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Hiroyuki Komatsu (1) Junichiro Sugimoto (1) Ken Goto (1) Kiyoshi Kushima (1) Naohisa Tsutsui (1) Shigeru Hisada (1) Shiho Ito (1) Tadashi Kosaka (1) Takumi Ohishi (1) Yasuharu Otsubo (1) Yoshihiro Takahashi (1)
(1) AOP Working Group, Testing Methodology Committee, The Japanese Society of Immunotoxicology
Corresponding author: Kiyoshi Kushima (firstname.lastname@example.org)
Point of Contact
Kiyoshi Kushima (email point of contact)
- Kiyoshi Kushima
|Author status||OECD status||OECD project||SAAOP status|
|Open for comment. Do not cite||EAGMST Under Review||1.38||Included in OECD Work Plan|
This AOP was last modified on November 09, 2017 02:44
|Interference, nuclear localization of NFAT||November 09, 2017 03:00|
|Reduction, NFAT complex formation||March 15, 2018 04:15|
|Impairment, T-cell dependent antibody response||November 09, 2017 03:17|
|Suppression, IL-2 and IL-4 production||November 09, 2017 03:11|
|Inhibition, Calcineurin Activity||November 09, 2017 02:51|
|Interference, nuclear localization of NFAT leads to Reduction, NFAT complex formation||November 09, 2017 03:38|
|Reduction, NFAT complex formation leads to Suppression, IL-2 and IL-4 production||November 09, 2017 03:52|
|Suppression, IL-2 and IL-4 production leads to Impairment, T-cell dependent antibody response||November 09, 2017 04:03|
|Inhibition, Calcineurin Activity leads to Interference, nuclear localization of NFAT||November 13, 2017 18:27|
|Tacrolimus||November 29, 2016 18:42|
|Cyclosporin||May 18, 2017 08:31|
Calcineurin (CN) is a type of protein phosphatase that is known to impair immune function when its phosphatase activation is inhibited. The relationship between CN and immune functions is well understood, and immunosuppressants that work by inhibiting CN have been developed.
CN inhibitors (CNIs) suppress many kinds of immune functions leading to increased susceptibility to infections. T-cell dependent antibody response (TDAR) is considered to be the most important endpoint on evaluating immunotoxicity of chemicals; therefore, this AOP describes the linkage between the inhibition of CN activity and impairment of TDAR.
CN activity is inhibited when stressors of CNIs bind to immunophilins, which interferes with the nuclear localization of nuclear factor of activated T cells (NFAT), a substrate of CN. As a result, the formation of functional NFAT complexes that bind at the site of IL-2, IL-4 and other T cell -derived cytokine promoters is reduced, thereby suppressing production of these cytokines. Thus, TDAR is impaired mainly by the suppression of production of IL-2 and IL-4, which affects the proliferation and differentiation of B-cells.
We have identified a number of key events along this pathway and determined the key event relationships, based on which we have created an AOP for inhibition of CN activity leading to impaired TDAR.
Since CN expresses in cells among vast variety of species, this AOP is applicable to many mammal species, including humans and rodents.
Although there are numerous stressors that inhibit CN activity, this AOP is mainly based on an understanding of immunosuppression caused by FK506 and FKBP12 complexes, on which a significant body of scientific literature has been published.
We look forward to future amendments to this AOP with up-to-date information on other stressors, which will more clarify the linkage between inhibition of CN activity and impairment of TDAR.
