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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 Do not cite||EAGMST Under Review||1.38||Included in OECD Work Plan|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 January 29, 2019 18:11
|Interference, nuclear localization of NFAT||January 30, 2019 19:22|
|Reduction, NFAT/AP-1 complex formation||January 30, 2019 19:22|
|Impairment, T-cell dependent antibody response||January 30, 2019 19:21|
|Suppression, IL-2 and IL-4 production||January 30, 2019 19:20|
|Inhibition, Calcineurin Activity||January 30, 2019 07:16|
|Inhibition, Calcineurin Activity leads to Interference, nuclear localization of NFAT||May 31, 2018 01:27|
|Interference, nuclear localization of NFAT leads to Reduction, NFAT/AP-1 complex formation||January 30, 2019 07:33|
|Reduction, NFAT/AP-1 complex formation leads to Suppression, IL-2 and IL-4 production||January 30, 2019 07:35|
|Suppression, IL-2 and IL-4 production leads to Impairment, T-cell dependent antibody response||January 30, 2019 07:37|
|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) inhibit CN phosphatase activity to suppress many kinds of immune functions and have been used to prevent hyper immune reactions such as rejection and graft versus host disease (GVHD), and treat allergic disorders such as psoriasis and atopic dermatitis. On the other hand, CN inhibitors are reported to induce immunosuppression-derived adverse effects such as increased frequency and/or severity of infections and increased tumor incidences. CN inhibitors might affect kinds of T-cell derived immune functions to induce compromised host. Among the affected immune functions. T-cell dependent antibody response (TDAR) is the most important factor to resist infections and thought to be the most useful 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 CN with their respective 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 with activator protein-1 (AP-1) 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. Among the affected cytokines from each of the helper T cell subsets, reduced production of IL-2 and IL-4 affects the proliferation and differentiation of B-cells to suppress TDAR.
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 might be 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/AP-1 complex formation||Reduction, NFAT/AP-1 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
Inhibition of CN might induce suppression of cytokines production from all the T helper cell subsets as well as other immune functions of other immune cells. Suppression of cell-mediated immunity is involved in the pharmacology of preventing hyper immune reactions such as rejection and GVHD, and treatment of allergic disorders such as psoriasis and atopic dermatitis. On the other hand, CN inhibition might induce immunosuppression-derived adverse outcomes. One of the effects is increased frequency and/or severity of infections. Compromised host might be related with impairment of multiple immune functions; however, impaired TDAR deems to be usually related. Moreover, TDAR is the frequently used measurable endpoint in immunotoxicity testing according the ICH S8 immunotoxicity testing guideline. Therefore, the present AOP focus on CN inhibition-induced impairment of TDAR.
CN phosphatase activity is inhibited when stressors bond to Calcineurin-A (CnA) with immunophilins, which interferes with the nuclear localization of NFAT, a substrate of CN. As a result, the formation of functional NFAT/ AP-1 complexes that bind at the site of IL-2 , IL-4 and other cytokine promoters in each of the T helper cell subsets is reduced, thereby suppressing production of these cytokines. Among the affected cytokines TDAR is impaired mainly by the suppression of production of IL-2 and IL-4, which affect the proliferation and differentiation of B-cells to lower TDAR. 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 KE involving MIE and AO is quantifiable, and shows similar dose responses with the CNIs in vitro, this AOP is useful for understanding immunosuppression due to inhibition of CN activity. In addition, each KER is based on sufficient scientific evidence and exhibits no contradiction with dose responses of adjacent KEs.
Since CN/NFAT system expresses in cells among vast variety of species and the function in immune system is common in at least human and mice, this AOP might be applicable to many mammalian species, including humans and rodents.
