Murine cytomegalovirus reactivation concomitant with acute graft-versus-host disease is controlled by antibodies

Research ArticleHematologyImmunology Open Access | 10.1172/jci.insight.149648

Martina Seefried,1 Nadine Hundhausen,2 Irena Kroeger,3 Maike Büttner-Herold,4 Petra Hoffmann,5 Matthias Edinger,5 Evelyn Ullrich,3,6,7 Friederike Berberich-Siebelt,2 William J. Britt,8 Michael Mach,9 and Thomas H. Winkler1

1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

Find articles by Mach, M. in: JCI | PubMed | Google Scholar

1Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

2Institute of Pathology, University of Würzburg, Würzburg, Germany.

3Department of Internal Medicine 5, Hematology and Oncology, University Hospital, Erlangen, Germany.

4Department of Nephropathology, Institute of Pathology, FAU, Erlangen, Germany.

5Department of Internal Medicine III, Hematology and Oncology, University Hospital, Regensburg, Germany and LIT - Leibniz Institute for Immunotherapy, University Regensburg, Regensburg, Germany.

6Experimental Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

7Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany.

8Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama, USA.

9Institute for Clinical and Molecular Virology, University Hospital, Erlangen, Germany.

Address correspondence to: Thomas H. Winkler, Nikolaus-Fiebiger-Center for Molecular Medicine, Department of Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany. Phone: 49.9131.85.29136; Email: thomas.winkler@fau.de.

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Published January 31, 2023 - More info

Published in Volume 8, Issue 5 on March 8, 2023
JCI Insight. 2023;8(5):e149648. https://doi.org/10.1172/jci.insight.149648.
© 2023 Seefried et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published January 31, 2023 - Version history
Received: March 16, 2021; Accepted: January 27, 2023 View PDF Abstract

Reactivation of human cytomegalovirus (HCMV) from latency is a frequent complication following hematopoietic stem cell transplantation (HSCT). The development of acute graft-versus-host disease (GVHD) is a significant risk factor for HCMV disease. Using a murine GVHD model in animals latently infected with murine CMV (MCMV), we studied preventive and therapeutic interventions in this high-risk scenario of HSCT. Mice latently infected with MCMV experienced reactivated MCMV and developed disseminated MCMV infection concomitant with the manifestations of GVHD. Dissemination was accompanied by accelerated mortality. We demonstrate that MCMV reactivation and dissemination was modulated by MCMV-specific antibodies, thus demonstrating in vivo protective activity of antiviral antibodies. However, the efficacy of serum therapy required repetitive doses of high-titer immune serum secondary to the shortened serum half-life of IgG in animals with GVHD. In a complementary approach, treatment of GVHD by adoptive transfer of donor-derived Tregs facilitated production of MCMV-specific antibodies from newly developing donor-derived B cells. Together, our findings strongly suggest that antibodies play a major role in controlling recurrent MCMV infection that follows GVHD, and they argue for reassessing the potential of antibody treatments as well as therapeutic strategies that enhance de novo antibody development against HCMV.

Graphical Abstractgraphical abstract Introduction

Human cytomegalovirus (HCMV) is an important and ubiquitous human pathogen that is found throughout all geographic areas and socioeconomic groups. Initial infection with HCMV is followed by life-long persistence characterized by episodes of periodic reactivation. Most infections are subclinical in immunocompetent hosts since the virus is controlled by a multilayered and redundant innate and adaptive immune response (1). However, in immunocompromised patients, loss of immune control and dissemination of the virus can result in severe clinical disease. Thus, HCMV remains the most important viral infection after hematopoietic stem cell transplantation (HSCT), especially in high-risk patients (seronegative donor and seropositive recipient), and can lead to life-threatening HCMV disease in ~10% of HSCT recipients (2).

In addition to complications associated with infections, graft-versus-host disease (GVHD) — caused primarily by infusion of mature donor-derived T cells — continues to be a major cause for morbidity and nonrelapse mortality after HSCT (3). Multiple studies identified acute GVHD and its therapy as significant risk factors for HCMV reactivation in seropositive patients with HSCT (4, 5). Moreover, extensive T cell depletion for prevention of GVHD and cases of mismatched or haploidentical HSCT create additional clinical challenges in the management of HCMV infection.