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||980||Inhibition, Calcineurin Activity||Inhibition, Calcineurin Activity|
|2||KE||979||Interference, nuclear localization of NFAT||Interference, nuclear localization of NFAT|
|3||KE||981||Reduction, NFAT complex formation||Reduction, NFAT complex formation|
|4||KE||1202||Suppression, IL-2 and IL-4 production||Suppression, IL-2 and IL-4 production|
|5||AO||984||Impairment, T-cell dependent antibody response||Impairment, T-cell dependent antibody response|
Relationships Between Two Key Events
(Including MIEs and AOs)
Life Stage Applicability
|All life stages||Moderate|
|Homo sapiens||Homo sapiens||High||NCBI|
|Mus musculus||Mus musculus||High||NCBI|
|Macaca fascicularis||Macaca fascicularis||High||NCBI|
|Rattus norvegicus||Rattus norvegicus||NCBI|
Overall Assessment of the AOP
CN activity is inhibited when stressors bond with immunophilins, which interferes with the nuclear localization of NFAT, a substrate of CN. As a result, the formation of functional NFAT complexes that bind at the site of IL-2 and other cytokine promoters is reduced, thereby suppressing production of these cytokines. Thus TDAR is impaired mainly by the suppression of production of IL-2 and IL-4, which affect the proliferation and differentiation of B-cells. We have identified a number of key events (KEs) along this pathway, and based on these key event relationships (KERs), created an AOP for inhibition of CN activity leading to impaired TDAR.
Since each of the KEs involving MIE and AO is measurable quantitatively and shows clear dose responses with the CNIs, this AOP is useful for understanding of immunosuppression derived from CN activity inhibition. In addition, each of the KERs is based on sufficient scientific evidence and there is no contradiction found between dose responses of the adjacent KEs.
Since CN expresses in cells among vast variety of species, this AOP is applicable to many mammal species, including humans and rodents.
Domain of Applicability
The proposed AOP of inhibited CN activity leading to impaired TDAR is not associated with life stage-, sex-, or age-dependency. The relevant life stages for the AOP are from child to adult, and since tacrolimus (FK506) ointment (Protopic) is approved for pediatric atopic dermatitis, the MOA for immunosuppression appears to be applicable to all of life stages. Since FK506 or CsA -induced outcomes in humans are mimicked by similar responses in a variety of animal models, immunosuppression induced by immunophilin-CNI complexe is considered to be preserved across a variety of mammalian species.
Essentiality of the Key Events
MIE and later events: CnA-knockout (KO) mice
The CN molecule consists of two regions, CnA and CnB, and CnA holds phosphatase activity. In CnA KO mice, T cell proliferation in response to ovalbumin stimulation is lower than for wild-type mice and is not complemented by normal antibody producing cells1. In addition, when stimulated with ovalbumin, CnA KO mice produce less IFN-γ, IL‑2, and IL‑4 than those in wild-type mice.1 However, primary antibody response in CnA-/- mice is normal in response to TNP-ovalbumin.
Stressor: FKBP12-KO mice
There is no evidence of a relationship between FKBP12 KO and the immune system in the FKBP12 KO mouse model. Steric structure of FKBP12/FK506 complex is the key factor for inhibition of CN phosphatase activity, but not the enzymatic activities of FKBP12.
KE1 and later events: NFAT-KO mice
The following phenotypes are observed in NFAT KO mice2 : moderate hyperproliferation with splenomegaly; moderately enhanced B- and T-cell responses, with bias towards Th2-cell responses; decreased IFN-γ production in response to TCR ligation; reduced proliferative responses by T cells; impaired repopulation of the thymus and lymphoid organs; impaired Th2- cell responses and IL-4 production; grossly impaired T-cell effector functions, profound defects in cytokine production and cytolytic activity; B-cell hyperactivity; impaired development of CD4 and CD8 single-positive cells, increased apoptosis of double -positive thymocytes; mild hyperactivation of peripheral T cells.
Therefore, the study of NFAT-KO mice shows that NFAT is involved in a wide range of immune-cell phenomenon, and some of these phenomenon are known to be regulated by CN. This indicates that the production of T-cell derived cytokine is regulated by the CN-NFAT system.
T cell functions are regulated by CN-NFAT system and the suppression of CN activity in T cell is well known to induce multiple types of immunosuppression including TDAR.