Domain of Applicability
The proposed AOP regarding inhibition of CN activity leading to impaired TDAR is not dependent on life stage, sex, or age. Since tacrolimus (FK506) ointment (Protopic) is approved for pediatric atopic dermatitis, the MOA for immunosuppression appears to be applicable to all life stages. Since FK506 or Cyclosporine A (CsA)-induced outcomes in humans are mimicked by similar responses in a variety of animal models including non-human primates and rodents, immunosuppression induced by inhibition of CN activity is considered to occur 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, of which CnA exhibits phosphatase activity. In CnA-KO mice, T-cell proliferation in response to ovalbumin stimulation is lower than that for wild-type mice and is not complemented by normal antibody producing cells. In addition, when stimulated with ovalbumin, CnA-KO mice produce less IFN-γ, IL‑2, and IL‑4 than wild-type mice. However, primary antibody response in CnA-KO mice is normal in response to TNP-ovalbumin, which means that CnA deficiency affects only on T cell-dependent antibody response (TDAR).
Stressor: FKBP12-KO mice
FK506 induces suppression of immune responses; however, there is no evidence of a relationship between FKBP12 knockout 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 for the enzymatic activities of FKBP12.
KE1 and later events: NFAT-KO mice
The following phenotypes are observed in NFAT-KO mice: moderate hyperproliferation with splenomegaly, moderately enhanced B- and T-cell responses, with bias towards Th2-cell response, decreased IFN-γ production in response to T-cell receptor (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, and mild hyperactivation of peripheral T cells.
Therefore, the study of NFAT-KO mice shows that NFAT is involved in a wide range of immune responses, and some of these phenomenon are known to be regulated by CN. Suppression of T-cell-derived cytokines is noted both in CnA-knockout and NFAT-knockout mice, which indicates that the production of T-cell derived cytokines such as IL-2 and IL-4 is regulated by the CN-NFAT system.
T-cell functions are mainly regulated by the CN-NFAT system and suppression of CN activity in T cells is known to induce multiple types of immunosuppression, including T cell-dependent antibody response (TDAR).
Experiments with T cells indicate that TCR stimulation brings about increases in intracellular concentrations of Ca2+ that trigger CN activity, thereby inducing nuclear localization of substrate NFAT per dephosphorylation. The localized NFAT 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 known to be inhibited by the formation of immunophilin-CN inhibitor (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 amino-acid sequences of the two classes of immunophilins. The three-dimensional structures of immunophilin complexes are essential to the inhibition of CN phosphatase activity, even though their enzymatic activities are not.
It is also known that one of the effects on immune function when CNI forms complexes with its respective immunophilin and inhibits CN activity is the suppression of IL-2 and other T-cell derived cytokine production. It is further 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. At the time of our review of the literature, however, we did not find any reports of a direct effect of CN inhibition on B‑cells, such as changes in proliferation, 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 in which it regulates the expression of IL-2 receptors, there are no reports of effects on the production of T cell-dependent antibodies due to CNI-induced alteration in expression of IL-2 receptors in these cells.
CN-NFAT system-mediated immunosuppression is well understood based on the pharmacology of some CNI drugs; therefore, AOP of CN inhibition-induced suppression of TDAR is useful for prediction of CN-mediated immunotoxicity.
|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.
CN phosphatase activation through TCR stimulation dephosphorylates NFAT, thereby promoting nuclear localization of NFAT.
CN phosphatase activity in T cells could be inhibited by CNI/immunophilin complexes, thus interfering with dephosphorylation and nuclear localization of NFAT.
|KER2||Interference, nuclear localization to reduction, NFAT/AP-1 complex formation||Strong||
CN activity dephosphorylates NFAT, thereby promoting 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/AP-1 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, which promotes production of cytokines from T cells. Of these cytokines, IL-2 and IL-4 have a major role in promoting proliferation, maturation and class-switching of B cells, and development of TDAR.
Reduction of NFAT/AP-1 complex formation in the nucleus due to inhibition CN activity 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||
T cell-derived cytokines play important roles in TDAR. Among them, IL-2 promotes proliferation of B cells, and IL-4 affects maturation and class switching of B cells as well as proliferation.
