Method Description

Assessment of cytomegalovirus (CMV)-immune competence measures 3 different components of the immune response: 1) numerical analysis of the CMV-specific CD8 T-cell population (enumeration), which provides absolute counts of the antigen-specific CD8 T cells, 2) functional, and 3) cytotoxic analysis of the CMV-specific CD8 T cells (functionality and cytotoxic activity).

Enumeration:

Enumeration of CD8 T cells is performed on whole blood using Beckman Coulter MHC class I tetramers, specific for 5 major histocompatibility complex (MHC) class I alleles-A1, A2, B7, B8, and B35.

Panel I consists of whole blood stained with CD3, CD8, and CD4 fluorochrome conjugated antibodies followed by incubation at ambient temperature in the dark. The sample is then treated with a tetramer lyse reagent. The flow count bead solution is then thoroughly mixed and added at a volume exactly equal to the volume of whole blood added. After the addition of beads, the sample is analyzed on a flow cytometer.

In Panel II, whole blood is stained with CD3 and CD8 fluorochrome conjugated antibodies and incubated with the relevant MHC class I tetramer at ambient temperature in the dark. The sample is then treated with a tetramer lyse reagent and washed with wash buffer. The sample is then analyzed on a flow cytometer an the CMV-specific CD8 T cells are analyzed as a percentage of the total CD8+ T cells calculated in Panel I.

The final absolute counts for CD3 T cells, CD8 T cells, and CMV-specific CD8 T cells are expressed as cells/mL of whole blood.

Functionality (Intracellular Interferon-gamma: IFN-gamma) and Cytotoxic Activity (Surface CD107a/b):

Peripheral blood mononuclear cells (PBMC) are isolated from the same sample of whole blood used for enumeration. PBMC are stimulated with human leukocyte antigen (HLA) allele-specific CMV peptides and costimulatory molecules (CD28/CD49d) in a polypropylene tube. Antihuman CD107a/b conjugated with fluorochrome is added along with the peptides to capture the transient expression of the CD107a and CD107b, which are markers for cytotoxic activity. A mixture of Brefeldin A (BFA) and monensin is also added during the stimulation to facilitate the intracellular accumulation of IFN-gamma, which is detected using an antihuman IFN-gamma antibody conjugated with fluorochrome. A similar tube is prepared for the patient sample with the absence of exogenous CMV peptide and this tube serves as the unstimulated control (back-ground stimulation). Thus, there are 2 tubes for every HLA allele. After peptide stimulation, EDTA is added to the sample to arrest activation and to remove adherent cells from the activation tube. Antihuman CD8 antibody and the relevant MCH-class I tetramer are added and incubated at ambient temperature in the dark. This step is followed by a simultaneous lysis and fixation of the cells to prepare them for permeabilization. Cells are then washed and permeabilized with BD FACS Permeabilizing Solution 2. The antihuman IFN-gamma antibody is added, and the sample is incubated at ambient temperature in the dark. Finally, the cells are washed and analyzed by flow cytometry. The delta percent (stimulation in the presence of CMV-specific peptide stimulation in the absence of specific peptide) CMV-specific CD8 T cells expressing IFN-gamma and CD107a/b is used to calculate the absolute count of activated and functionally cytotoxic CMV-specific CD8 T cells.(Functionality: IFN-gamma assay, Unpublished Mayo method; Cytotoxic Activity: Betts MR, Brenchley JM, Price DA, et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods. 2003;281 (102):65-78; Betts MR, Price DA, Brenchley JM, et al. The functional profile of primary human antiviral CD8 T cell effector activity is dictated by cognate peptide concentration. J Immunol. 2004;172(10):6407-6417)

Global CD8 T-Cell Immune Competence:

Peripheral blood mononuclear cells (PBMC), which contain CD8 T cells, are stimulated with a mixture of phorbol myristate acetate (PMA) and ionomycin, and with stimulatory signals derived using antibodies against the costimulatory molecules CD28/CD49d. The cells are simultaneously treated with a mixture of BFA and monensin, which blocks extracellular secretion of IFN-gamma, enabling intracellular retention and detection of the protein. PBMCs that have not been stimulated are used as a control to determine the background levels of IFN-gamma and CD107a and CD107b. The cells are analyzed on a flow cytometer and analysis involves gating (defining) of the CD8 T cells using an antihuman CD8 antibody. Specific IFN-gamma and CD107a and CD107b signals are determined within the "gated" CD8 T cell population. Global CD8 T cell immune competence is measured by the amount of IFN-gamma produced (CD8 T-cell functional activity) and surface expression of CD107a/b (cytotoxicity assessment) relative to the unstimulated control and is interpreted on the basis of the reference range determined from healthy adult donors.(Unpublished Mayo method)