Clinical Information

Cytomegalovirus (CMV), a double-stranded DNA virus, belongs to the Herpesviridae family of viruses and is structurally similar to other herpes viruses. Although many human strains of CMV exist, there is little genetic homology between human CMV and CMV of other species. The reported seroprevalence rates of CMV range from 40% to 100% in the general population. In the urban United States, the seroprevalence of CMV has been reported to be 60% to 70%.(1) However, data from Mayo Clinic's laboratory indicate that the seroprevalence in the Midwestern US population is closer to 30% (unpublished observations).

Once CMV infection occurs, the virus spreads hematogenously to almost every organ. After acute infection, the virus enters a latent phase. Activation from this phase can be seen after acute illness, immunosuppression in allogeneic hematopoietic stem cell transplantation (HSCT) or solid organ transplantation, or use of chemotherapy agents. CMV infection or reactivation has been implicated in allograft rejection in renal(2) and cardiac transplantation.(1) In cardiac transplants, CMV infection has been shown to contribute to accelerated development of transplant atherosclerosis (cardiac allograft vasculopathy). CMV remains a significant cause of morbidity and mortality after HSCT. Of allogeneic HSCT patients who are CMV-seropositive, 60% to 70% will experience reactivation and, without ganciclovir or other preemptive therapy, 20% to 30% will develop end-organ disease.(3)

CD8 T cells play a critical role in viral immunity, and CD8 T-cell effector functions include cytotoxicity and cytokine production. Cytotoxicity occurs after CD8 T-cell activation, causing target cell apoptosis. Cytotoxic T-cell responses mediate killing of target cells via 2 major pathways, granule-dependent (perforin and granzymes) and granule-independent (Fas and Fas ligand [FasL]) mechanisms. The granule-dependent pathway does not require the de novo synthesis of proteins by effector CD8 T cells, but rather it utilizes preformed lytic granules located within the cytoplasm. Among the proteins in these preformed lytic granules are the lysosomal-associated membrane proteins (LAMP), including LAMP-1 (CD107a) and LAMP-2 (CD107b). These proteins are not normally found on the surface of T cells. Degranulation of activated CD8 T cells occurs rapidly after T-cell receptor (TCR) stimulation, exposing CD107a and CD107b. The cytokines produced by activated T cells include interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), macrophage inflammatory protein 1 alpha (MIP-1 alpha), macrophage inflammatory protein 1 beta (MIP-1 beta), and interleukin-2 (IL-2). Several studies have shown the importance of cytotoxic T-cell responses to CMV in conferring protection from subsequent CMV disease.

Antiviral drugs have helped reduce the incidence of early CMV infection, and acyclovir and ganciclovir have been the mainstay of antiviral treatment for a number of years, although these drugs have poor bioavailability.(4) The development of the new antiviral drugs valacyclovir and valganciclovir (by the addition of a valine ester) has increased the bioavailability of these drugs by 10-fold.(4) There is some data to suggest valganciclovir prophylaxis may be considered for HSCT patients who are at high risk for infection and disease, though there is a need for further study in this area.(5)

Two main strategies have been used for the prevention of early CMV infection and disease in CMV-seropositive patients and in seronegative recipients who receive a seropositive graft-preemptive therapy:

-Patient monitoring for CMV infection and treatment only when CMV viremia is present.

-Prophylactic management-where all patients receive treatment after transplantation with the goal of preventing CMV disease.(5) The disadvantage of prophylactic therapy is that it requires monitoring for myelosuppression and infections-side effects of antiviral drug therapy. Despite this disadvantage, there are several reasons to consider prophylaxis, including the fact that the incidence of recurrent infections after treatment is 30% to 40%,(5) patients receiving preemptive therapy have a 5% CMV disease break-through, and prophylaxis reduces the risk of viral reactivation.

Late CMV infection occurs 3 months after transplantation and is now recognized as a significant cause of morbidity after allogeneic HSCT.(6) These complications usually occur in the setting of continued immunosuppression for chronic graft-versus-host disease (GVHD). The clinical manifestations of late CMV disease differ slightly from those seen early after transplantation. Within the first 100 days after HSCT, almost all patients with CMV disease have CMV pneumonia or gastrointestinal disease. In late CMV disease, the more unusual manifestations of CMV infection (eg, CMV retinitis, CMV-associated bone marrow failure, or CMV encephalitis) tend to occur.(7) These late manifestations occur in a setting of continued CMV-specific T-cell immunodeficiency. Therefore, it is necessary to monitor CMV-specific CD8 T-cell responses, in addition to viral load, to effectively identify patients at higher risk of CMV disease.