Inhibition of CN activity by CNIs is known to suppress production of multiple cytokine species from T cells.
Of these cytokines and receptors, suppression of IL-2 and IL-4 production mainly leads to impairment of TDAR.
Suppressed production of other cytokines due to inhibition of CN activity exhibits only minor effects, if any, on TDAR.
|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.
MIE: CN phosphatase activity is inhibited by CNI of FK506 with IC50 values of 0.5 nM (FK506) and 5nM (CsA) after 1 hours treatment (Fruman et al.1992).
KE1: Concentration-dependent reduction of in vitro nuclear localization of NFAT was evident at the concentration from 0.1 nM (Jurkat T cells) or 10nM (human CD4+ T cells) and up to 1 μM (1000 nM) under the conditions of 2 hours treatment (Maguire et al. 2013).
CN phosphatase activity and nuclear translocation of NFAT seems to be suppressed by CNIs at the similar ranges of doses and reaction times of 1 to 2 hours.
|KE1=>KE2：Interference, nuclear localization of NFAT leads to reduction, NFAT/AP-1 complex formation||
Empirical support of the KE1 => KE2 is strong.
KE1: Concentration-dependent reduction of in vitro nuclear localization of NFAT was evident at the concentration from 0.1 nM (Jurkat T cells) or 10nM (human CD4+ T cells) and up to 1 μM (1000 nM) under the conditions of 2 hours treatment (Maguire et al. 2013).
KE2: Treatment of activated T cells with FK506 at 100ng/mL (124nM) or CsA at 500ng/mL (416nM) for 2 hours hinders the formation of functional NFAT/AP-1 in the nucleus (Flanagan et al. 1991).
Quantitative data on NFAT/AP-1 complex formation in the nucleus is insufficient; however, inhibition of nuclear localization of NFAT and following NFAT/AP-1 complex formation in the nucleus are simultaneously detected by gel mobility shift assay at the concentration of FK506 within the range for inhibition of nuclear translocation of NFAT using imaging flowcytometry after 2 hours culture of T cells.
|KE2=>KE3：Reduction, NFAT/AP-1 complex formation leads to suppression, IL-2 and IL-4 production||
Empirical support of the KE2 => KE3 is moderate.
KE2: Gel mobility shift assay revealed that treatment of activated T cells with FK506 at 100ng/mL (124nM) or CsA at 500ng/mL (416nM) for 2 hours hinders NFAT nuclear translocation and following formation of NFAT/AP-1 complexes in the nucleus (Flanagan et al. 1991).
KE3: 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 after 22 to 24 hours culture 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).
Therefore, concentration of CNI needed for inhibition of NFAT/AP-1 complex formation in the nucleus is higher than that for inhibition of IL-2 and IL-4 production.Time lag is found between the two KEs; 2 hours for KE2 and 22 to 48 hours for KE3.
|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.
KE3: In CD3/PMA-activated human T cells, FK506‑suppressed production of IL-2, IL-4, and IFN-γ at concentrations of 1.2 to 12.5 nM after 22 to 24 hours cultures 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).
AO: After a 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 (0.37 and 1.24 nM) of FK506 or 50 and 100 ng/mL (41.6 and 83.2 nM) of CsA (Heidt et al, 2009).
After a 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 concentrations of 0.01 to 100 ng/mL (0.012 to 124 nM) of FK506 or 0.1 to 1000 ng/mL (0.083 to 83.2 nM) of CsA (Sakuma et al. 2001b).
Rats were treated with FK506 for over four weeks and immunized with KLH, after which serum concentration of anti-KLH IgM and IgG was reduced at the dose level of 3 mg/kg/day (Ulrich et al. 2004).
Mice were treated with FK506 or CsA for 4 days, and immunized with SRBC, after which antigen-specific plaque-forming splenocytes were reduced at dose levels of 3.2, 10, 32 and 100 mg/kg of FK506 or 32 and 100 mg/kg of CsA (Kino et al. 1987b).
