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Relationship: 2027

Title

The title of the KER should clearly define the two KEs being considered and the sequential relationship between them (i.e., which is upstream and which is downstream). Consequently all KER titles take the form “upstream KE leads to downstream KE”. More help

Suppression of IL-4 production leads to Impairment, TDAR

Upstream event

Upstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help

Suppression of IL-4 production

Downstream event

Downstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help

Impairment, TDAR

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

This table is automatically generated upon addition of a KER to an AOP. All of the AOPs that are linked to this KER will automatically be listed in this subsection. Clicking on the name of the AOP in the table will bring you to the individual page for that AOP. More help

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Inhibition of JAK3 leading to impairment of T-Cell Dependent Antibody Response	adjacent	High	High	Yasuhiro Yoshida (send email)	Under development: Not open for comment. Do not cite	Under Development

Taxonomic Applicability

Select one or more structured terms that help to define the biological applicability domain of the KER. In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. Authors can indicate the relevant taxa for this KER in this subsection. The process is similar to what is described for KEs (see pages 30-31 and 37-38 of User Handbook) More help

Term	Scientific Term	Evidence	Link
Homo sapiens	Homo sapiens	High	NCBI
Mus musculus	Mus musculus	High	NCBI

Sex Applicability

Authors can indicate the relevant sex for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of the User Handbook). More help

Sex	Evidence
Mixed	High

Life Stage Applicability

Authors can indicate the relevant life stage for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of User Handbook). More help

Term	Evidence
All life stages	High

Key Event Relationship Description

Provide a brief, descriptive summation of the KER. While the title itself is fairly descriptive, this section can provide details that aren’t inherent in the description of the KEs themselves (see page 39 of the User Handbook). This description section can be viewed as providing the increased specificity in the nature of upstream perturbation (KEupstream) that leads to a particular downstream perturbation (KEdownstream), while allowing the KE descriptions to remain generalised so they can be linked to different AOPs. The description is also intended to provide a concise overview for readers who may want a brief summation, without needing to read through the detailed support for the relationship (covered below). Careful attention should be taken to avoid reference to other KEs that are not part of this KER, other KERs or other AOPs. This will ensure that the KER is modular and can be used by other AOPs. More help

IL-2 induces T cell proliferation Therefore, the suppression of IL-2 production leads to the impairment of TDAR. The IL-2-JAK3-STAT5 axis regulates Th1 cell differentiation, suggesting that IL-2 mediated JAK3-STAT5 signaling may generically operate in the production of Th1-related cytokines (Shi, et al. 2008).

IL-2 is produced and secreted by helper T cells. IL-2 has important roles in the development of TDAR. IL-2 promotes differentiation of B cells by stimulating differentiation of activated T cells to Th2 T cells. Therefore, suppressed production of IL-2 impairs T cell dependent antibody production.

In T cells, binding of IL-4 to its receptor induces proliferation and differentiation into Th2 cells. Th2 cells assist B cells and promote class switching from IgM to IgG1 and IgE. Therefore, the suppression of IL-4 production leads to impairment of TDAR.

After treatment with FK506 or CsA, production of IL-2, IL-4, and other cytokines decreases in T cells (Dumont, et al. 1998, Dumont, et al. 1998). This reduces stimulation of B cells as well as proliferation, activation, and class switching, leading to impairment of TDAR. Therefore, FK506 and CsA are potent inhibitors of T cell dependent antibody production. Suppression of the production of these B cell related cytokines appears to be the main factor in the impairment of TDAR (Heidt, et al. 2010).

Evidence Supporting this KER

Assembly and description of the scientific evidence supporting KERs in an AOP is an important step in the AOP development process that sets the stage for overall assessment of the AOP (see pages 49-56 of the User Handbook). To do this, biological plausibility, empirical support, and the current quantitative understanding of the KER are evaluated with regard to the predictive relationships/associations between defined pairs of KEs as a basis for considering WoE (page 55 of User Handbook). In addition, uncertainties and inconsistencies are considered. More help

Biological Plausibility

Define, in free text, the biological rationale for a connection between KEupstream and KEdownstream. What are the structural or functional relationships between the KEs? For example, there is a functional relationship between an enzyme’s activity and the product of a reaction it catalyses. Supporting references should be included. However, it is recognised that there may be cases where the biological relationship between two KEs is very well established, to the extent that it is widely accepted and consistently supported by so much literature that it is unnecessary and impractical to cite the relevant primary literature. Citation of review articles or other secondary sources, like text books, may be reasonable in such cases. The primary intent is to provide scientifically credible support for the structural and/or functional relationship between the pair of KEs if one is known. The description of biological plausibility can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured (see page 40 of the User Handbook for further information). More help

FK506 and rapamycin suppress the mRNA expression levels of IL-2 and IL-4 in T cells, which stimulate the proliferation of B cells (Heidt, et al. 2010).

