1 exon 4-specific PCR. Plasmid pIE111 was added to pulmonary DNA from uninfected BMT recipients (10,000 copies in 3 μg) and was titrated in parallel. Amplification products were analyzed by gel electrophoresis. Lane M contains the 100-bp size marker kit. An ethidium bromide-stained gel is shown on the left, and the corresponding Southern blot autoradiograph obtained after hybridization with a γ-³²P-end-labeled internal oligonucleotide probe is shown on the right.

FIG. 4

Quantitation of latent CMV DNA in the lungs after CD8 T-cell immunotherapy. Lung cell DNA pooled at 12 months after BMT and infection from the superior lobe, middle lobe, and inferior lobe of five mice per experimental group was serially diluted in duplicate in log₂ steps and subjected to ie1 gene exon 4-specific PCR in a microplate format. (Top) Autoradiograph obtained after dot blotting of the amplification products and hybridization with a γ-³²P-end-labeled internal oligonucleotide probe. ⊘, Group with no CD8 T-cell therapy; BMT ⊘ CMV, Uninfected BMT recipients; Standard, plasmid pIE111 added to pulmonary DNA from uninfected BMT recipients. (Bottom) Computed phosphorimaging results of the same blot. Log-log plots of radioactivity (mean of duplicates) measured as phosphostimulated luminescence (PSL) units (ordinate) versus the dilutions (abscissa) are shown. The lower and upper rules relate the dilutions to the amount of lung cell DNA and to the number of plasmids in the standard (S, open circles), respectively.

FIG. 5

Determination of the latent CMV DNA load by comparative amplification of sequences from distant regions of the viral genome. For salivary gland cell DNA of the infected groups with no therapy (⊘) and with high-dose therapy (10⁷ CD8 T cells), latent viral DNA was quantitated by PCR amplification of an ie1 gene sequence (left panel) and a gB gene sequence (right panel), with virion DNA serving as a common standard for both PCRs. Shown are the autoradiographs obtained after dot blotting of the amplification products and hybridization with the respective γ-³²P-end-labeled internal oligonucleotide probes. The loads were calculated, as in Fig. 4, from the linear portions of the log-log plots of radioactivity (mean of duplicates) versus the DNA dilutions (not depicted). For the groups given no therapy and given therapy, the loads were determined to be ca. 3,200 and 1,250 copies per 10⁶ cells, respectively, regardless of whether the calculation was based on ie1 gene-specific amplification or on gB gene-specific amplification.

FIG. 6

Effect of CD8 T-cell immunotherapy on the load of latent CMV DNA in different organs. The viral DNA loads in the indicated organs are expressed as viral copies per 10⁶ cells of the respective tissues. ⊘, Control group given no CD8 T-cell immunotherapy.

FIG. 7

Effect of CD8 T-cell immunotherapy on the incidence of recurrence. Latently infected mice of the group with no therapy (left) (mice 1 to 5) and the group with immunotherapy by 10⁷ CD8 T cells (right) (mice 1∗ to 5∗) were subjected to immunoablative γ-ray treatment with 6.5 Gy. Recurrence of viral infectivity was measured 14 days later in separate lobes of the lungs (see the scheme in Fig. 3) by an RT-PCR-based focus expansion assay. For each lobe, the result of one culture infected with one 2-ml aliquot of the respective homogenate is depicted. The ie1 exon 3/4-specific RT-PCR was performed with 100 ng of poly(A)⁺ RNA.