In total, 20%–40% of HCMV-seronegative patients who receive grafts from HCMV-seropositive donors will develop primary HCMV infection (6). Untreated, 50% of patients with HSCT with HCMV reactivation will develop HCMV disease; CMV pneumonia is the most clinically significant manifestation, with a fatality rate of approximately 50% (7). Thus, even in the era of antiviral therapy, CMV infection and subsequent CMV disease still occurs in a significant fraction of patients.

Reconstitution of adaptive and innate immunity plays a pivotal role in the control of HCMV infection after HSCT, and poor postengraftment immune reconstitution represents a major risk factor for the development of severe HCMV infection. A number of studies have identified the presence of antiviral T cell immunity as a crucial factor associated with successful HSCT, and protocols involving adoptive T cell therapy have been successfully implemented in the treatment of transplant recipients (8). In contrast, the impact of the humoral immune response on the clinical outcome of HCMV infections in patients with HSCT remains controversial (9, 10).

Due to the strict species specificity of CMVs, there is a lack of animal models for study of infections with HCMV. However, infection of mice with murine CMV (MCMV) represents a well-characterized and extensively used animal model HCMV infections (11). Reports derived from studies in his model have demonstrated the relevance of antibodies in limiting and controlling viral infection. In immunocompromised mice, several studies showed that primary and recurrent infections are efficiently controlled by transfer of sera from MCMV-immune donors or monoclonal antibodies (1214). Moreover, Cekinović and colleagues demonstrated that, in MCMV-infected newborn mice, antibody treatment resulted in the clearance of virus from the central nervous system and reduction of virus-related neuropathology (15).

Preclinical as well as clinical studies established an adoptive immunotherapy regimen with CD4+FOXP3+ Tregs to significantly ameliorate GVHD (reviewed in ref. 16). Nothing is known on the influence of such an adoptive Treg transfer on the development of HCMV-specific antibodies, however. One might envisage a negative role on antibody responses exerted by the regulatory function on T cell help for B cells (17).

In the current study, we have used a mouse model for the analysis of the humoral immune response in an experimental protocol that mimics key aspects of GVHD and MCMV reactivation in recipients of MHC-mismatched allogeneic BM transplantation (allo-BMT). Our results demonstrate that viral reactivation was modulated by MCMV-specific antibodies and that, by repetitive administration of MCMV-immune serum, viral reactivation and disseminated infection could be significantly reduced. Furthermore, adoptive transfer of Tregs allows de novo antibody production by newly generated B cells from the donor while mitigating GVHD. Our studies suggest that antibodies play a major role in controlling recurrent MCMV infection after allo-BMT and suggest reassessing the value of antibody therapy as well as therapeutic strategies that enhance de novo antibody development against HCMV for preventing severe HCMV infection after stem cell transplantation.

Results

BALB/c mice latently infected with MCMV demonstrate reactivation of viral infection concomitant with manifestations of GVHD. Previously, small-animal models for HCMV infections after allogeneic stem cell transplantation have used sublethally irradiated mice and acute infection with MCMV (11, 18). To establish a mouse model for reactivation from latent infection, BALB/c mice were infected with 1 × 105 pfu of a luciferase-expressing MCMV strain (MCMV157luc; ref. 12), and viral replication was monitored in vivo using bioluminescence imaging. In the MCMV157luc strain, the m157 gene is replaced by the firefly luciferase gene. thus destroying the ligand of the Ly49H activation receptor on NK cells in the C57BL/6 strain, resulting in increased virulence (19). When assayed at 25 days postinfection (dpi), viral replication was not detectable in the animals, thus indicating that acute infection had been resolved in these animals (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.149648DS1). Four weeks after infection BALB/c mice were lethally irradiated with a single dose of 8 Gy and transplanted with 5 × 106 T cell–depleted BM cells from C57BL/6 donors. In addition, 1 group received purified splenic C57BL/6 T cells for induction of acute GVHD. Transfer of splenic MHC mismatched T cells resulted in a severe GVHD beginning 10–15 days after cell transfer. Using in vivo bioluminescence imaging, viral replication was detectable on day 14 after BMT in T cell recipient mice concomitant with the development of clinically apparent GVHD and increased significantly at later time points in these animals (Figure 1A). In contrast, mice that received BM alone and remained free of signs of GVHD had little to no detectable bioluminescence signal. To correlate the in vivo bioluminescence data with viral load in selected organs, animals were sacrificed on day 25 after transplantation, and infectious virus was quantified in different organs. Mice with GVHD had a high viral burden in all organs, consistent with disseminated virus infection (Figure 1B). In mice without GVHD, the quantity of infectious virus was below the level of detection in almost all mice and in most organs (Figure 1B).