Several in vivo studies in rodents have shown decreased TDAR following treatment with FK506 (Kino, et al. 1987, Ulrich, et al. 2004). In vitro tests examined antibody production in blood samples obtained from blood bank donors and PBMCs treated with FK506 and CsA. The suppressed production of immunoglobulin (Ig) M and G antibodies to T cell dependent antigens was demonstrated (Heidt, et al. 2010).

T cells, B cells, and antigen-presenting cells, such as dendritic cells, are involved in the induction and development of TDAR. Thus, changes in any of these immune cell populations can influence TDAR. However, concerning the suppression of humoral immunity induced by the inhibition of CN phosphatase activity, CNIs do not affect B cells directly. Rather, the effect is indirect via T cells. FK506 and CsA are capable of inhibiting immunoglobulin production when B cells are cultured with non-pre-activated T cells, but FK506 and CsA fail to inhibit immunoglobulin levels when pre-activated T cells are used to stimulate B cells. Hence, the inhibition of B-cell response by FK506 and CsA appears solely due to inhibition of T helper cells (Heidt, et al. 2010).

Therefore, it is concluded that decreased amounts of IL-4, in addition to IL-2, secreted from helper T cells, is the main factor in the suppression of TDAR.

Empirical Evidence

In this section authors are encouraged to cite specific evidence that supports the idea that a change in the upstream KE (KEupstream) will lead to, or is associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. Given the likelihood that new empirical support will be developed over time, particularly as various AOPs are tested and applied, it is most practical to provide empirical support in the form of bulleted lists or tables that include a short description of the nature of the empirical support along with the corresponding reference(s).In particular, it is useful to cite evidence showing that stressors that perturb KEupstream also perturb KEdownstream. Because this section of the KER description cites evidence from specific studies, it is also helpful to provide as much detail about the toxicological and biological context in which the measurements were made, as is feasible, including the stressor(s) tested, the effective doses at each KE, etc. While the KER itself is not intended to be stressor-specific, those details can aid the overall assessment of the individual AOPs that include that KER. These details also help inform the question of consistency of supporting data, consistency across different biological contexts for which the KER is relevant, and the applicability domain of the KER. However, authors are cautioned that this evidence should focus on data that only relate KEupstream to KEdownstream, and should avoid reference to other KEs, KERs and AOPs as much as possible in order to maintain modularity of the KER. Please see pages 40-41 of the User Handbook for more information. More help

Empirical support for the suppression of IL-4 production leads to impairment, and the T cell dependent antibody response is strong.

Rationale：

In CD3/PMA activated human T cells, FK506 suppressed the production of IL-2, IL-4, and IFN-γ at concentrations of 1.2 to 12.5 nM and inhibited the expression of IL-2, IL-4, and IFN-γ mRNA at concentrations of 10 nM (Dumont, et al. 1998).

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. The FK506 levels were 0.3 and 1.0 ng/mL (0.37 and 1.24 nM) and the CsA levels were 50 and 100 ng/mL (41 and 83 nM) (Heidt, et al. 2010).

After a 4-day culture of SKW6.4 IL-6-dependent IgM-secreting human B cells and anti-CD3/CD28 stimulation of the 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 (0.01 to 124 nM) of FK506 and 0.1 to 1000 ng/mL (0.08 to 832 nM) of CsA (Sakuma, et al. 2001).

Rats were treated with FK506 for over 4 weeks and immunized with KLH. The serum concentrations of anti-KLH IgM and IgG were reduced at a dose of 3 mg/kg/day (Ulrich, et al. 2004).

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 between these two KEs, which showed earlier onset of cytokine production and delayed onset of antibody production.