Experiments with T cells indicates that T cell receptor (TCR) stimulation brings about intracellular increases in concentrations of Ca2+, which triggers CN activity, thereby inducing nuclear localization of its substrate NFAT per dephosphorylation, which forms complexes with activator protein 1 (AP-1) at the promoter sites of the T‑cell cytokine genes and induces production of the cytokines.
CN phosphatase activity is well known to be inhibited due to the formation of immunophilin-CNI complexes, such as CsA/cyclophilin complexes or FK506/FK506- binding protein (FKBP) 12 complexes. Immunophilins are a general class of proteins that exhibit peptidyl-propyl isomerase (PPIase) activity, but there is no commonality between their two amino acid sequences. The three-dimensional structures of immunophilin complexes, but not these enzyme activities, are essential to the inhibition of CN phosphatase activity.
It is also well known that one of the effects on immune function when FK506 forms complexes with immunophilins and inhibits CN activity is the suppression of IL-2 and other T-cell derived cytokine production. It is further well known that inhibition of CN leads to suppression of TDAR because IL‑2 and IL‑4 mainly promote the proliferation, class switching, differentiation, and maturation of B-cells.
Furthermore, CN-NFAT also exists in B‑cells and it has been reported that CNIs do suppress production of certain cytokines from them; though, at the time of our review of the literature, we did not find any reports of a direct effect of CN inhibition on B‑cells such as changes in proliferations, class switching, differentiation, or maturation of B‑cells.
Also, although CN-NFAT is known to exist in dendritic cells, natural killer T (NKT) cells, and other types of cells, where it regulates the expression of IL-2 receptors, there are no reports of effects on the production of antibodies due to altered expression of IL-2 receptors in these cells.
|KER||KEup-KEdown||Plausibility||Rationales supported by literatures|
|KER1||CN inhibition to interference, NFAT nuclear translocation||Strong||
T cell functions are regulated by CN-NFAT system.
Activated CN through TCR stimulation dephosphorylates NFAT to promote its nuclear localization.
CN in T cells is inhibited by CNI/immunophilin complexes, which does not induce dephosphorylation of NFAT and following its nuclear localization.
|KER2||Interference, nuclear localization to reduction, NFAT complex formation||Strong||
Activated CN dephosphorylates NFAT to promote its nuclear translocation. Nuclear-located NFAT binds with AP-1 at the promoter regions of the cytokine genes to promote T-cell cytokine production.
Inhibition of dephosphorylation of NFAT by CNIs prevents nuclear localization of NFAT and resultant binding with AP-1 at the promoter region of the T cell cytokine genes.
|KER3||Reduction, NFAT complex formation to suppression of IL-2 and IL-4 production||Strong||
NFAT/AP-1 complexes bind to the promoter regions of the cytokine genes leading to produce these cytokines from T cells. Among these cytokines, IL-2 and IL-4 have a major role in promoting proliferation, maturation and class-switching of B cells, and induction of TDAR.
Reduced NFAT complex formation in the nucleus induced by CNIs suppresses production of T-cell derived cytokines including IL-2 and IL-4.
|KER4||Suppression of IL-2 and IL-4 production to impaired TDAR||Strong||
Inhibition of CN by CNIs is known to suppress production of multiple cytokine species from T cells and other populations of immune cells and in some cases IL-2 receptor expression.
Among these cytokines and receptors, suppression of IL-2 and IL-4 production by CNIs are known to affect proliferation, maturation and class switching of B cells leading to impairment of TDAR..
Other cytokines show only minor effects, if any, on TDAR in cases where their production is suppressed through inhibition of CN activity.
MIE：Inhibition, calcineurin activity
Phosphatase activity of CN can be measured quantitatively using a phosphatase assay.
