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AIDS VACCINE RESEARCH LABORATORYDavid Watkins, P.I. |
Immunology Core
Eva Rakasz, Ph.D. – Associate Scientist, leads the Immunology core. She has experience in all aspects of flow cytometry and development of diverse immunological assays. Kimberly Weisgrau, B.S. is responsible for the day-to-day operation of the flow cytometry facility. Shari Piaskowski, B.S. is the lead technician in the ELISPOT group. She and Jessica Furlott, B.S. is responsible for performing ELISPOT assays, and maintaining our extensive peptide library that covers the entire length of the SIVmac239 genome.
Routine activities performed by the Immunology Core Unit to monitor SIV infection, pathogenesis and infection/vaccination elicited cellular immune responses are: 1. quantification of SIVmac239 epitope-specific CD8+ T cell responses by tetramer staining; 2. detection of SIVmac239 epitope-specific cellular recognition utilizing intracellular cytokine staining and FACS analysis; 3. detection of CD8+ antigen-specific responses using ELISPOT; 4. in vitro CD4 proliferative responses; 5. measurement of the changes of absolute CD4 count in the peripheral blood; 6. analysis of CD4 and CD8 memory subsets in peripheral blood and mucosal tissues.
1. Tetramers
Tetramers are used to enumerate T cells responding to a particular MHC-peptide combination. We have identified several MHC class I alleles that are present at high frequency in the Indian rhesus macaque population (Mamu-A*01 (Allen 1998; Sidney 2000; Allen 2001), Mamu-A*02 (Vogel 2002; Loffredo 2004) and Mamu-B*17 (Mothe 2002)) and determined which SIV-derived epitopes are bound by these alleles. We are working to produce tetramers to all of the identified epitopes for these alleles, so that we can use tetramers to follow as many immune responses as possible in MHC-defined macaques.
Tetramers are purified on a size exclusion column, then concentrated to a stock solution of 1 mg/ml or greater. Before use, they are diluted to 100 mg/ml and 1-5 ml of this dilution is used to stain 5 x 105 cells for 1.5 hours at 37°C. During the last half hour of staining, surface stains (CD3 and CD8) are added. The cells are fixed and tetramers visualized using a FACSCalibur flow machine. Data are processed using the FlowJo program. A tetramer is considered positive if there is little to no tetramer in the CD8- (CD4+) population and the tetramer positive, CD8+ population is reasonably well separated from the negative population. Stricter criteria are used if the tetramer is to be used for sorting. We currently have 27 different MHC class I tetramers and are developing tetramers for additional MHC class I alleles, as well as MHC class II tetramers to follow HTL responses.
2. Intracellular cytokine analysis (ICS)
To assess which cytokines are secreted by SIV-specific cells we routinely use freshly isolated mononuclear cells. The cell source is most frequently peripheral blood, but our facility is proficient in performing ICS on diverse types of tissues including lymphoid (lymph nodes, spleen, bone marrow, thymus) and non-lymphoid origin (intestine, female reproductive tract). Depending on the number of cells available and the availability of defined SIVmac239 epitopes, we maximize the scope of immune response measurement by using peptide pools covering the whole SIV proteome, selected regions of viral proteome, and/or defined dominant and subdominant epitopes.
ICS is a powerful and flexible technique to look at T cell activation. PBMC are isolated from fresh blood and 5 x 105 cells per test activated by specific peptide or by peptide pools. Brefeldin A (BFA) or monensin are added after 1.5 hours of stimulation, to keep any cytokine produced on the inside of the cell, rather than being exported to the supernatant. After 6.5 hours of stimulation, cells are surface stained, fixed, perforated with saponin, stained for various cytokines, then fixed again for flow analysis. Cells are visualized on a FACSCalibur and FlowJo is used for data analysis.
We typically surface stain for CD4 and CD8, so that responses can be delineated between these two subsets. Cytokines chosen for observation are limited only by the availability of antibodies that stain rhesus cytokines. We typically stain with interferon gamma (IFNg) and either interleukin-2 (IL-2) or tumor necrosis factor alpha (TNFa). In addition, cells can be stained for granzyme A or B, which is an indication that the cells are ready to perform killing, or for CD107a or CD107b, which is a measure of degranulation in response to activation. SEB is used as a positive control and either no peptide or irrelevant peptide are used as negative controls.
For an ICS spanning the whole SIVmac239 proteome, we will utilize 10 µM peptide pools containing ten 11-mers overlapping by 10. Other than minimal optimal peptides, the most sensitive stimulation for CD8 T cells has been observed to be pools of ten 11-mers overlapping by 10. These also work for detection of CD4+ responses (data not shown). We will be synthesizing 11-mers overlapping by 10 that would cover the entire proteome of SIV.
