Table 3 Summary of current immunological methodologies to assess the immunogenicity of cellular products in vitro and in vivo.
From: Immunological considerations and challenges for regenerative cellular therapies
Immune compartment | Immunological assay | ||||
|---|---|---|---|---|---|
In vitro | In vivo | ||||
Innate immunity | • NK cell degranulation/activation (CD107 flow cytometric analysis) and cytotoxicity (51Cr/111In release, impedance measurements)23,26,27,28,55,56 | • Immunohistochemical/flow cytometric quantification of graft immune phenotype and infiltration with NK cells (CD56+), neutrophils (CD11b+, CD66b+, CD33+), macrophages (RAM11+)23 | |||
• Evaluation of inhibitory or activation ligands by flow cytometric/immunofluorescence analysis in grafted cells (e.g., HLA-ABC, HLA-C, HLA-E, NKG2D, MIC-A/B, CD155, CD112, PCNA, CD47, CD55, CD59)20,23,56 | • Deposition of complement molecules in the graft (e.g., CL-11, C3d, C4b-9), collectin-11, DAMP secretome32 | ||||
• Complement molecule concentration in serum (e.g., C3a, C5a, CFB, MAC, CFH, CFI)60 | |||||
Adaptive Immunity | Humoral | B Cell Activation | • No available assays at present | • Flow cytometric characterization of transitional, naïve, memory and plasma cell phenotypes (e.g., CD10, CD19, CD20, CD23, CD27, CD38, IgD, IgM, IgG)40,167 | |
• Ex vivo B cell ELIOSPOT assays167 | |||||
• Splenic germinal center formation167 | |||||
Antibody Production | • No available assays at present | • Total serum human IgG and IgM levels | |||
• Incubation of serum from rejecting animals with donor cells followed by fluorescently-labeled anti-human IgG or/and IgM staining for flow cytometric analysis23,40 | |||||
• IgG and B immunohistochemistry analysis32 | |||||
• Production of HLA-specific anti-donor antibodies with Luminex assay | |||||
Cellular | CD8+ T Cells | • Co-culture with cellular product and characterization of T cell proliferation (CFSE-labeled T cells; production of IFN-γ) and the main CD8+ T cell subsets by flow cytometry23,25,40,41,42,45,46,55,62,64,65 | • Immunohistochemical/flow cytometric quantification of graft immune phenotype and infiltration with lymphocytes (CD3+, CD8+)4,5,23,39,40,41,46,64,68 | ||
• Cytotoxic activity by 51Cr/111In release assay55 | |||||
• Ex vivo activation in Granzyme B ELISPOT assay | |||||
CD4+ T Cells | Common | • Co-culture with the cellular product and characterization of T cell proliferation (CFSE-labeled T cells; production of IFN-γ)74 and the main CD4+ T cell subsets by flow cytometry23,25,40,41,42,45,46,55,62,63,64,65,66,67,68 | • Immunohistochemical/flow cytometric quantification of graft immune phenotype and infiltration with lymphocytes (CD3+, CD4+)4,5,23,40,41,46,66,83 | ||
Indirect Pathway | • Ex vivo proliferation (CFSE-labeled T cells; production of IFN-γ in ELIOSPOT assays) in response to DCs from the donor used for reconstitution of the immune response/recipient pulsed with preparations of the grafted cells46 | ||||
Direct Pathway | • Ex vivo proliferation (CFSE-labeled T cells; production of IFN-γ in ELIOSPOT assays) in response to DCs from the donor used for generation of grafted cells (or third-party control DCs) cells with/without specific cytokines and ligand/antibody-specific antibodies46 | ||||
Common | • Evaluation of cytokine production/proliferation from in vitro co-cultures, sera/tissue from transplanted cells (e.g., Multiplex Luminex of IFN-γ, IL-1b, IL-6, IL-17 or by tissue immunofluorescence)23,25,39,40,41,42,45,46,55 | ||||
Cellular products | • Evaluation of the of inhibitory or activation ligands by flow cytometric/immunofluorescence analysis of cellular product (e.g., CD80, CD86, PD-L1, PD-L2)23,25,41,42,48,64,65,67,74 and evaluation of anti and pro-inflammatory cytokine production63,67,73,74 | ||||
