Ex vivo generation of effective Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocytes from the peripheral blood of immunocompetent Epstein Barr virus-seronegative individuals.
BACKGROUND
Although readily accomplished from immunocompetent Epstein-Barr virus- (EBV) seropositive individuals, the effective ex vivo generation of EBV-specific cytotoxic T lymphocytes (CTL) from the peripheral blood mononuclear cells (PBMC) of EBV-seronegative subjects has proven to be a challenge. The focus of our study was to ascertain optimized culture conditions required for the ex vivo generation of EBV-reactive autologous CTL from the PBMC of EBV-seronegative volunteers.


METHODS
Freshly isolated PBMC obtained from immunocompetent EBV-seronegative and -seropositive individuals were used to generate EBV-specific autologous CTL lines using both conventional and a novel, modified ex vivo culture technique.


RESULTS
In contrast to responses observed in EBV-seropositives after two to three rounds of ex vivo stimulation, gamma-irradiated autologous lymphoblastoid cell lines (LCL) were incapable of eliciting an effective anti-EBV cytotoxic response when freshly-isolated PBMC from EBV-seronegative individuals were used as responders. Under these culture conditions, CD4+ T cells with preferential expression of the Th2-type cytokine IL-4 were predominantly expanded in the PBMC obtained from EBV-seronegative individuals. However, the addition of recombinant human (rh) IL-12 during the primary phase of ex vivo stimulation resulted in augmentation of EBV-specific cytolysis of autologous LCL by CD8+ T cells. Furthermore, there was down-regulation in the secretion of IL-4 and up-regulation in that of the Th1-type cytokine IFN-gamma by responder CD4+ and CD8+ T cells.


CONCLUSIONS
Taken together these data suggest that the addition of rhIL-12 during the primary phase of ex vivo stimulation of freshly isolated PBMC from EBV-seronegative individuals results in skewing of the immune response predominantly towards a CD4+ Th1-type (IFN-gamma) with the generation of an efficacious CTL-mediated anti-EBV reactivity. This novel ex vivo approach for generating effective autologous EBV-specific CTL could be adopted to treat refractory post-transplant lymphoproliferative disorders, which may be encountered in EBV-seropositive-->EBV-seronegative organ transplant recipients. Additionally, these ex vivo generated anti-EBV T cells could also be infused perioperatively to enhance prophylactically immunity against EBV infection in high-risk EBV-seronegative organ allograft recipients.
Metes
D
D
Thomas E. Starzl Transplantation Institute and the Department of Surgery, School of Medicine, University of Pittsburgh, PA 15213, USA.
Storkus
W
W
Zeevi
A
A
Patterson
K
K
Logar
A
A
Rowe
D
D
Nalesnik
M A
MA
Fung
J J
JJ
Rao
A S
AS
D000914:Antibodies, Viral / Q000097:blood*; D000954:Antigens, Surface / Q000235:genetics; D016026:Bone Marrow Transplantation / Q000009:adverse effects*; D018414:CD8-Positive T-Lymphocytes / Q000821:virology*; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D018664:Interleukin-12 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000821:virology; D008232:Lymphoproliferative Disorders / Q000209:etiology; D010641:Phenotype; D011994:Recombinant Proteins / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000821:virology
D000914:Antibodies, Viral; D000954:Antigens, Surface; D011994:Recombinant Proteins; D018664:Interleukin-12