GVHD results in recurrent infection in MCMV-infected BMT recipients.Figure 1

GVHD results in recurrent infection in MCMV-infected BMT recipients. MCMV-infected BALB/c mice were lethally irradiated and received 5 × 106 T cell-depleted bone marrow cells (BM) ± 6 × 105 to 8 × 105 T lymphocytes from C57BL/6 donors. (A) Bioluminescence imaging of 5 mice per group at 7, 14, 21, and 28 days after BMT. (B) MCMV viral load in mice transplanted with BM (red square) or BM + T cells (red triangle) 25 days after BMT. Median values are depicted as horizontal bars and detection limit as dashed lines. Representative data from 3 experiments. (C) Survival curve of 12–15 infected recipients per group transplanted with BM (red squares) or BM + T cells (red triangles). For comparison of survival, 13 uninfected recipients per group were transplanted in parallel with BM (black squares) or BM + T cells (black triangles). MCMV-infected mice that received BM + T cells showed significantly accelerated mortality compared with uninfected recipients receiving BM + T cells. ***P < 0.001; log-rank test. Results of 2 independent experiments were combined. (D) IHC stainings of sections from rectum (1 and 2) and liver (3 and 4) from BALB/c infected mice 30 days after transplantation without (1 and 3) or with (2 and 4) GVHD. Immediate-early protein 1 (IE) protein from MCMV was detected in the nucleus of infected cells, as labeled by the arrows. (E) Quantification of IE+ nuclei in liver and rectum of transplanted mice. (F) Double stainings of rectum of GVHD mice for glial fibrillary acidic protein (GFAP) in blue and MCMV IE protein in brown showing colocalization as labelled by the arrows.

MCMV-infected mice with GVHD exhibited an accelerated mortality compared with uninfected mice with GVHD (P < 0.001, Figure 1C), suggesting that viral infection contributed to accelerated mortality in animals with GVHD, as the clinical GVHD scores were comparable in infected and uninfected mice (Supplemental Figure 2). Significant and mostly focal infection in the livers of MCMV-infected mice with GVHD was observed 30 days after transplantation (Figure 1, D and E). In addition, MCMV-infected cells identified by nuclear expression of the immediate early antigen (IE) were readily detectable in the rectum of mice with GVHD (Figure 1, D and E). Interestingly, MCMV infection was detected predominantly in the muscularis propria of the rectum (Figure 1D). Double labeling of sections from the rectums of these mice confirmed a colocalization of MCMV and the glial cell marker glial fibrillary acidic protein (GFAP) in the infected cells (Figure 1F). This finding raised the possibility that MCMV persisted in the ganglia of the enteric nervous system in latently infected mice. MCMV IE+ cells were not detectable in matched tissues from any of the infected mice that received only BM and did not develop GVHD (Figure 1, D and E). Previous reports in renal transplant recipients and, more recently, descriptions of tissue from aborted fetuses infected with human HCMV have demonstrated the presence of HCMV in myenteric ganglia from the large intestines (20, 21).