Uncertainties and Inconsistencies

In addition to outlining the evidence supporting a particular linkage, it is also important to identify inconsistencies or uncertainties in the relationship. Additionally, while there are expected patterns of concordance that support a causal linkage between the KEs in the pair, it is also helpful to identify experimental details that may explain apparent deviations from the expected patterns of concordance. Identification of uncertainties and inconsistencies contribute to evaluation of the overall WoE supporting the AOPs that contain a given KER and to the identification of research gaps that warrant investigation (seep pages 41-42 of the User Handbook).Given that AOPs are intended to support regulatory applications, AOP developers should focus on those inconsistencies or gaps that would have a direct bearing or impact on the confidence in the KER and its use as a basis for inference or extrapolation in a regulatory setting. Uncertainties that may be of academic interest but would have little impact on regulatory application don’t need to be described. In general, this section details evidence that may raise questions regarding the overall validity and predictive utility of the KER (including consideration of both biological plausibility and empirical support). It also contributes along with several other elements to the overall evaluation of the WoE for the KER (see Section 4 of the User Handbook). More help

IL-2 affects multiple populations of immune cells expressing IL-2 receptors, while IL-4 mainly acts on B cells. Additional suppression of other immune functions may also be possible.

Quantitative Understanding of the Linkage

The quantitative understanding section of the KER description is intended to capture information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. Empirical support addresses whether data between the two KEs are consistent with the patterns that are expected if the upstream event is causing the downstream event to occur, while the quantitative understanding section helps to define the precision with which the state of the downstream KE can be predicted from knowledge of the state of the upstream KE. These quantitative relationships may be defined in terms of correlations, response-response relationships, dose-dependent transitions or points of departure (i.e., a threshold of change in KEupstream needed to elicit a change in KEdownstream), etc. They may take the form of simple mathematical equations or sophisticated biologically-based computational models that consider other modulating factors such as compensatory responses, or interactions with other biological or environmental variables. Regardless of form, the idea is to briefly describe what is known regarding the quantitative relationship between the KEs and cite appropriate literature that defines those relationships and/or provides support for them.Data that confer quantitative understanding of a KER are not necessarily mutually exclusive from those addressing other weight of evidence considerations. In that respect, the quantitative understanding section of the KER description is not intended to be redundant with the other WoE sections. Rather, it is intended to aid application of the AOP by allowing a reader to rapidly identify the relationships that would support a quantitative prediction of the probability or magnitude of change in KEdownstream based on a known state of KEupstream. For transparency, the toxicological and biological context in which the quantitative relationships were defined should be indicated within the description. However, the ultimate goal is to identify quantitative relationships that generalize across the entire applicability domain of the two KEs being linked via the KER. More help

CsA treatment achieved 100% maximal inhibition of the ex vivo IL-2 response on Days 0, 9, and 16. CsA treatment achieved 82 [± 10]%, 68 [± 25]%, and 82 [± 9]% maximal inhibition of the ex vivo IL-4 response on Days 0, 9, and 16, respectively.

Response-response Relationship

This subsection should be used to define sources of data that define the response-response relationships between the KEs. In particular, information regarding the general form of the relationship (e.g., linear, exponential, sigmoidal, threshold, etc.) should be captured if possible. If there are specific mathematical functions or computational models relevant to the KER in question that have been defined, those should also be cited and/or described where possible, along with information concerning the approximate range of certainty with which the state of the KEdownstream can be predicted based on the measured state of the KEupstream (i.e., can it be predicted within a factor of two, or within three orders of magnitude?). For example, a regression equation may reasonably describe the response-response relationship between the two KERs, but that relationship may have only been validated/tested in a single species under steady state exposure conditions. Those types of details would be useful to capture. More help

In a rat T cell proliferation assay, IL-2-induced T cell proliferation was inhibited by peficitinib in a concentration-dependent manner with an IC50 of 10 nM and by tofacitinib with a similar IC50 of 24 nM (Gianti and Zauhar 2015). In addition, cynomolgus monkeys treated with CsA showed suppression of IL-2 and TDAR using SRBCs in a dose-dependent manner (Gaida, et al. 2015).\

In the human T-B-cell co-culture stimulated with anti-CD3 monoclonal antibody, CNIs of FK506 and CsA lowered the mRNA levels of T cell cytokines at 8 h post-stimulation including IL-2 and IL-4 at 1.0 ng/mL (1.24 nM) FK506 or 100 ng/mL (90.7 nM) CsA, and inhibited IgM and IgG productions after 9 days at 0.3 and 1.0 ng/mL FK506 and 50 and 100 ng/mL CsA (Heidt, et al. 2010).

Time-scale

This sub-section should be used to provide information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). This can be useful information both in terms of modelling the KER, as well as for analyzing the critical or dominant paths through an AOP network (e.g., identification of an AO that could kill an organism in a matter of hours will generally be of higher priority than other potential AOs that take weeks or months to develop). Identification of time-scale can also aid the assessment of temporal concordance. For example, for a KER that operates on a time-scale of days, measurement of both KEs after just hours of exposure in a short-term experiment could lead to incorrect conclusions regarding dose-response or temporal concordance if the time-scale of the upstream to downstream transition was not considered. More help

In human T cell culture, suplatast tosilate (an inhibitor of the production of cytokines by Th2 cells) inhibited IL-4 production after 3 days and antigen-specific IgE production after 10 days (Taiho 2013).

