Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ T lymphocytes
Introduction
Human cytomegalovirus (CMV) infection continues to be one of the most important infections in patients undergoing allogeneic stem cell transplantation [1]. It may also affect as many as 60% of recipients after liver or renal transplantation [2, 3]. Whereas early-onset human CMV disease—that occurring during the first 100 days after transplantation—can be significantly reduced by introduction of preemptive or prophylactic antiviral therapy, CMV disease occurring after day 100 is now one of the leading causes of death after allogeneic stem cell transplantation [4, 5]. Prolonged prophylaxis with antiviral drugs, such as ganciclovir and foscarnet, can lead to antiviral resistance and severe side effects. The duration of ganciclovir treatment for CMV infection determines the risk of secondary neutropenia and secondary infections [5, 6, 7, 8, 9].
Prospective monitoring of CMV viral load in post-transplant patients with polymerase chain reaction or CMV antigenemia to guide preemptive antiviral therapy can reduce the incidence of CMV disease and CMV-associated mortality, but the high sensitivity of these assays leads to treatment of many patients who would never have progressed to active disease [10]. Novel criteria to help with the decision regarding antiviral therapy after allogeneic stem cell or organ transplantation would therefore be of great value. Various arguments strongly suggest that CD8+ T lymphocytes play a major role in the control of CMV infection and disease. Infusion of CMV-specific CD8+ T cell clones after allogeneic stem cell transplantation prevents the development of CMV disease [11, 12, 13, 14]. Recovery of CMV-specific cytotoxic T lymphocytes (CTLs) is associated with protection from CMV disease after allogeneic stem cell transplantation [15]. There is an inverse correlation between the CMV-directed CTL response and CMV antigenemia after renal transplantation [16]. Clinically significant CMV disease and death are observed exclusively in renal transplant recipients without a demonstrable CTL response [17]. The detection of CMV-specific CD8+ T cells may therefore provide additional information about CMV virus detection to predict the risk of infection and development of CMV disease.
Various techniques have been used to detect CMV-specific CD8+ T cells in humans. The chromium release assay is time-consuming, requires a large number of peripheral blood mononuclear cells (PBMCs), uses radioactive materials, and has a low sensitivity [16].
Most recent studies have identified and quantitated CMV-specific CD8+ T lymphocytes by the CMH-peptide tetramer staining [18, 19, 20, 21, 22, 23]. This technique presents the advantage of detecting both functional and anergic T cells but is unable to distinguish between these two types of cells. Effector CMV-specific CD8+ T cells, which may more accurately reflect anti-CMV immunity, are not well correlated with the number of tetramer-positive cells [24]. The detection of intracytoplasmic interferon-γ (IFN-γ) within tetramer-positive CD8+ T cells represents a possible surrogate marker of specific effector T cells, but the coupling of intracellular cytokine staining with major histocompatibility complex (MHC) tetramer remains difficult and cannot be easily performed for routine analysis [24].
The enzyme-linked immunospot (Elispot) assay is based on solid-phase immunoenzyme technology [25]. This test allows the detection of functional specific CD8+ T cells secreting cytokines at a single cell level [26]. Until now, few groups have used Elispot to detect CMV-specific CD8+ T lymphocytes [27, 28, 29, 30]. We compared and tested various experimental conditions to optimize an Elispot assay for the detection of CMV-specific CD8+ T lymphocytes. We demonstrated its high sensitivity, its ease of use for routine analysis, and its application to transplant recipients with lymphopenia.
Section snippets
Study population
This study was performed on blood from healthy donors obtained from the Etablissement Français du Sang (Rungis, France). CMV serology was performed by this blood transfusion center for each subject.
Peripheral blood mononuclear cell preparation and fluorescence activated cell sorter analysis
PBMCs were extracted from peripheral blood by a standard Ficoll density-gradient centrifugation and were immediately tested by fluorescence activated cell sorter (FACS) analysis for the expression of HLA-A2 or HLA-B7 or HLA-A1. The combined frequencies of these three alleles encompassed 70% to 80% of
Comparative analysis of direct or indirect Elispot to detect CMV-specific CD8+ T cells
Fourteen HLA-A2 healthy individuals with positive CMV serology were tested for the presence of CMV-specific CD8+ T cells by direct or indirect Elispot using either PBMC or purified CD8+ T cells.
As shown in Table 1, the most sensitive method to detect CMV-specific CD8+ T cells was the indirect Elispot with PBMC after one in vitro sensitization step for six days, because this method was able to detect specific anti-CMV-specific CD8 T cells in all tested patients with positive CMV serology. The
Discussion
We have developed a highly sensitive indirect Elispot for the detection of CMV-specific CD8+ T cells. We have demonstrated that an in vitro sensitization step with a specific CMV peptide allows better detection of CMV-specific CD8+ T cells compared to direct Elispot with unfractionated PBMCs or purified CD8+ T cells. Most groups have analyzed CMV-specific CD8+ T cells with direct Elispot using unfractionated PBMC [28, 29, 34, 41], which may explain why the frequency of CMV-specific CD8+ T-cell
Acknowledgments
This work was supported by grants from Fondation de France, Agence Nationale de Recherche sur le SIDA (RIVAC program), Ligue Nationale Contre le Cancer, INSERM (ATC Biotherapy), Association pour la Recherche sur le Cancer, and Canceropole.
References (63)
- et al.
Late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplantsimportance of viral load and T-cell immunity
Blood
(2003) - et al.
Effects of changing immunosuppressive regimen on the incidence, duration, and viral load of cytomegalovirus infection in renal transplantationa single center report
Transpl Infect Dis
(2002) - et al.
Risk factors for cytomegalovirus and severe bacterial infections following liver transplantationa prospective multivariate time-dependent analysis
J Hepatol
(1993) - et al.
Successful modification of a pp65 antigenemia-based early treatment strategy for prevention of cytomegalovirus disease in allogeneic marrow transplant recipients
Blood
(1999) - et al.
Risk factors for treatment failures in patients receiving PCR-based preemptive therapy for CMV infection
Bone Marrow Transplant
(2000) - et al.
Recovery of HLA-restricted cytomegalovirus (CMV)-specific T-cell responses after allogeneic bone marrow transplantcorrelation with CMV disease and effect of ganciclovir prophylaxis
Blood
(1994) - et al.
Immune reconstitution to cytomegalovirus after allogeneic hematopoietic stem cell transplantationimpact of host factors, drug therapy, and subclinical reactivation
Blood
(2003) Ganciclovir-resistant cytomegalovirus in organ transplant recipients
Clin Infect Dis
(2002)- et al.
Rapid development of ganciclovir-resistant cytomegalovirus infection in children after allogeneic stem cell transplantation in the early phase of immune cell recovery
Bone Marrow Transplant
(2002) - et al.
Plasma polymerase chain reaction for cytomegalovirus DNA after allogeneic marrow transplantationcomparison with polymerase chain reaction using peripheral blood leukocytes, pp65 antigenemia, and viral culture
Transplantation
(1997)