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Immunology and Gene Therapy

1A. IFN-γ ELISPOT (mouse and NHP)

The ELISPOT detecting interferon (IFNγ) has become widely used as a means of quantization of T cell-mediated immune responses (CD4+ or CD8+) and is becoming the assay of choice for clinical trials for detecting T cell responses to gene therapy product and candidate vaccines. It is relatively rapid; sensitive, and allows the screening of multiple samples on a single day. The ELISPOT assay is based on the principle of the ELISA detecting antigen-induced secretion of cytokines trapped by an immobilized antibody and visualized by an enzyme-coupled second antibody.

Figure 1 : IFNg ELISPOT of mice splenocytes. Host T cell responses to AAV2, 7, or 8 capsid studied in C57BL/6 mice injected i.m. with 1x1011 GC of rAAV2, 2/7, or 2/8 vectors. 


1B. Validated human IFN-γ ELISPOT for Clinical Trials

A properly conducted trial requires the assay to be validated. Validation is ensures that an assay is:
- Specific: measures in a proven way what it is supposed to measure
- Sensitive: has determined upper and lower limits of quantitation
- Precise: has determined maximum intra- and inter assay variations
- Robust: is independent of the operator and the laboratory in which is carried out

For our validation assay we used human PBMC samples from The University of Pennsylvania Hospital. As stimulants we used CEF (MHC class I restricted T- cell epitopes from CM, Epstein Barr and influenza virus), SEB (Staphylococcal enterotoxin B) and PMA+ION (calcium ionophore)

An automated ELISPOT reader (AID) was used to minimize the operator dependent influence on the quantitative output.

1C. Intracellular cytokine (ICS), tetramer staining and immunophenotyping

Used routinely to quantify cytotoxic T cells by virtue of their IFNg secretion and binding capacity to MHC class I tetramers and also with cell surface markers to identify the cell subsets. Recently, we have included cytokines such as IL2 as well as TNFa to the staining panel to be able to quantify polyfunctional T cells (cells that co-secrete IFNγ, IL2 and TNFα).  Recognition and quantification of the different cytotoxic T cell subsets yield a better understanding of the quality and magnitude of ongoing T cell responses in both Gene therapy and vaccine settings.

Phenotyping of antigen specific T cells elicited is extremely important in the evaluation of a T cell response generated in gene transfer and vaccine applications.  Using a combination of cell surface markers, we can now differentiate naïve, central memory and effector/effector memory T cells in both non-human primates and mice. The memory phenotyping panel in NHPs is: CD95/CD28/CD8/CD4/IFNγ, while that used in mice is: CD8/Gag-tetramer/CD127/CD62L.

Fig 2
Figure 2 : T cell response in NHP monitored for secretion of IFNγ, TNFα and IL2 by ICS as well as memory phenotyping using combinations of cell surface markers CD28 and CD95.

Fig 3
Figure 3 : T cell response in mice monitored for MHC class I Gag tetramer staining as well as memory phenotyping using combinations of cell surface markers CD62L and CD127

1D. Cultured ELISPOT and ICCS

The IFNg ELISPOT after “in vitro” expansion in the presence of IL2 and IL7 increases the sensitivity of the assay allowing us to detect even a small central memory-mediated T-cell response.  

Fig 4
Figure 4 : Cultured ELISPOT to detect central memory capsid T cells in human. PBMCs from human were expanded in vitro prior to assay as follows. Freshly isolated cells are cultured for 7 days in medium with peptides pools in the presence of hIL2 and hIL-7 at 2x106 cells/ml. Cells are harvested and analyzed by ELISPOT measuring production of IFN-γ. The data are presented for two samples, one that showed high frequencies in most peptide pools and one in which the frequencies were intermediate. Indicated are the peptide pools for the different serotypes 2, 7, 8 and 9. SEB and PMA are positive controls.