GVHD after allo-BMT impedes lymphoid reconstitution. The adaptive immune system plays a critical role for the clearance of acute MCMV infection in immunocompetent hosts (22). In contrast to immunocompetent C57BL/6 WT mice, immunocompromised Rag1–/– C57BL/6 mice, which lack mature B and T cells, succumb to a MCMV infection with MCMV157luc (12). This is most likely due to the deletion of m157 in this virus strain; this deletion impedes disease control by NK cells that is otherwise observed in C57BL/6 hosts (19). Thus, it was conceivable that a delayed or inadequate reconstitution of lymphoid cells in BMT recipients with GVHD favored the disseminated MCMV infection. Therefore, we analyzed the reconstitution of cells of the adaptive immune system in peripheral blood 30 days after transplantation and the kinetics of MCMV-specific IgG2a titers of recipient (IgG2aa) and donor (IgG2ab) origin. BALB/c recipients with GVHD had very low to no detectable B cells, CD4 T cells, or CD8 T cells in peripheral blood as compared with transplanted control mice without GVHD (Figure 2A), suggesting that loss of MCMV control in recipient mice with GVHD was caused by profound GVHD-induced immunodeficiency. In agreement with these data, within 3 weeks after transplantation, MCMV-specific IgG2aa (recipient) titers decayed rapidly in animals developing GVHD, and no newly generated antiviral IgG2a antibody responses from B cells of graft origin (IgG2ab) were detected (Figure 2B). In contrast, BMT recipients of T cell–depleted BM that did not develop GVHD generated MCMV-specific serum titers from the C57BL/6 BM graft. Interestingly, the loss of detectable virus-specific IgG2a host antibodies coincided with the time of MCMV reactivation (Figure 1A).

Impact of GVHD on lymphoid immune reconstitution and MCMV-specific antibodyFigure 2

Impact of GVHD on lymphoid immune reconstitution and MCMV-specific antibody kinetics. MCMV-infected BALB/c mice were lethally irradiated and received 5 × 106 T cell–depleted BM cells ± 6 × 105 to 8 × 105 T cells from C57BL/6 donors. (A) Flow cytometric analysis of B cells, CD4, and CD8 T cells in peripheral blood. Blood samples were taken from mice transplanted with BM (red square, n = 6) or BM + T cells (red triangle, n = 5) on day 30 after BMT and stained with fluorochrome conjugated anti-CD19, anti-CD3, anti-CD4, anti-CD8, and anti-CD45.2 antibodies. Mean values are depicted as horizontal bars and mean values of age-matched untreated BALB/c mice (n = 4) as dashed lines. **P < 0.01; Mann-Whitney U test. Representative data from 3 independent experiments are shown. (B) MCMV-specific serum IgG2a titers of mice transplanted with BM (n = 7) or BM + T cells (n = 4) were analyzed on the days indicated. Detection of IgG2a allotypes a and b allowed the distinction between recipient (IgG2aa, gray) or graft (IgG2ab, black) origin. Data are shown as mean ± SD.

Anti-MCMV antibodies in the transplant recipient protect from MCMV reactivation and disseminated infection. As shown above, there was incomplete lymphoid reconstitution in mice with GVHD and an accelerated and nearly complete decline of MCMV-specific IgG2a titers. Moreover, the decline of MCMV antibodies correlated with progression of MCMV infection. Previous studies have demonstrated the protective capacity of MCMV-specific antibodies in prophylactic (23, 24) and a more limited number of therapeutic immune serum transfer protocols (12, 14, 15). To evaluate the impact of antibodies on MCMV reactivation and disease in BMT recipients, we used B cell–deficient BALB/c mice (25). Latent viral DNA load had been shown to be comparable in B cell–deficient mice compared with WT mice (23). In addition, we analyzed MCMV infection in B cell–deficient BALB/c mice and found no difference in MCMV infection by bioluminescence imaging (Supplemental Figure 3). These data rule out that absence of antibodies in the primary infection leads to higher numbers of infected cells.

