Immunosuppression for in vivo research: state-of-the-art protocols and experimental approaches

Abstract

Almost every experimental treatment strategy using non-autologous cell, tissue or organ transplantation is tested in small and large animal models before clinical translation. Because these strategies require immunosuppression in most cases, immunosuppressive protocols are a key element in transplantation experiments. However, standard immunosuppressive protocols are often applied without detailed knowledge regarding their efficacy within the particular experimental setting and in the chosen model species. Optimization of such protocols is pertinent to the translation of experimental results to human patients and thus warrants further investigation. This review summarizes current knowledge regarding immunosuppressive drug classes as well as their dosages and application regimens with consideration of species-specific drug metabolization and side effects. It also summarizes contemporary knowledge of novel immunomodulatory strategies, such as the use of mesenchymal stem cells or antibodies. Thus, this review is intended to serve as a state-of-the-art compendium for researchers to refine applied experimental immunosuppression and immunomodulation strategies to enhance the predictive value of preclinical transplantation studies.

Figure 1

Cellular pathways of commonly used clinical immunosuppressive agents. GCs reach the nucleus via diffusion through the cell membrane and form a complex after binding to a steroid receptor protein following separation from Hsp 90. The complex binds to specific DNA sequences and affects the transcription of a variety of genes. MTX inhibits DHR, which is necessary for nucleotide synthesis, thereby constraining cell division. MMF blocks the IMDH, which is also required for nucleotide synthesis. CY is metabolized by CYP450 to 4HCY, which interconverts to AP. Both tautomers are able to passively diffuse into cells. Then, AP is converted to AC and PHM, which possesses DNA-crosslinking properties. CsA binds to an intracellular immunophilin and blocks calcineurin to enable NFATs, whereas tacrolimus (Tcr) binds to the intracellular FK506 binding protein (FKBP) and also inhibits NFAT activation, ultimately preventing cell proliferation. AC, acrolein; AP, aldophosphamide; CsA, cyclosporin A; CY, cyclophosphamide; CYP450, cytochrome P450; DAG, diacylglycerol; DHR, dihydrofolate reductase; ERK, extracellular signal-regulated kinase; Fyn, tyrosine-protein kinase; GEF, guanine-nucleotide exchanging factor; GH, glucocorticoid; 4HCY, 4-hydroxyphosphamide; Hsp 90, heat-shock protein 90; IP₃, inositol triphosphate; IMDH, inosine monophosphate dehydrogenase; JNK, c-Jun N-terminal kinase; JNKK, c-Jun N-terminal kinase kinase; RAC, guanosine triphosphate; RAS, guanosine-nucleotide-binding protein; Lck, lymphocyte-specific protein tyrosine kinase; MEK, mitogen-activated protein kinase kinase; MEKK, serine/threonine-specific protein kinase; MMF, mycophenolate mofetil; MTX, methotrexate; NF-κB, nuclear factor 'κ-light-chain enhancer' of activated B cells; NFAT, nuclear factor of activated T cell; PHM, phosphoramide mustard; Pip₂, phosphatidyl inositol bisphosphate; PKC, protein kinase C; PLCγ, phospholipase C-γ RAF, serine/threonine-specific protein kinase; TCF, transcription factor; TCR, T-cell receptor; Zap-70, zeta-chain-associated protein kinase 70.

Figure 2

Mechanisms of action of the experimental approaches. Experimental approaches for immunosuppression comprise MSCs, T_regs, anti-CD4 antibodies and substances blocking costimulatory pathways. MSCs act on CD4⁺ T cells via IFN-γ and TGF-β. First, the IFN-γ concentration is reduced by MSCs, inhibiting proliferation and inducing the apoptosis of T cells. Second, the TGF-β concentration is increased by MSCs, driving the differentiation of naive CD4⁺ T cells into T_regs. In turn, T_regs directly inhibit CD4⁺ T-cell proliferation via the suppression of Ca²⁺-dependent pathways and indirectly act by downregulating costimulatory molecules such as CTLA4. Finally, anti-CD4 antibodies and substances blocking costimulatory pathways (belatacept) impair T-cell activation. CTLA4, cytotoxic T-lymphocyte-associated protein 4; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; Fyn, tyrosine-protein kinase; Ido, indolamine-2,3-dioxygenase; GEF, guanine-nucleotide exchanging factor; IFN-γ, interferon-γ IP₃, inositol triphosphate; Jak, Janus kinase; JNK, c-Jun N-terminal kinase; JNKK, c-Jun N-terminal kinase kinase; Lck, lymphocyte-specific protein tyrosine kinase; MEK, mitogen-activated protein kinase kinase; MEKK, serine/threonine-specific protein kinase; MSC, mesenchymal stem cell; NF-κB, nuclear factor 'κ light-chain enhancer' of activated B cells; Pip₂, phosphatidyl inositol bisphosphate; PI3K/Pi3 kinase, phosphoinositide 3-kinase; PLCγ, phospholipase; PKC, protein kinase C; RAC, guanosine triphosphate; RAF, serine/threonine-specific protein kinase; RAS, guanosine-nucleotide-binding protein; STAT, signal transducer and activator of transcription; TCF, transcription factor; TCR, T-cell receptor; TGF-β, tumor growth factor-β T_regs, regulatory T cells; Zap-70, zeta-chain-associated protein kinase 70.