It has been shown that ganciclovir may delay the recovery of the protective CMV-specific T-cell response, which may contribute to the occurrence of late CMV disease.(8) The use of ganciclovir as early treatment after detection of CMV in blood or other body fluid and as a prophylaxis for CMV infection in bone marrow transplant (BMT) and heart transplant recipients has dramatically reduced the incidence of CMV in these immunocompromised hosts. Yet, early treatment and prophylaxis have not been uniformly successful, with up to 15% of BMT recipients developing CMV disease after discontinuation of antiviral therapy. Similarly, patients undergoing lung transplantation have been shown to be only transiently protected with antiviral agents. These data suggest that the CMV-specific responses necessary for protection may not recover during the time the host is receiving antiviral therapy.

Ganciclovir exerts its antiviral effects at the stage of viral DNA replication and, therefore, in the presence of the drug, infected cells may express some of the immediate early and early gene products, but not the full complement of CMV genes required for replication and virion formation. In latently infected CMV-seropositive individuals, the human leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocyte response to CMV is predominantly specific for epitopes derived from structural virion proteins. Therefore, in patients receiving ganciclovir, the viral antigens may be inadequate to activate host T-cell responses, resulting in the failure to reconstitute CMV-specific immunity. In fact, a prospective, randomized placebo-controlled study of ganciclovir prophylaxis revealed that when ganciclovir therapy is discontinued, a larger fraction of patients (who received the drug) are deficient in CMV-specific T-cell immunity at day 100 than in the placebo group.(8) That study also showed that bone marrow donor serology has an important influence on the early detection of virus-specific T-cell responses.(8) Not all medical centers use ganciclovir for prophylaxis; some use acyclovir and the same findings may apply in this case as well.

In a more recent study, it was shown that impaired CD8 function was associated with the use of high-dose steroids, bone marrow as a source of stem cells, and CD8 T-cell lymphopenia.(3) In the absence of high-dose steroids, low-level subclinical CMV antigenemia was found to stimulate both CD4 and CD8 functional recovery in recipients of ganciclovir prophylaxis, suggesting that subclinical CMV reactivation while on antiviral therapy can be a potent stimulator of T-cell function.(3) Regardless of antiviral therapy, immunologic reconstitution remains the key element in protection from late-onset CMV disease.

This test assesses the number of CMV-specific CD8 T cells and their function (activation via production of the cytokine IFN-gamma and cytotoxic potential via CD107a and CD107b as markers of degranulation) using a panel of 5 major histocompatibility complex (MHC) class I alleles (HLA A1, A2, B7, B8, and B35) along with their respective immunodominant CMV epitopes. This 3-part assay allows a comprehensive assessment of CMV-specific CD8 T cell immunity and, when combined with evaluation of viremia using molecular analyses, provides a more accurate picture of the nature of CMV reactivation and the corresponding immune response than evaluating viremia alone (9).

Assessment of Global CD8 T-Cell Function:

CD8 T cell activation occurs either through the TCR-peptide-MHC or by use of a mitogen (eg, phorbol myristate acetate and the calcium ionophore ionomycin). Mitogen-mediated activation is antigen nonspecific.

Impairment of global CD8 T cell activation (due to inherent cellular immunodeficiency or as a consequence of immunosuppression by therapeutic agents) results in reduced production of interferon-gamma and other cytokines, reduced cytotoxic function, and increased risk for developing infectious complications. Agents associated with over-immunosuppression include the calcineurin inhibitors (eg, cyclosporine A, FK506 [Prograf/tacrolimus], and rapamycin [sirolimus]), antimetabolites (eg, mycophenolate mofetil), and thymoglobulin.

The incorporation of global CD8 T cell function in this assay is helpful in determining if the lack of CMV-specific (antigen-specific) response is due to a global impairment in CD8 T cell function, due to immunosuppression or other causes, or whether the lack of CMV CD8 T cell immunity is unrelated to overall CD8 T cell function.

The absolute counts of lymphocyte subsets are known to be influenced by a variety of biological factors, including hormones, the environment, and temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells increase between 8:30 a.m. and noon, with no change between noon and afternoon. Natural killer cell counts, on the other hand, are constant throughout the day.(10) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(11-13) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(11) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening(13), and during summer compared to winter.(14) These data, therefore, indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.