3. ELISPOT
We currently use PBMC from SIV-infected rhesus macaques to map CD8+ T-lymphocyte epitopes using overlapping peptide pools (10 peptides per pool). 96-well flat bottom plates are coated with 5 µg/well of anti-IFNg monoclonal antibody and incubated overnight at 4˚C. The plates are then washed and blocked with 2% PBSA (PBS containing 2% bovine serum albumin) for 2 hours at 37˚C. Plates are washed just before use, then we add 10 µM test peptide in duplicate wells and add 1.0 x 105 freshly isolated PBMC resuspended in 100 µl R5 per well.
As positive control, one set of duplicate wells per plate contains 5 µg/ml Concanavalin A and a second set contains 10 µM of either Gag181-189CM9 (Mamu-A*01), Nef159-167YY9 (Mamu-A*02), or Nef165-173IW9 (Mamu-B*17) as a positive peptide control, since responses against these epitopes consistently arise in SIV-infected animals expressing these alleles. Several duplicate wells containing no peptide stimulation are used as the negative control.
For an ELISPOT spanning the whole SIVmac239 proteome, 10 µM peptide pools containing ten 11-mers overlapping by 10 will be tested in duplicate. In addition we include plates containing minimal optimal peptide epitopes corresponding to the genotype of the animal.
The plates are incubated 18 hours at 37˚C, in a 5% CO2 incubator. Cells are washed from the plates, and 200 µl/well of cold deionized water is added to lyse any remaining PBMC. Rabbit polyclonal biotinylated detector antibody solution (1 µg/well) is added and the plates are incubated for 1 hour at 37˚C, 5% CO2. The plates are washed and 50 µl/well of a gold-labeled anti-biotin IgG solution is added. The plates are once again incubated for 1 hour at 37˚C, 5% CO2, washed and 30 µg/well of activator mix is added, and the plates are developed for approximately 15 minutes. The activator solution consists of a silver salt solution that precipitates at the sites of gold clusters (from the gold-labeled anti-biotin solution), visualizing the sites where the IFN-g is secreted. Once precipitates are seen in the wells under microscope, the wells are washed 5 times with distilled water to stop the development process. The plates are then air-dried.
Wells are imaged with the AID ELISPOT Reader System v3.1.1 (Cell Technologies Inc, Columbia, MD). A Spot Forming Cell (SFC) is defined as a large spot with a fuzzy border. We use a profile to initially identify SFCs, then confirm identification manually. A positive result has a number of SFCs that are two standard deviations above the negative control wells. The calculation is done by our database and the results stored there for additional analysis or for comparison of results of many animals.
4. CD4 proliferation using CFSE staining
CFSE is a dye that binds covalently to proteins inside a cell. When a cell divides, each daughter cell contains half the amount of CFSE as the parent cell, which can be read in the FL-1 channel of a flow cytometer. CFSE has therefore been used to measure proliferation of lymphocytes in response to antigen stimulation. Briefly, cells are labeled with CFSE at 37°C for 7 minutes, washed and distributed to 96 well U-bottom plates. Peptides or peptide pools are added to each well in duplicate, and tests include positive controls such as SEB and animals with known proliferative responses, and negative controls including CFSE labeled unstimulated cells. After incubation at 37°C for 6-7 days, cells are surface stained with CD3 PE, CD4 APC and CD8 PerCP, analyzed on a FACSCalibur and the data analyzed in FlowJo. Proliferation is seen as a decrease in CFSE compared with unstimulated controls. We are also testing other proliferation methods, such as BrdU staining in parallel experiments.
5. Monitoring the changes of absolute CD4 count in the peripheral blood
The CD4 count/ml peripheral blood is quantified at least every month after virus inoculation to follow progression of the disease. A two-platform method using data derived from CBC and FACS analysis is employed. Samples are routinely stained to quantify CD3, CD4, CD8 and CD20 antigen positive cells.
6. Analysis of CD4+ and CD8+ memory subset populations
Memory subsets will be analyzed prior to vaccination, and in particular during the early acute phase of infection. Perturbations of the CD4+ memory subset populations are seen during the first few weeks of infections and may constitute the immune system dysfunction that will eventually result in immune system collapse (Li 2005, Mattapallil 2005). We will continue to monitor these populations at least monthly to see how well the vaccine can protect these critical subsets, and to monitor their recovery from the initial hit during early infection. PBMC or mucosal samples are stained according to the method of Picker 2004 with CD95 FITC, CD28 PE, CD4 PerCP, CCR5 APC, CD8 APC-Cy7, and either CD3 PE-Cy7 or CD3 Pacific blue, then samples are analyzed on the BD LSR II flow cytometer. Samples are analyzed the same day or within 24 hours so that the labile CCR5 marker can be reliably detected. CBC analysis will also be performed so that data can be reported either as percent of the CD4+ or CD8+ subsets or as absolute numbers.