1E. Lymphoproliferation

A key feature of the adaptive immune response is the ability of clones of antigen-specific lymphocytes to rapidly proliferate and differentiate into effector cells. The intracellular fluorescent dye, CFSE, has been found to be particularly effective at monitoring lymphocyte division in many experimental situations. The tracking of lymphocyte division using CFSE has become a routine procedure in many laboratories. In particular, the procedure has been used for the analysis of cell division in vivo in mice and in vitro using mouse and human lymphocytes.

Fig 5
Figure 5 : PBMCs isolated from a Cynomolgus macaque were stained with CFSE and stimulated with PHA for 3 days. PBMCs were also stained for CD4 and CD8.

1F. In-vivo CTL assay (mouse)

This assay allows the detection of cytolytic activity specific for a peptide epitope in-vivo in mice. One group of target cells is labeled with a higher concentration of CFSE dye and pulsed with the antigenic peptide epitope. While the other group of target cells is labeled with a lower concentration of CFSE and pulsed with an irrelevant peptide. They are mixed in a 1:1 ratio and injected i.v. into immunized and naïve mice. A deviation from the 1:1 ratio when the mice are sampled indicates specific lysis of the cells with the relevant epitope on the surface. Normal spleen cells are used as target cells.

Fig 6
Figure 6: In-vivo CTL assay in Balb/c mice immunized against Hiv-1 gag. Balb/c mice were immunized IM with AdH5-Gag. On Day 35, two populations of naïve target cells were labeled with CFSE followed by pulsing with relevant Gag and irrelevant RT epitopes respectively. Target cells were introduced in a 1:1 ratio into the immunized mice and naïve control mice. Eighteen hrs post transfer, splenocytes were harvested and subjected to flow cytometry to measure the relative proportion of CFSE hi-vs CFSE lo cells.

1G Lymphocyte isolation from various NHP and Human tissues

Isolation of lymphocytes from the different compartments is critical to the study and understanding of the immune response to viral vectors and transgene products.

Blood collection and isolation of peripheral blood mononuclear cells (PBMCs). PBMCs are isolated from whole blood collected in EDTA-containing Vacutainer tubes after percoll density-gradient centrifugation. Cells are collected from the interphase and washed with PBS. PBMCs are incubated with of ACK lysing buffer to lyse RBCs. Cells are washed again and resuspended in complete RPMI medium (Mediatech) containing 10% FBS and 2 mM glutamine.

Isolation of lymphocytes from liver. Tissue sections of the liver weighting 20 grams are placed in RPMI 1640 medium, diced into 5 mm peaces and washed with PBS. Tissue fragments are diced is smaller fragments of 1mm3 in RPMI+ Collagenase type 1a, crushed on stainless steel mesh and incubated at 37oC. Digested tissue is passed thru a 40 um nylon mesh, washed with PBS and resuspended in RPMI. Lymphocytes are isolated after after 30-70% percoll density-gradient. Cell are collected from the interphase, washed with PBS and resuspended in complete RPMI medium

.Isolation of lymphocytes from lymphoid nodes and spleen. Tissue sections of the spleen and lymph nodes are crushed on 40 um stainless steel meshes. Filtered cells are washed with PBS, incubated with ACK lysing buffer, washed again and resuspended in complete RPMI medium.

Isolation of lymphocytes from Bone Marrow. Bone marrow samples from crashed Femure and humerus are colleted in PBS containing heparin. Lymphocytes are isolated after percoll density-gradient centrifugation. Cells are collected from the interphase, washed and resuspended in complete RPMI medium.

Isolation of lymphocytes from gut. Biopsy samples are washed with EDTA and incubated with HBSS in continuous agitation. Supernatant is removed and intraepithelial lymphocytes (IELs) are isolated. The settled biopsies are incubated with a collagenase solution on a rocker. Collagenase digestion is used to isolate Lamina Propria Lymphocytes (LPLs). IELs and LPLs are enriched using Percoll density gradient centrifugation.

Fig 7
Figure 7 : Isolation and immunophenotyping of lymphocytes from blood, gut and liver and immunophenotyping using 2 different panels of memory markers: CD28-CD95 and CD62L-CD45RA. CM: central memory, EM: effector memory, TD: terminally differentiated.