In the phosphatase assay for CN, addition of 1 molar equivalent of FKBP-FK506 complex inhibited over 80% of the CN protein phosphatase activity (mixed with 300 nM calmodulin-CN and 30 µM FK506) (Liu et al. 1992). Dose-response analysis of the effects of FK506 on CN-mediated phosphatase activity in KiSVMC4W cells showed that increased expression of FKBP12 resulted in a greater than ten-fold increase in the sensitivity of the KiSV-MC4W cells containing human FKBP12 cDNA to FK506-mediated inhibition of CN phosphatase activity, as indicted by an IC50 value of ~3 nM (Fruman et al.1995). The phosphatase assay showed that FK506 inhibition of CN activity was concentration-dependent and that IC50 values for CN inhibition were approximately 0.5 nM for FK 506 (Fruman et al.1992).
KE1:Interference, nuclear localization of NFAT
Interference with the translocation of NFAT to the nucleus can be detected quantitatively using a gel mobility shift assay.
Dose-dependent interference with nuclear translocation of NFAT1 was observed with increasing CNI concentrations up to 1 μM (1000 nM). Higher concentrations induced cellular toxicity and resulted in cell death. Dose-dependent interference of nuclear NFAT1 translocation per CN inhibition was also observed in CD4+ T cells from healthy donors, again at maximal concentrations of 1 μM (Maguire et al. 2013).
KE2:Reduction, NFAT complex formation
Activated NFAT that has localized to the nucleus binds cooperatively at the site of the IL-2 promoter with activator protein AP-1, which is a heterodimer comprising a Fos and a Jun protein (Schreiber and Crabtree 1992, Jain et al. 1992 , thereby inducing transcription of IL-2 (Jain et al. 1993). FK506 hinders the formation of the functional NFAT complexes necessary to binding at the site of IL-2 promoters by interfering with nuclear localization of NFAT (Flanagan et al. 1991). Concentration-dependent reduction of in vitro nuclear localization of NFAT by FK506 was evident at the maximum concentration of 1µM (Maguire et al. 2013).
KE3:Suppression, IL-2 and IL-4 production
Quantification of cytokine content can be measured using a Sandwich ELISA kit, and cytokine mRNA levels can be determined using a RiboQuant MutiProbe RPA system (PharMingen, San Diego, CA).
FK506 suppressed production of IL-2, IL-4, and IFN-γ in human T cells stimulated with anti- CD3 mAb in the presence of PMA at 1.2 and 12.5 nM, and inhibited expression of IL-2, IL-4, and IFN-γ mRNA expression in anti-CD3/PMA-activated cells at 10 nM (Dumont et al. 1998). FK506 or CsA suppressed production of IL-2 in mouse and human MLR from 0.1 to 10 nM of FK506 or 10 to 100 nM of CsA (Kino et al. 1987a).
KE4:Impairment, T-cell dependent antibody response
Total IgM and IgG levels as well as antigen-specific antibodies can be determined in vitro and in vivo. The effects on immunoglobulin class switching can also be evaluated in vitro. These all parameters can be determined quantitatively.
In vitro: T cells and B cells isolated from human PBMC were co-cultured with a CNI for nine days in the presence of polyclonal T cell stimulation, after which supernatants were tested for immunoglobulin IgM and IgG levels using a Sandwich ELISA kit. FK506 or CsA reduced the levels of IgM and IgG at the concentrations of 0.3 and 1.0 ng/mL or 50 and 100 ng/mL (Heidt et al, 2009).
SKW6.4 cells (IL-6-dependent IgM-secreting human B-cell line) were cultured with anti-CD3/CD28 antibody-stimulated PBMC culture supernatant. After culturing for four days, IgM produced in the culture supernatants was measured using an ELISA kit. FK506 or CsA reduced the levels of IgM at the concentrations of 0.01 to 100 ng/mL or 0.1 to 1000 ng/mL (Sakuma et al. 2001b).
In vivo: FK506 reduced serum concentration of anti-KLH antibodies IgM and IgG in rats treated with FK506 at 3 mg/kg for over four weeks and immunized with KLH (Ulrich et al. 2004). FK506 or CsA reduced antigen-specific plaque -forming splenocytes in mice treated with FK506 or CsA for 4 days and immunized with SRBC from 3.2 to 100 mg/kg of FK506 or 32 to 100 mg/kg of CsA (Kino et al. 1987b).
