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Immunology Core (Penn)

Director: Calcedo de Hoyo, Roberto, PhD

Summary:

Immune responses to products of viral vectors have posed formidable barriers to efficient gene therapies. The important immune effectors of the immune response include CD4+ T helper cells, CD8+ cytotoxic T cells, which are responsible for mediating elimination of transgene expression and B cells which produce neutralizing antibodies that block effective readministration of vector. In addition immune responses directed to neoantigens expressed by the transgene in vector-transduced cells, are also responsible for the rapid elimination of transgene expression. The Immunology Group is responsible for performing various assays to evaluate both cell mediated- as well as humoral immune responses in animal models of gene therapies. In this respect, the Group has undertaken analyses of immune responses in pre-clinical trials in gene therapy in mice, rats, rhesus monkeys, and dogs and in several clinical trials. These assays monitor adenovirus-, adeno-associated virus- and transgene-induced cell mediated and humoral immune responses.

The following schematic illustrates the various immunological processes for which the Immunology Group has developed methodologies:
schematic from core website
Figure Legend: Antigen taken by antigen presenting cells (APC) is processed and presented by MHC class I to CD8 T cells , and MHC class II to CD4 T cells . Recognition of the antigen, along with costimulatory molecules (B7-CD28; CD40-CD40 ligand) results in activation of antigen-specific CD4 T cells, which leads to lymphoproliferation and cytokine secretion . Depending on several conditions (e.g. strength of antigen signaling, costimulation, cytokines secreted by APC, etc.) CD4 T cells differentiate into either TH1 or TH2 type cells. TH1 cells secrete predominantly IFNg (interferon-gamma), which plays a role in activation of cell mediated immune responses which culminates in activation of cytotoxic T lymphocytes . CTL have been shown to be responsible for elimination of transduced cells in vivo by effector mechanisms involving Fas-FasL and perforin-granzymes. TH2 cells on the other hand secrete IL-4, which helps B cells differentiate into antibody secreting plasma cells. Secretion of neutralizing antibodies results in blocking of vector readministration. The nature of the neutralizing antibody response can be measured by determining the antigen (by Western blot) and isotype of the immunoglobulin.

Understanding the molecular mechanisms involved in the cascade of events from antigen uptake by antigen presenting cells to differentiation of T and B cells, will allow development of therapeutic immunosuppressive agents to allow persistent transgene expression and ability to readminister viral vectors.

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Services

  • Cultured ELISPOT and ICCS ( Material analysis service )

    "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.
    Figure 4 from core website
    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."

  • IFNγ ELISPOT (mouse and NHP) service ( Material analysis service )

    "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 from core website
    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."

  • In vivo CTL assay (mouse) service ( Analysis service )

    "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.
    Figure 6 from core website
    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."

  • Infection inhibition based neutralizing antibody assay (Adv wt virus) ( Analysis service )

    "The principle of this assay is the same as the transduction inhibition neutralizing antibody assay but in the infection inhibition assay we use wild type virus and A549 cells. Infection is quantified by immunostaining using a goat anti-hexon antibody raised against a conserve region present in all identified Adeno types.
    bottom figure on core website
    Figure : Infection inhibition neutralizing antibody assay to HAdv5 and SAdV24 using polyclonal rabbit antisera to HAdv5, SAdV23, SAdV24 and Human pooled IgG. Neutralization titers are highlighted."

  • Intracellular cytokine, tetramer staining and immunophenotyping service ( Material analysis service )

    "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.
    Figure 2 from core website
    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.
    Figure 3 from core website
    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"

  • Lymphocyte isolation from various tissues (mouse, NHP and human) ( Material processing service )

    "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.
    Figure 7 from core website
    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."

  • Lymphoproliferation assay (CFSE) service ( Material analysis service )

    "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.
    Figure 5 on core website
    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."

  • Transduction inhibition based neutralizing antibody assay (AAV and Adv vectors) ( Analysis service )

    top image on core website

    "Neutralizing antibodies

    Principle: Virus neutralization assays are performed to assess the potential of antibody to block virus transduction of susceptible cell lines. The ability of antibodies to block adenovirus infection of Hela cells or AAV2 infection of 84-31 (293 cells stably expressing Ad-E4) in vitro is analyzed utilizing Green Fluorescent Protein as a reporter*. For the assay, various dilutions of antibodies pre-incubated with moi of 100 for adenovirus and 1000 for AAV reporter viruses for 1 hour at 37°C, is added to 90% confluent cell cultures. Cells are incubated for 20 hours. Expression of GFP is quantitated by manually counting under a UV-microscope and LacZ by an automated luminometer. The neutralizing titer of antibody is calculated by the highest dilution of the sera where 50% of the cells are green.

    *or LacZ"

  • Validated human IFNγ ELISPOT for Clinical Trials ( Material analysis service )

    "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."


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Last updated: 2015-02-12T15:43:44.438-05:00

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The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016