Niche Perivascular Stromal Cells As a Potential Reservoir and a Source of HCMV Reactivation

Abstract 3464

Cytomegalovirus (HCMV) infection or reactivation is an important cause of morbidity and mortality among hematopoietic cell transplantation (HCT) recipients. HCMV infection in these patients may result from a new HCMV infection, reactivation of pre-existing latent virus, or both. Regardless, HCMV causes end organ diseases including pneumonia, gastroenteritis, retinitis, hepatitis, and encephalitis, and can contribute to graft failure. Although the use of antiviral drugs has decreased the incidence, morbidity, and mortality of HCMV disease in transplant recipients, identification of cells serving as the reservoir and/or that are responsible for reactivation of the disease could potentially lead to the development of novel targeted therapies. Although hematopoietic cells, particularly myeloid progenitors, have been identified as reservoirs for HCMV reactivation, it is possible that within the bone marrow environment there are cells that are able to serve as a reservoir for this virus. Perivascular mesenchymal/stromal cells (MSC) are part of the microenvironmental niche and play a role in hematopoietic stem cell (HSC) maintenance and differentiation. Because MSC have an increased resistance to chemo- and radio-therapy, allowing the survival of resident MSC during HCT, they express CD29, a main receptor for HCMV capture and entry, and they directly interact with HSC within the niche, we hypothesized that MSC could serve as a potential reservoir and a source of HCMV reactivation in transplantation patients. To this end, we collected bone marrow (BM) from 10 different healthy adult donors, who had tested positive for CMV antibodies, and investigated whether MSC (phenotypically defined as cells positive for CXCL12, CD90, CD44, CD29, CD73, CD105, and CD146, but negative for both CD34 and CD45) were positive for CMV, by testing for the presence of Immediate Early (IE) HCMV gene products by PCR with subsequent confirmation by sequencing. MSC sorted from 7 out of 10 adult donors tested positive for the presence of IE HCMV DNA, demonstrating persistence of the virus in these cells in the absence of disease. In order to further evaluate the permissivity of these cells to HCMV infection and to determine whether the virus was able to establish a lytic infection, we infected MSC from HCMV-negative donors in vitro, with the clinical isolate Davis strain of HCMV (MOI of 3). At 1, 3, 5, and 7 days post-infection (dpi), the cells were positive for IE HCMV DNA. They were also positive, at one or more of the tested time points, for UL-83, a late gene associated with lytic infection. IE protein expression was also detected by immunohistochemistry. The mock infected cells remained negative for both IE and UL-83 throughout the duration of the experiment. In order to determine whether MSC were able to produce and release infectious viral particles, supernatants were collected at different times post-infection, and evaluated for the presence of infectious units (IU) using HFF. At 1, 3, 5, and 7 dpi, MSC produced 5167±809, 6867±104, 3800±200, and 3467±788 IU, respectively, demonstrating that MSC are able to produce and release infectious particle and infect neighboring cells. We have therefore shown that a highly defined population of human stromal cells (MSC) naturally harbors latent HCMV. Since MSC reside within/comprise the HSC niche, they share an intimate relationship with hematopoietic progenitors, and they participate in homing and retention of HSC, our data support the conclusion that MSC may serve as a previously unrecognized reservoir for this clinically important virus in transplant recipients. Moreover, upon infection with HCMV, MSC are able to produce infectious virus, thereby propagating HCMV infection to adjacent cells. Studies are underway to determine whether productive HCMV infection of this stromal cell population also alters the expression of proteins that regulate HSC maintenance.