Class switching: T cells derived from human PBMCs were cultured with CNI, and cytokine mRNA levels of IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13, and other B cell stimulatory cytokines produced in T cells were measured by quantitative PCR. FK506 suppressed production of IL-2, IL-4, and IFN-γ in human T cells stimulated with anti-CD3 mAb in the presence of PMA at 1.2 and 12.5 nM, and inhibited expression of IL-2, IL-4, and IFN-γ mRNA expression in anti- CD3/PMA-activated cells at 10 nM (Dumont et al. 1998).
|KER||Empirical support of KERs|
MIE=>KE1：Inhibition, calcineurin activity leads to interference, nuclear localization of NFAT
Empirical support of the MIE => KE1 is strong.
Many experimental data support the inhibition of CN activity induced by CNI - immunophilin complexes and following suppression of nuclear localization.
CN phosphatase activity is inhibited by CNI of FK506 with IC50 values of 0.5 – 30nM and 80% suppression at 30 µM.
Concentration-dependent reduction of in vitro nuclear localization of NFAT was evident at the maximum concentration of 1µM.Dose responses and temporality deem to be similar between these two key events.
|KE1=>KE2：Interference, nuclear localization of NFAT leads to reduction, NFAT complex formation||
Empirical support of the KE1 => KE2 is strong.
The relationship between interfered nuclear localization of NFAT and resultant reduced NFAT complex formation is well known by the sufficient experiments.
There have been no data on the direct measurement of NFAT/AP-1 complex bound at the promoter sites of cytokine genes in the presence of CNIs; however, the amounts of NFAT/AP-1 complexes and the transcribed mRNAs are expected to be the alternative parameters.
Concentration-dependent reduction of in vitro nuclear localization of NFAT was evident at the maximum concentration of 1µM.
NFAT/AP-1 complexes at the site of cytokine promoters is dependent on CNI dosage.
Dose responses and temporality deem to be similar between these two key events.
|KE2=>KE3：Reduction, NFAT complex formation leads to suppression, IL-2 and IL-4 production||
Empirical support of the KE2 => KE3 is strong.
It is well known that inhibition of NFAT complex formation composed of NFAT, AP-1 at the promoter sites reduces the production of T cell cytokines including IL-2 and IL-4, which are mainly involved in T cell -dependent antibody response.
NFAT/AP-1 complexes at the site of cytokine promoters is dependent on CNI dosage.
In CD3/PMA-activated human T cells, FK506 suppressed production of IL-2, IL-4, and IFN-γ at the concentrations of 1.2 to 12.5 nM as well as inhibited expression of IL-2, IL-4, and IFN-γ mRNA in a dose-dependent (10 nM) manner. (Dumont et al. 1998).Dose responses and temporality deem to be similar between these two key events.
|KE3=>AO: Suppression, IL-2 and IL-4 production leads to Impairment, T-cell dependent antibody response||
Empirical support of the KE3 => AO is strong.
CN-NFAT system functions in many cell types throughout the body including T cells, B cells and other immune cells, and inhibition of CN-NFAT by CNIs affects production of cytokines from these immune cells. Among these cytokines, it is well known that reduced production of IL-2 and IL-4 from T cells plays a major role in CNI-induced suppression of TDAR, and our present literature research showed that decreased production of cytokines other than the two showed only minor effect on TDAR.
After 9-day culture of B cells and non-pre-activated T cell stimulation with FK506 or CsA, the levels of IgM and IgG in the culture supernatant were reduced at 0.3 and 1.0 ng/mL of FK506 or 50 and 100 ng/mL of CsA.
