Mutual Interference between Cytomegalovirus and Reconstitution of Protective Immunity after Hematopoietic Cell Transplantation

Abstract

Hematopoietic cell transplantation (HCT) is a therapy option for aggressive forms of hematopoietic malignancies that are resistant to standard antitumoral therapies. Hematoablative treatment preceding HCT, however, opens a "window of opportunity" for latent Cytomegalovirus (CMV) by releasing it from immune control with the consequence of reactivation of productive viral gene expression and recurrence of infectious virus. A "window of opportunity" for the virus represents a "window of risk" for the patient. In the interim between HCT and reconstitution of antiviral immunity, primarily mediated by CD8(+) T cells, initially low amounts of reactivated virus can expand exponentially, disseminate to essentially all organs, and cause multiple organ CMV disease, with interstitial pneumonia (CMV-IP) representing the most severe clinical manifestation. Here, I will review predictions originally made in the mouse model of experimental HCT and murine CMV infection, some of which have already paved the way to translational preclinical research and promising clinical trials of a preemptive cytoimmunotherapy of human CMV disease. Specifically, the mouse model has been pivotal in providing "proof of concept" for preventing CMV disease after HCT by adoptive transfer of preselected, virus epitope-specific effector and memory CD8(+) T cells bridging the critical interim. However, CMV is not a "passive antigen" but is a pathogen that actively interferes with the reconstitution of protective immunity by infecting bone marrow (BM) stromal cells that otherwise form niches for hematopoiesis by providing the structural microenvironment and by producing hematopoietically active cytokines, the hemopoietins. Depending on the precise conditions of HCT, reduced homing of transplanted hematopoietic stem- and progenitor cells to infected BM stroma and impaired colony growth and lineage differentiation can lead to "graft failure." In consequence, uncontrolled virus spread causes morbidity and mortality. In the race between viral BM pathology and reconstitution of antiviral immunity following HCT, exogenous reconstitution of virus-specific CD8(+) T cells by adoptive cell transfer as an interventional strategy can turn the balance toward control of CMV.

Keywords: CD8 T cells; Cytomegalovirus; adoptive cell transfer; bone marrow stroma; hematopoietic cell transplantation; hemopoietins; immunotherapy; reconstitution.

Figures

Figure 1

Negative impact of CMV infection on survival rates after HCT demonstrated in the murine model. (Top) Sketch of the experimental model, transplanting increasing numbers of hematopoietic cells (HC) to BALB/c recipients immunocompromised by hematoablative total-body γ-irradiation with single doses increasing from 5 to 7 Gy. (Bottom) Kaplan–Meier survival curves depending on the two variables HC dose (increasing reconstitution) and dose of γ-irradiation (increasing ablation). (Green lines) HCT, no infection. (Red lines), HCT and intraplantar infection with a constant dose of purified mCMV. Green- and red-shaded panels correspond to the bone marrow histology shown in Figures 3A,B, respectively. Reproduced from Ref. (59) with permission from Caister Academic Press, Norfolk, UK.

Figure 2

Infection of BM stromal cells in primary cell culture. (A) Images of the typically flattened, uninfected myofibroblastic BM stroma cells co-expressing smooth muscle cell marker α-SM-actin (a1, red fluorescence) and endothelial cell marker von Willebrand factor concentrated in Weibel–Palade bodies (a2, green fluorescence). (a3) merge. (B) Cytopathic effect of mCMV infection in the BM stroma cells. Detection of intranuclear E1 protein (spotty green fluorescence) indicates that infection has proceeded to the early (E) phase of the viral replicative cycle. Bar markers: 10 μm. Images illustrate information reported in Ref. (59).

Figure 3

Bone marrow aplasia caused by infection. (A) Overview image of BM histology performed on day 14 after HCT, revealing a beginning repopulation of the BM of the HCT recipients with myeloid lineage colonies homing to the BM stroma in a femoral diaphysis in the absence of infection. The image corresponds to survival shown in Figure 1, green-shaded panel. (B) Images document absence of hematopoiesis in infected HCT recipients under conditions otherwise identical to those in (A), corresponding to high mortality shown in Figure 1, red-shaded panel. (b1) Overview, showing empty stromal network in a femoral epiphysis. (b2) Detail, with immunohistological staining of viral intranuclear protein IE1 (brown staining), detecting in situ infected, network-forming stromal cells. Bar markers: 25 μm, throughout. Note that the size and shape of the stromal cells in situ matches their size and shape in cell culture (compare Figure 3B, b2 with Figure 2A). Images reproduced in rearranged form from Ref. (59, 84, 86) with permissions from Caister Academic Press, Norfolk, UK and from the Journal of Virology, American Society for Microbiology.

Figure 4

Infection inhibits HC engraftment. (A) Sketch of the experimental design of transplanting male (XY, gene sry+) HC to female (XX, gene sry−) BALB/c recipients, resulting in transplantation chimeras with sry+ HC and sry− stromal cells. Flash symbol: total-body γ-irradiation (B) PCR quantitation of sry+ HC engraftment on day 14 after HCT without infection (Ø) or with infection (CMV). Modified from Ref. (86) with permission from the Journal of Virology, American Society for Microbiology.