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 (Dumont et al. 1998).
In vitro suppression 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. Time gaps were found, however, between these two events, which showed earlier onset of cytokine production and delayed onset of antibody production.
Based on these findings of empirical support, each KE involving MIE and AO except for KE2 shows similar dose responses to the CNIs in vitro; however, culture time lag is noted, in that, 1 hour for MIE, 2 hours for KE1 and KE2, 22 to 24 hours for KE3 and more than days for AO.
There have been no literature available to show clear quantitative relationship between the inhibition of CN phosphatase activity and nuclear translocation of NFAT; however, the dose responses of CN phosphatase activity and nuclear translocation of NFAT to CNI deem to be the same.
Gel mobility shift assay of activated T cells showed that NFAT/AP-1 complexes are only found in nuclear extract, which indicates a strong relationship between the nuclear translocation of NFAT and simultaneous complex formation with AP-1 in the nucleus. CNI treatment clearly suppresses the complex formation of nuclear located NFAT and AP-1 in the nucleus, which also shows the solid relationship between these adjacent two KEs although quantitative data on suppressed NFAT/AP-1 complex formation is insufficient (Flaganan W.M. et al. 1991).
The quantitative relationship between the decreased formation of NFAT/AP-1 complexes and the production of IL2/IL-4 formation induced by CNIs has not been reported.
However, as mentioned in the empirical support, nuclear localization of NFAT is strongly related to NFAT/AP-1 complex formation in the nucleus based on the fact that these two events are detected simultaneously by gel mobility shift assay, and the dose responses of IL2/IL-4 production and nuclear translocation of NFAT inhibited by CNI are similar; therefore, dose ranges of CNI in the inhibitions of IL2/IL-4 production and NFAT/AP-1 complex formation in the nucleus might also be the same.
In addition, T-5224 and ursolic acid inhibit AP-1 DNA binding activity or production of NF-κB, NFAT and AP-1, respectively, and both suppress the IL-2 and/or IL-4 production with dose dependent manner including the doses of inhibiting NFAT-AP-1 system (Yoshida et al. 2015, Checker et al. 2012).
Inhibition of IL-4 production in mice treated with oral administration of suplatast tosilate suppresses antigen-specific IgE production with a dose-dependent manner (Taiho Pharmaceutical 2013). In the inhibition of IL-4 production in human cell culture by suplatast tosilate at the concentration of 10 μg/mL for 10 days, antigen specific IgE production was suppressed from 56 to 72% and IL-4 production was suppressed from 58 to 76% (Taiho Pharmaceutical 2013).
As for IL-2 and antibody production, in vitro T-cell-induced polyclonal B cell activation to produce antibody was inhibited with anti-IL-2 and anti-IL-2R antibodies. T (Owens T, 1991). In addition, cynomolgus monkeys treated wth CsA showed suppression of IL-2 and TDAR using sheep red blood cells with a dose dependent manner (Gaidal K. 2015).
In the human T-B cell co-culture stimulated with anti-CD3 monoclonal antibody, CNIs of FK506 and CsA lowered the levels of T-cell cytokines including IL-2 and IL-4 and inhibited IgM and IgG productions with a dose-dependent manner (Heidt S. 2010).
These results show the quantitative relationships between the inhibition of IL-4 or IL-2 by specific antibodies or CNI and suppression of antibody production.
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 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. Thus, changes in any of these immune cell populations can influence TDAR.
Moreover, when evaluating the immunotoxicity of pharmaceuticals, the ICH S8 immunotoxicity testing guideline recommends that TDAR be evaluated whenever the target cells of immunotoxicity are not clear based on pharmacology and findings in standard toxicity studies.
The present AOP could be used to predict whether or not a compound that potentially acts on T cells could also affect TDAR. On the other hand, it would be inappropriate to use the present AOP as an alternative to TDAR measurement in the ICH S8 immunotoxicity testing guideline.
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