After 4-day culture of SKW6.4 cells (IL-6-dependent IgM-secreting human B-cell line) and anti-CD3/CD28 stimulated PBMC culture supernatant with FK506 or CsA, the level of IgM in the culture supernatant was reduced at the concentrations of 0.01 to 100 ng/mL of FK506 or 0.1 to 1000 ng/mL of CsA.
Rats were treated with FK506 for over four weeks and immunized with KLH, after which serum concentration of anti-KLH IgM and IgG reduced at the dose levels of 3 mg/kg/day.
Mice were treated with FK506 or CsA for 4 days, and immunized with SRBC, after which antigen-specific plaque -forming splenocytes reduced at the dose levels of 3.2, 10, 32 and 100 mg/kg of FK506 or 32 and 100 mg/kg of CsA.
In vitro class switching; in CD3/PMA-activated human T cells, FK506 suppressed production of IL-2, IL-4, and IFN at the concentrations of 1.2 to 12.5 nM as well as inhibited expression of IL-2, IL-4, and IFN-γ mRNA at the concentrations of 10 nM. FK506 or CsA suppressed production of IL-2 in mouse mixed lymphocyte reaction (MLR) at 0.1 to 10 nM of FK506 and 10 to 100 nM of CsA as well as in human MLR at 0.1 to 10 nM of FK506 and 10 to 100 nM of CsA.
In vitro suppressions of T cell -derived cytokines and T cell -dependent antibody production or antibody production after polyclonal T-cell stimulation showed similar dose responses to CNIs, however, time gaps were found between the two events, which showed earlier onset of cytokine production and delayed onset of antibody production.
In the present AOP, information on the effects of CN inhibition depends mainly on the findings for one of the CNIs, FK506; therefore, the relationships between two KEs in each of the KERs could be evaluated based on the dose responses to FK506.
As described in the Empirical Support, each of the MIE, KEs and AO except for KE2, are able to be measured directly and quantitatively; accordingly, clear dose responses to CNIs could be found in each of the KEs.
As for KE2, there have been little data on the amounts of NFAT complex formation at the cytokine promoter sites influenced by CNIs; however, NFAT/AP-1 complex formation or mRNA levels of related cytokines are measurable and served as the alternative parameters to NFAT complex formation at the promoter sites. The changes in the amounts of NFAT/AP-1 complex or mRNA levels of cytokines also showed clear dose-responses to CNIs.
In vitro dose responses are found to be similar between all KEs from MIE to AO, and temporality is also similar from MIE to KE3; however, in KER4, the temporality showed that delayed onset of AO (in vitro antibody responses) compared with KE3 of cytokine production; therefore, no contradictions in dose response and temporality are found in each of the KERs.
Each of the KEs and KERs of the present AOP is supported by sufficient scientific evidence and each KE shows clear dose response relations without any contradiction in dose response and temporality; therefore, measurement of each of the KEs could serve as an appropriate predictor of the adverse outcome of CN inhibition-induced impairment of TDAR.
Considerations for Potential Applications of the AOP (optional)
CN is expressed in T cells as well as other types of immune cells like B cells and dendritic cells. CNIs suppress many kinds of immune functions leading to increased susceptibility to infections and decreased hyper immune reactions such as rejection and graft versus host disease (GVHD). Among these, TDAR is considered to be the most important endpoint of immunotoxicity, because T cells, B cells and antigen-presenting cells such as dendritic cells are involved in inducing and developing of TDAR and changes in at least one of these immune cell populations influencing TDAR.
Moreover, on evaluation of immunotoxicity of pharmaceuticals, the ICH S8 immunotoxicity testing guideline recommends to evaluate TDAR in cases where target cells of immunotoxicity are not clear based on its pharmacology and findings in standard toxicity studies.
The present AOP could be applied to predict whether a compound might affect TDAR in cases where the compound shows a possibility to act on T cells, meanwhile, it is inappropriate that the present AOP would be used as an alternative method to TDAR measurement in the ICH S8 immunotoxicity testing guideline.
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