Other authors described that in human T-B-cell co-cultures, FK506 and CsA lowered the mRNA levels of IL-2 and IL-4 at 8 h post-stimulation and inhibited IgM and IgG production after 9 days (Heidt, et al. 2010).

Treatment with CsA (50 mg/kg) twice daily in cynomolgus monkeys resulted in reduction of IL-4 cytokine production from PMA/ionocycin stimulation of whole blood starting on day 0 and continuing through the end of the study on day 16. CsA treatment achieved 82 [±10]%, 68 [± 25]%, and 82 [± 9]% 100% maximal inhibition of ex vivo IL-4 response on days 0, 9, and 16. SRBC-specific IgM and IgG were significantly lower in animals dosed with CsA than in animals dosed with the vehicle control on days 9, 12, and 16 post-immunization. There was ≥80% or greater reduction in SRBC-specific IgM on days 9–16. SRBC-specific IgG was decreased by ≥95% on days 9–16 (Gaida, et al. 2015). This was similar to the degree of inhibition observed in rats using an KLH immunization model (Smith, et al. 2003).

Known modulating factors

This sub-section presents information regarding modulating factors/variables known to alter the shape of the response-response function that describes the quantitative relationship between the two KEs (for example, an iodine deficient diet causes a significant increase in the slope of the relationship; a particular genotype doubles the sensitivity of KEdownstream to changes in KEupstream). Information on these known modulating factors should be listed in this subsection, along with relevant information regarding the manner in which the modulating factor can be expected to alter the relationship (if known). Note, this section should focus on those modulating factors for which solid evidence supported by relevant data and literature is available. It should NOT list all possible/plausible modulating factors. In this regard, it is useful to bear in mind that many risk assessments conducted through conventional apical guideline testing-based approaches generally consider few if any modulating factors. More help

Treatment with CsA (cyclosporin A) at 50 mg/kg BID (bis in die) resulted in reduction of IL-2, IL-4 cytokine production from PMA/ionomycin stimulation of whole blood in synomolgus monkey starting on Day 0 and continuing through the end of study on Day 16. In addition, Tacrolimus concentration was 1.0 ng/ml. Tacrolimus inhibited IL-2 and IL-4 mRNA levels. Glycosylation-inhibiting factor (GIF) secreted from CD4 cells suppressed IL-4 mRNA levels of the same cells during the initial 24 h of CD3/CD28 stimulation.

Known Feedforward/Feedback loops influencing this KER

This subsection should define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits? In some cases where feedback processes are measurable and causally linked to the outcome, they should be represented as KEs. However, in most cases these features are expected to predominantly influence the shape of the response-response, time-course, behaviours between selected KEs. For example, if a feedback loop acts as compensatory mechanism that aims to restore homeostasis following initial perturbation of a KE, the feedback loop will directly shape the response-response relationship between the KERs. Given interest in formally identifying these positive or negative feedback, it is recommended that a graphical annotation (page 44) indicating a positive or negative feedback loop is involved in a particular upstream to downstream KE transition (KER) be added to the graphical representation, and that details be provided in this subsection of the KER description (see pages 44-45 of the User Handbook). More help

B cells are required for the generation and / or maintenance of Th2 responses. Germinal center B cells regulate Th2 development through an IL-4 dependent process. Type 2 immunity and allergic responses are initiated by T cells and DCs, this response may be sustained and potentially amplified by an IL-4-driven feedback loop between Ag-specific T and B cells (Harris, et al. 2005).

Domain of Applicability

As for the KEs, there is also a free-text section of the KER description that the developer can use to explain his/her rationale for the structured terms selected with regard to taxonomic, life stage, or sex applicability, or provide a more generalizable or nuanced description of the applicability domain than may be feasible using standardized terms. More help

The effects of FK506 on serum concentrations of anti-KLH antibodies IgM and IgG have been demonstrated in rats treated with FK506 for over 4 weeks and immunized with KLH (Ulrich, et al. 2004). The effects of FK506 and CsA on the levels of IgM and IgG in the culture supernatant have been demonstrated in human cells (Heidt, et al. 2010, Sakuma, et al. 2001). In thymectomized mice, the development of KLH-specific effector CD4 T cells was reportedly reduced and these cells were suppressed in their production of IL-4 (Bradley, et al. 1991). The effects of FK506 and CsA on the production of IL-2 have been demonstrated using mice and human cells. These facts suggest that there are no species differences between humans and rodents in the inhibition of IL-4 production and TDAR induction.