We first analyzed the role of antibodies in the transplant host in the absence of GVHD. To this end, C57BL/6 mice were treated with B cell–depleting anti-CD20 antibodies before infection and monthly thereafter. Latently infected mice were transplanted with syngeneic BM, and MCMV reactivation was measured by bioluminescence on day 8 after transplantation. A significant reactivation of MCMV was observed in anti-CD20–treated mice, and this negatively correlated with significantly less IgG anti-MCMV antibodies in the anti-CD20–treated mice (Supplemental Figure 4). These results show that antibodies in the infected host can significantly ameliorate MCMV reactivation already in a non-GVHD BM transplantation situation, extending the observations of Jonjic et al. (23) to a syngeneic BM transplant setting.

B cell–deficient mice that were infected > 25 days prior to BMT succumbed significantly earlier after the development of GVHD when compared with WT mice (Figure 3A). Three weeks after transplantation, < 10% of B cell–deficient mice were alive as compared with 100% of mice in the control group (Figure 3A). There was no difference in survival secondary to GVHD-mediated mortality in the uninfected control groups, suggesting that the increased mortality of MCMV-infected B cell–deficient mice could not be attributed to divergent courses of GVHD in the presence or absence of B cells (Figure 3A). Bioluminescence data of infected B cell–deficient mice revealed a more rapid dissemination of MCMV in the B cell–deficient group compared with WT recipients with GVHD (Figure 3B). These results suggest that antibodies present in the WT but not in B cell–deficient recipients could modify the course of MCMV infection and prolong the survival of recipient mice.

Endogenous recipient antibody titer delayed viral dissemination and prolongFigure 3

Endogenous recipient antibody titer delayed viral dissemination and prolonged overall survival of recipient mice. Uninfected or MCMV-infected B cell–deficient BALB/c mice were lethally irradiated and received 5 × 106 T cell–depleted BM cells and 8 × 105 T cells from C57BL/6 donors. (A) Survival curve of infected (red diamond, n = 8) or uninfected (black diamond, n = 7) B cell–deficient recipients transplanted with BM + T cells. For comparison, a control group of MCMV-infected (red triangle, n = 6) or uninfected (black triangle, n = 8) BALB/c WT recipients transplanted with BM + T cells is depicted. **P < 0.01; log-rank test. (B) Bioluminescence imaging of 5 infected B cell–deficient mice at 7, 14, 21, and 28 days after BMT. For comparison, a control group of 5 MCMV-infected BALB/c WT recipients transplanted with BM + T cells is depicted. Data presented are representative of 2 independent experiments.

Therapeutic transfer of immune serum protects from recurrent MCMV infection. Thus far, our results have shown that MCMV-specific antibodies might have a protective effect, even in the presence of GVHD. We therefore determined whether this protective effect of MCMV-specific antibodies could be corroborated by passively transferring MCMV antibodies into MCMV-infected animals in the posttransplant period. Previously, we reported that a single dose of immune serum can completely control MCMV infection in infected C57BL/6 Rag1–/– mice for at least 14 days when given at day 3 of infection (12). In a modification of this protocol, we induced lethal GVHD in latently MCMV-infected BALB/c recipients and treated them either with a single dose of C57BL/6-derived (IgG2ab) immune serum (200 μL) on day 8 after BMT or with repetitive injections of immune serum (200 μL on day 8 after BMT and 100 μL every second day thereafter). On day 23 after transplant, recipients were sacrificed and viral load was determined in selected organs (Figure 4A). Mice receiving repetitive serum application showed a significantly lower viral burden as compared with untreated mice that reached a 77%–99% reduction of viral load in target organs. Importantly, the viral load in organs of mice given a single serum application remained comparable with mice that did not receive serum. Analysis of MCMV-specific serum IgG2aa titers revealed only a short-term increase of MCMV-specific IgG2a after single serum transfer, whereas repetitive application of immune serum maintained an IgG2aa titer at a constant level that correlated with protection from virus infection (Figure 4B).