Figure 5

Sketch of serial transplantation and re-transplantation protocols. (A) Serial transplantation of HC for quantitating BM-repopulating hematopoietic stem cells (HSC) depending on a past productive infection of primary HCT recipients. Infected primary HCT recipients become lastingly deficient in hematopoiesis reflected by reduced numbers of HSC. (B) Localization of the cause of enduring hematopoietic deficiency in primary HCT recipients with a past productive infection. Failure to cure by re-transplantation with normal HC localizes the hematopoietic deficiency to the BM stroma. This Figure illustrates the experimental regimens used to demonstrate enduring bone marrow stroma deficiency reported in Ref. (82, 83).

Figure 6

Model of “closed” and “open” hematopoietic niches. (Left) Low-dose HCT: in the model example, a single transplanted HSC has a 20% chance to occupy a single “open niche” (green) out of a total of five niches of which four are closed (red) due to CMV infection of the BM stromal cells that form the niche. (Right) High-dose HCT: high probability of occupying the single “open niche” out of five niches when five HSC are transferred. Note that the probability for a successful lodging event is <100% since more than one HSC may try to lodge to a “closed niche,” but the overall probability for the event that at least one out of five transplanted HSC lodges to the single “open niche” is increased. Importantly, the rate of stroma cell infection is not notably influenced by the dose of transplanted HC over the range tested. Stop symbol: hematopoiesis is not supported. Bent arrow: successful HSC lodging and self-renewal. This Figure provides a graphical new explanation for data published in Ref. (86), corresponding to Figure 4B.

Figure 7

Infection of HCT recipients impedes the reconstitution of CD8+ T cells. (Top) Sketch of the experimental protocol, transplanting HC from BALB/c mice to immunocompromised (flash symbol: sublethal γ-irradiation with a dose of 6 Gy) mutant mice not expressing the MHC-I molecule Ld. (Bottom) Cytofluorometric analysis of Ld expression in the recipients revealed chimerism within the spleen-derived CD8+ T-cell population that shifted toward donor-type reconstitution with increasing doses of transplanted HC (left panel, HCT). This shift is impeded by infection (right panel, HCT + infection). *100% mortality. Percentages of donor-derived CD8+ T cells are indicated. Modified from Ref. (96) with permission from the Journal of Virology, American Society for Microbiology.

Figure 8

CD8+ T-cell infiltration of infected lungs after HCT coincides with control of the infection in nodular inflammatory foci (NIF). HCT was performed with the high dose of 107 HC, a condition under which infected recipients survive (recall Figure 1). (A) Coincidence of the peak of CD8+ T-cell infiltration (top panel) and the onset of decline in titers of infectious virus (bottom panel). Symbols represent data from individual mice with the median values marked. Reproduced from Ref. (59, 109) with permissions from Caister Academic Press, Norfolk, UK and from the Journal of Virology, American Society for Microbiology. (B) Confinement of pulmonary infection in NIF; shown here is a NIF with peribronchiolar localization. Two-color IHC with red-staining of IE1 protein in infected lung cells and black staining of T cells. The bar marker represents 25 μm. This image has been the cover photograph of Journal of Virology, volume 74, issue no. 16 (August 2000), accompanying the publication cited here as Ref. (61). Reproduced with permission from the American Society for Microbiology.

Figure 9

Survival of infected HCT recipients depends on the reconstitution of CD8+ T cells. In the process of an ongoing reconstitution after HCT, T-cell subsets were depleted on days 7 and 14. Shown are Kaplan–Meier survival curves that reveal 100% mortality only when CD8+ T cells were depleted. Reproduced from Ref. (59, 61) with permissions from Caister Academic Press, Norfolk, UK and from the Journal of Virology, American Society for Microbiology.

Figure 10

CD8+ T cells are essential for confining the infection to nodular inflammatory foci (NIF) during reconstitution after HCT. Two-color IHC images of viral pathology in the liver correspond to the survival/mortality data shown in Figure 9. (A) Uncontrolled virus spread in liver tissue after HCT and depletion of CD8+ T cells. The red arrow representatively points to an infected hepatocyte identified by red-staining of intranuclear IE1 protein. The black arrow representatively points to an infiltrating CD4+ T cell identified by black staining of cell surface CD3ε. Note that the CD4+ T cells are randomly distributed, do not form NIF, and apparently fail to limit the virus spread. (B) Lack of liver tissue infiltration, absence of NIF, and uncontrolled spread of the infection after depletion of both T-cell subsets, which excludes participation of other CD3ε-expressing immune cells, such as γ/δ T cells and NKT cells, in the control of infection. As the infection is not controlled, cells of innate immunity (unstained) apparently are also not functional in antiviral protection after HCT. (C) Confinement of infection to NIF in the absence of T-cell depletion. (D) Confinement of infection to NIF is maintained after depletion of CD4+ T cells, indicating that formation of NIF and control of the infection by CD8+ T cells do not critically require CD4+ T cell help. Bar markers represent 25 μm. Reproduced in rearranged form from Ref. (59) with permission from Caister Academic Press, Norfolk, UK.

Figure 11

Synopsis of the mutual interference between Cytomegalovirus and reconstitution of protective immunity after HCT. See the body of the text for explanation. HC, hematopoietic cells; MC, mast cells; SCF, stem cell factor; G-CSF, granulocyte colony-stimulating factor; IL-6, interleukin-6; NIF, nodular inflammatory focus/foci. Red TCR symbol: CMV-specific. Blue TCR symbol: unrelated TCR specificity. Red-colored nuclei: infected cells.