References

List of the literature that was cited for this KER description using the appropriate format. Ideally, the list of references should conform, to the extent possible, with the OECD Style Guide (OECD, 2015). More help

Bradley LM, Duncan DD, Tonkonogy S, Swain SL. 1991. Characterization of antigen-specific CD4+ effector T cells in vivo: immunization results in a transient population of MEL-14-, CD45RB- helper cells that secretes interleukin 2 (IL-2), IL-3, IL-4, and interferon gamma. J Exp Med 174:547-559. DOI: 10.1084/jem.174.3.547.

Dumont FJ, Koprak S, Staruch MJ, Talento A, Koo G, DaSilva C, Sinclair PJ, Wong F, Woods J, Barker J, Pivnichny J, Singer I, Sigal NH, Williamson AR, Parsons WH, Wyvratt M. 1998. A tacrolimus-related immunosuppressant with reduced toxicity. Transplantation 65:18-26. DOI: 10.1097/00007890-199801150-00005.

Dumont FJ, Staruch MJ, Fischer P, DaSilva C, Camacho R. 1998. Inhibition of T cell activation by pharmacologic disruption of the MEK1/ERK MAP kinase or calcineurin signaling pathways results in differential modulation of cytokine production. J Immunol 160:2579-2589.

Gaida K, Salimi-Moosavi H, Subramanian R, Almon V, Knize A, Zhang M, Lin FF, Nguyen HQ, Zhou L, Sullivan JK, Wong M, McBride HJ. 2015. Inhibition of CRAC with a human anti-ORAI1 monoclonal antibody inhibits T-cell-derived cytokine production but fails to inhibit a T-cell-dependent antibody response in the cynomolgus monkey. J Immunotoxicol 12:164-173. DOI: 10.3109/1547691X.2014.915897.

Gianti E, Zauhar RJ. 2015. An SH2 domain model of STAT5 in complex with phospho-peptides define "STAT5 Binding Signatures". J Comput Aided Mol Des 29:451-470. DOI: 10.1007/s10822-015-9835-6.

Harris DP, Goodrich S, Mohrs K, Mohrs M, Lund FE. 2005. Cutting edge: the development of IL-4-producing B cells (B effector 2 cells) is controlled by IL-4, IL-4 receptor alpha, and Th2 cells. J Immunol 175:7103-7107. DOI: 175/11/7103 [pii]10.4049/jimmunol.175.11.7103.

Heidt S, Roelen DL, Eijsink C, Eikmans M, van Kooten C, Claas FH, Mulder A. 2010. Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help. Clin Exp Immunol 159:199-207. DOI: 10.1111/j.1365-2249.2009.04051.x.

Kino T, Hatanaka H, Hashimoto M, Nishiyama M, Goto T, Okuhara M, Kohsaka M, Aoki H, Imanaka H. 1987. FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. J Antibiot (Tokyo) 40:1249-1255. DOI: 10.7164/antibiotics.40.1249.

Sakuma S, Kato Y, Nishigaki F, Magari K, Miyata S, Ohkubo Y, Goto T. 2001. Effects of FK506 and other immunosuppressive anti-rheumatic agents on T cell activation mediated IL-6 and IgM production in vitro. Int Immunopharmacol 1:749-757.

Shi M, Lin TH, Appell KC, Berg LJ. 2008. Janus-kinase-3-dependent signals induce chromatin remodeling at the Ifng locus during T helper 1 cell differentiation. Immunity 28:763-773. DOI: 10.1016/j.immuni.2008.04.016.

Smith HW, Winstead CJ, Stank KK, Halstead BW, Wierda D. 2003. A predictive F344 rat immunotoxicology model: cellular parameters combined with humoral response to NP-CgammaG and KLH. Toxicology 194:129-145. DOI: 10.1016/j.tox.2003.07.002.

Taiho PC, Ltd. 2013. Drug interview form IPD capsule 50 and 100. . Revised 5th edition.

Ulrich P, Paul G, Perentes E, Mahl A, Roman D. 2004. Validation of immune function testing during a 4-week oral toxicity study with FK506. Toxicol Lett 149:123-131. DOI: 10.1016/j.toxlet.2003.12.069