Cytomegalovirus-Associated Inhibition of Hematopoiesis Is Preventable by Cytoimmunotherapy With Antiviral CD8 T Cells

Abstract

Reactivation of latent cytomegalovirus (CMV) in recipients of hematopoietic cell transplantation (HCT) not only results in severe organ manifestations, but can also cause "graft failure" resulting in bone marrow (BM) aplasia. This inhibition of hematopoietic stem and progenitor cell engraftment is a manifestation of CMV infection that is long known in clinical hematology as "myelosuppression." Previous studies in a murine model of sex-chromosome mismatched but otherwise syngeneic HCT and infection with murine CMV have shown that transplanted hematopoietic cells (HC) initially home to the BM stroma of recipients but then fail to further divide and differentiate. Data from this model were in line with the hypothesis that infection of stromal cells, which constitute "hematopoietic niches" where hematopoiesis takes place, causes a local deficiency in essential hematopoietins. Based on this understanding, one must postulate that preventing infection of stromal cells should restore the stroma's capacity to support hematopoiesis. Adoptively-transferred antiviral CD8⁺ T cells prevent lethal CMV disease by controlling viral spread and histopathology in vital organs, such as liver and lungs. It remained to be tested, however, if they can also prevent infection of the BM stroma and thus allow for successful HC engraftment. Here we demonstrate that antiviral CD8⁺ T cells control stromal infection. By tracking male donor-derived sry⁺ HC in the BM of infected female sry^- recipients, we show the CD8⁺ T cells allow for successful donor HC engraftment and thereby prevent CMV-associated BM aplasia. These data provide a further argument for cytoimmunotherapy of CMV infection after HCT.

Keywords: bone marrow stroma; cytomegalovirus pathogenesis; graft failure; hematopoietic (stem) cell transplantation (HCT, HSCT); hematopoietic reconstitution; immunotherapy; murine cytomegalovirus; myelosuppression.

Figure 1

Control of CMV organ infection by immunotherapy with CD8⁺ T cells. (A) Sketch of the transplantation model defining group A (no therapy) and group B (therapy with virus-specific CTL). The flash symbol indicates hematoablative conditioning of the HCT recipients by total-body γ-irradiation. (B) Day-14 infection load in the organs of HCT recipients expressed as titers (plaque-forming units, PFU) of infectious virus (left panel) and as numbers of viral genomes (right panel). Symbols (red: group A; blue: group B) represent mice analyzed individually. Median values are marked. Differences between two experimental groups were determined by Student's t-test based on log-transformed, log-normally distributed data. Significance level: P-values of < 0.001 (***).

Figure 2

Immunohistological images of liver tissue infection and infiltration by T cells. Representative 2C-IHC images of liver tissue sections taken on day 14 after HCT and infection, corresponding to the infection load data shown in Figure 1B. (Red IHC staining) IE1 protein in nuclei of infected liver cells, which are predominantly hepatocytes. (Black IHC staining) CD3ε protein expressed by T cells. (Group A) No therapy. (A) Overview. (A*) Detail. (Group B) Therapy by transfer of CTL. (B) Overview. (B*) Detail. Frames in images (A,B) point to regions resolved to greater detail in images (A*,B*), respectively. Light counterstaining was done with hematoxylin. NIF (arrows in B*): nodular inflammatory foci that represent microanatomical structures where transferred CTL (black) congregate at infected liver cells (red) to prevent intra-tissue virus spread. Bar markers: 50 μm.

Figure 3

Immunohistological images of stromal infection in BM. Representative IHC images of vertebral BM sections taken on day 14 after HCT and infection. (Red staining) IE1 protein in nuclei of infected cells. Light counterstaining was done with hematoxylin. Arrows point to infected, very typically shaped “reticular stromal cells” (iRSC). (Group A) No therapy. (Group B) Therapy by transfer of CTL. Bar marker: 50 μm.

Figure 4

Inhibition of BM repopulation by CMV and its restoration by CTL transfer. (Control) HCT only. (Group A) HCT and infection. (Group B) HCT, infection, and immunotherapy with CTL. The indicated parameters of BM repopulation refer to day 14 throughout. (A) Hematopoietic cell (HC) counts refer to representative 2-mm² section areas of sternal and vertebral BM. All other parameters (B–D) refer to BM cell yields pooled from both femora and both tibiae of individual mice. Symbols represent mice analyzed individually. Median values are marked. Differences between two experimental groups were determined by Student's t-test based on log-transformed data (upper left panel) or on non-transformed data (remaining panels). Significance levels: P-values of <0.05 (*), and <0.01 (**). n.s., not significant.