Human CD32 (FcγRII) NFAT Reporter Jurkat Cells

Cell line descriptions

Jurkat-Lucia™ NFAT-CD32 cells were generated from the human T lymphocyte Jurkat cell line Jurkat-Lucia™ NFAT featuring an NFAT-inducible Lucia® luciferase reporter gene. They stably express the high-affinity allotype H131 of the cluster of differentiation 32 (CD32), an FcγR binding the constant region of immunoglobulin G (IgG) [1]. CD32-mediated NFAT activation is readily assessable in the supernatant using QUANTI-Luc™ 4 Lucia/Gaussia, a detection reagent. Of note, Jurkat cells naturally express a functional NFAT (nuclear factor of activated T cells) transcription factor, which is involved in the early signaling events of ADCC and ADCP [2-3].

ADCC & ADCP

ADCC and ADCP  - short for antibody-dependent cellular cytotoxicity & phagocytosis - are immune mechanisms through which Fc receptor-bearing effector cells can recognize and clear antibody (Ab)-coated microbes and target cells expressing specific antigens on their surface. Human IgGs bind to activatory (FcγRI (CD64), FcγRIIA (CD32A), FcγRIIa (CD16A), and inhibitory (FcγRIIb) receptors. The IgG-FcγR interaction is regulated by the Ab isotype and glycosylation [4, 5]. FcγRs differ in their cellular distribution and are often co-expressed. The high-affinity FcγRI (CD64) is expressed on myeloid cells, including monocytes and macrophages. The low-affinity FcγRIIA (CD32A) is expressed on myeloid cells, including monocytes, macrophages, and dendritic cells (DCs), whereas FcγRIIIa (CD16A) is expressed on macrophages and natural killer (NK) cells [1, 6].

ADCC and ADCP are initiated when multiple IgG molecules bind simultaneously to FcγRs. The binding of antibody-antigen complexes to activatory and inhibitory FcγRs induces their cross-linking and subsequent signaling through immunoreceptor tyrosine-based activation motifs (ITAMs) and inhibition motifs (ITIMs), respectively. Cytoplasmic signaling includes an increase in intracellular calcium concentration and calcineurin/calmodulin-mediated dephosphorylation of NFAT (nuclear factor of activated T cells), allowing its nuclear translocation and binding to promoter regions of ADCC and ADCP relevant genes [4, 5].

Balance in FcγR signaling

The balance in FcγR signaling controls the immune outcome.

• No response: inhibiting signals counterbalance activating signals.
• ADCC: an excess of engaged CD16A (FcγRIIIA) at the surface of Natural Killer (NK) cells induces the release of cytotoxic granules, which kill the target [6].
• ADCP: an excess of engaged CD32A (FcγRIIA) or CD64 (FcγRI) at the surface of monocytes, macrophages, and dendritic cells induces the phagocytosis of the microbe or target cells. This internalization is followed by phagolysosomal degradation, thus facilitating antigen presentation and stimulating inflammatory cytokine secretion [5, 6].

CD16A and CD32A allelic polymorphism

Single-nucleotide polymorphisms (SNPs) in human Fc receptors affect interactions with antibody Fc. Allelic variants of the same FcR can display lower or higher affinities for antibody-antigen immune complexes.

• CD16A features allelic polymorphisms among the human population, notably at position 158 in the mature protein (or position 161 in the full protein) [1]. The V158 allotype is reported to have a higher affinity for monoclonal immunoglobulin G (IgG) than the F158 allotype [1, 7].
• CD32A features allelic polymorphisms among the human population, notably at position 131 in the mature protein (or position 166 in the full protein) [1]. The H131 allotype is reported to have a higher affinity for monoclonal IgG than the R131 allotype [1].

References:

1. Nagelkerke S.Q. et al., 2019. Genetic variation in low-to-medium-affinity Fcγ receptors: functional consequences, disease associations, and opportunities for personalized medicine. Front. Immunol. 10:2237.
2. Shaw J-P. et al., 1998. Identification of a putative regulator of early T cell activation genes. Science. 241:202.
3. Leibson P.J., 1997. Signal transduction during natural killer cell activation: inside the mind of a killer. Immunity. 6:655.
4. Quast I. et al., 2016. Regulation of antibody effector functions through IgG Fc N-glycosylation. Cell. Mol. Life. Sci. 74(5):837-47.
5. Tay M.Z. et al., 2019. Antibody-Dependent Cellular Phagocytosis in Antiviral Immune Responses. Front Immunol. 10:332.
6. Holtrop T, et al. 2022. Targeting the high-affinity receptor, FcγRI, in autoimmune disease, neuropathy, and cancer. Immunother Adv.2(1):ltac011.
7. Bruhns P. et al., 2009. Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses. Blood. 113(16):3716.