Repetitive treatment of infected donors with immune serum protects from a rFigure 4

Repetitive treatment of infected donors with immune serum protects from a recurrent MCMV infection. MCMV-infected BALB/c mice were lethally irradiated and received 5 × 106 T cell–depleted BM cells ± 8 × 105 T cells from C57BL/6 donors. Eight days after BMT, 6–7 recipients transplanted with T cells received either a single application of immune serum (200 μL), repetitive treatment with immune serum (200 μL on day 8 postirradiation and 100 μL every second day) or no serum. (A) Relative viral load in untreated mice (black squares), in mice after a single treatment with serum (red squares), or after repetitive treatments with sera (blue squares). Mice transplanted only with BM (black circles) served as controls. Median values are depicted as horizontal bars and mean values of uninfected BALB/c (n = 3) as dashed lines. *P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney U test. (B) MCMV-specific serum IgG2a titers were analyzed at different time points after BMT. By using the IgG2a allotypes a and b, a distinction between recipient (IgG2aa, gray) or immune serum (IgG2ab, black) origin was possible. Data are shown as mean ± SD. Representative data from 2 independent experiments are shown.

Reduced absorption and shortened half-life of IgG in mice with GVHD. The finding that a single application of immune serum leads only to a short-term increase of the MCMV-specific IgG2a titer raised the possibility that IgG homeostasis could be altered in recipient mice with GVHD. To exclude the possibility that the observed deficiency of MCMV antibodies in recipient mice with GVHD was secondary to binding to MCMV antigens in the circulation or in the tissue, we injected uninfected BALB/c mice 14 days after BMT with serum containing antibodies reactive against the hapten 4-Hydroxy-3-Nitrophenyl Acetyl (NP), and we analyzed the serum kinetics of IgG anti-NP antibodies after transfer (Figure 5 and Table 1). Untreated and BM-transplanted mice exhibited an efficient absorption of IgG from the peritoneum to the bloodstream, whereas the efficiency IgG absorption following i.p. injection of mice with GVHD was reduced by nearly 50% (Table 1). Moreover, the half-life of IgG in mice with GVHD was reduced to 3 days, a more than 6-fold reduction when compared with untreated control mice and a 3-fold reduction compared with mice following BMT but without GVHD (Table 1). These results revealed that IgG levels were lower in animals with GVHD secondary to diminished absorption of IgG and, more importantly, an accelerated loss of serum antibodies in mice with GVHD. These findings may have important implications for the prophylaxis and/or therapy of HCMV in clinical HSCT.

Reduced serum half-life of serum IgG in mice with GVHD.Figure 5

Reduced serum half-life of serum IgG in mice with GVHD. Noninfected BALB/c mice were lethally irradiated and received 5 × 106 T cell–depleted BM cells ± 8 × 105 T cells from C57BL/6 donors. Fourteen days after BMT, recipients received anti–NIP-specific serum. Age-matched nontransplanted BALB/c mice injected with anti–NIP-specific serum served as controls. NIP-specific serum IgG titers were assessed at the indicated time points. Data are shown as mean ± SD. Day of serum injection is indicated by a vertical dotted line. Representative data from 2 independent experiments are depicted.

Table 1

Efficiency of absorption and serum half-life of IgG are reduced in mice with GVHD

Therapeutic intervention by adoptive transfer of Tregs ameliorates MCMV viral burden. Thus far, our data have demonstrated a therapeutic value of transfer of MCMV-specific antibodies in preventing and treating disseminated MCMV during GVHD. Since experimental data have shown a clear benefit of the transfer of CD4+CD25+ Tregs in the prevention and treatment of GVHD in mouse models (26, 27) and in human allograft recipients (28), we analyzed the impact of Treg suppression of GVHD on MCMV reactivation. To this end, we added donor-derived CD4+CD25+Foxp3+ Tregs to the transplantation protocol and studied their influence on GVHD and MCMV reactivation. First, we defined a dose of Tregs that could significantly reduce GVHD and prolong lifespan in uninfected BALB/c recipients (Supplemental Figure 5) (26). Transfer of the same number of Tregs into MCMV-infected BALB/c mice led to a significantly reduced GHVD index (Figure 6A) and a highly significant increase in survival of the animals (Figure 6B). When we analyzed MCMV reactivation in the Treg-treated mice, we observed a lower level of MCMV reactivation in Treg-treated mice that was significantly differen

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