AOP ID and Title:

Short Title: Immune dysfunction induced by JAK3 inhibition

Authors

Yasuhiro Yoshida (1) Takao Ashikaga (1) Tomoki Fukuyama (1) Ken Goto (1) Shinko Hata (1) Shigeru Hisada (1) Shiho Ito (1) Hiroyuki Komatsu (1) Sumie Konishi (1) Tadashi Kosaka (1) Kiyoshi Kushima (1) Shogo Matsumura (1) Takumi Ohishi (1) Yasuharu Otsubo (1) Junichiro Sugimoto (1)

(1) AOP Working Group, Testing Methodology Committee, The Japanese Society of Immunotoxicology

Corresponding author: Yasuhiro Yoshida (freude@med.uoeh-u.ac.jp)

Status

Abstract

Signal transduction between immune-related cells depends in many cases on cytokines. The transduction involves cell surface cytokine receptors as well as direct cell-to-cell interaction. Cytokines influence the movement, proliferation, differentiation, and activation of lymphocytes and other leukocytes in a variety of ways. Some cytokine receptors require an activation step through a Janus-kinase (JAK)/signal transducer and activator of transcription (STAT) system. When cytokines bind to specific cytokine receptors, the receptors form dimers, which more closely resemble JAK molecules. JAK is activated and phosphorylates adjacent cytokine receptors. STATs bind to the phosphorylated receptor sites and are in turn phosphorylated by the activated JAK. The phosphorylated STAT is dimerized and translocated into the nucleus. There it binds to the promoter regions of cytokine genes, which initiates the transcription of these genes in the nucleus.

 

In mammals, four JAK families of enzymes (JAK1, JAK2, JAK3, and TYK2) and seven STATs (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6) are utilized by more than 50 cytokines and growth factors to mediate intracellular signaling. In particular, pro-inflammatory cytokines such as interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4, IL-6, IL-13, IL-21, and IL-23 have been implicated in inflammatory diseases that utilize the JAK pathway. In addition, TH2 derived cytokines, including IL-31 and thymic stromal lymphopoietin (TSLP), are ligands for murine and human sensory nerves. These cytokines have critical roles in evoking itchiness. Because these cytokines also interact with JAK, several JAK inhibitors have received a lot of attention recently as therapeutic agents for major inflammatory diseases and pruritic diseases.

 

This proposed AOP consists of JAK3 inhibition as a MIE, blockade of STAT5 phosphorylation as the first key event (KE1), suppression of STAT5 binding to the promoter regions of cytokine genes as KE2, suppression of IL-4 production as KE3, and suppression of T cell dependent antibody response (TDAR) as an AO. This AOP especially focuses on the inhibition of JAK3, which is required for signal transduction by cytokines through the common γ chain of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In the proposed AOP, JAK3 selective inhibitors that include PF-06651600 (CAS No: 1792180-81-4) and the 4-aminopiperidine-based compound RB1 are stressors. STAT5 that is phosphorylated by JAK3 forms a homo-dimer that translocate to the nucleus and induces expressions of genes, such as IL-4. Therefore, JAK3 inhibition leads to the suppressed binding of STAT5 to the promoter regions of cytokine genes and the subsequent suppression of IL-4 production. Thus, JAK/STAT regulation plays an important role in the TDAR. TDAR is frequently affected by immunosuppressive conditions and is a major endpoint in many preclinical immunotoxicity studies.

Background

Although many stressors inhibit JAK3 activity, this AOP is based on immunosuppression caused by the recently developed and highly selective JAK3 inhibitors PF-06651600 and RB1. A significant body of scientific literature has been published concerning these two inhibitors. We look forward to future amendments to this AOP with up-to-date information on other stressors, which will clarify the link between inhibition of JAK activity and impairment of TDAR.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

JAKs are a family of nonreceptor tyrosine kinases and consist of four members: JAK1, JAK2, JAK3, and Tyk2 (Johnston, et al. 1994). All four mediate signals initiated by cytokines through interactions with receptors for IL-2, IL-5, IL-7, IL-9, and IL-15 via the common γ chain (Witthuhn, et al. 1994). Different studies have shown that JAK3 is widely expressed in different organs (Witthuhn, et al. 1994). Previous studies with IL-2Rγ-null mice showed that JAK3 is related to the development of spontaneous inflammatory bowel disease symptoms (Miyazaki, et al. 1994). Moreover, abnormal activation of JAK3 was associated with human hematology (Ihle, et al. 1997), indicating that a tight balance of its activity is essential for normal hematopoietic development.

Although JAK1, JAK2, and Tyk2 are widely expressed, JAK3 is predominantly expressed in hematopoietic cells and is associated only with the common γ chain of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors (Nosaka, et al. 1995). IL-4 is a very well-known cytokine that is crucial in the polarization of naïve T cells to type 2 helper T cells. IL-4 plays a major role in the growth and proliferation of many immune cells, such as natural killer (NK) cells and T cells (Dhupkar and Gordon 2017). Homozygous mutant mice harboring a disrupted JAK3 gene display profound reductions in thymocytes and severe B cell and T cell lymphopenia, similar to severe combined immunodeficiency disease (SCID), and functionally deficient residual T cells and B cells. Thus, JAK3 plays a critical role in γ chain signaling and lymphoid development.

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

The proposed AOP involves inhibition of JAK activity, which leads to suppression of TDAR independent of life stage, sex, or age. Since JAK3 inhibitors (PF-06651600, RB1) are currently under phase 2 clinical evaluation for the treatment of rheumatoid arthritis, the AOP appears to be applicable to all life stages. JAK3 inhibitor-induced outcomes in humans are mimicked by similar responses in a variety of animal models, including non-human primates and rodents. Thus, immunosuppression induced by inhibition of JAK3 activity is considered to occur across a variety of mammalian species. For example, PF-06651600 was reported to reduce paw swelling with an unbound EC50 of 169 nM in rat adjuvant-induced arthritis. Similarly, PF-06651600 significantly reduced disease severity in an experimental autoimmune encephalomyelitis mouse model at 30 or 100 mg/kg or prophylactically at 20 and 60 mg/kg. PF-06651600 will be evaluated in clinical trials (Telliez, et al. 2016).

Essentiality of the Key Events

MIE and later events: JAK3-knockout (KO) mice

JAK3 was initially identified (Johnston, et al. 1994, Witthuhn, et al. 1994) in studies designed to identify the JAK family member involved in the signaling of a group of cytokines with shared utilization of the γ chain first identified in the IL-2 receptor complex. It was subsequently demonstrated that JAK3 physically associates with the γ chain and is activated in a receptor complex that also contains JAK1, which associates with the ligand-specific α or β chain of the receptors (Miyazaki, et al. 1994). JAK3 is somewhat unique within the JAK family in that it is predominantly expressed in hematopoietic cells and is only activated in response to cytokines that use the γ chain (Ihle, et al. 1997). The phenotype of the JAK3 deletion mice is striking, with a range of deficiencies that collectively constitute SCID (Nosaka, et al. 1995, Thomis, et al. 1995). At the same time, two groups identified individuals that lacked JAK3 and exhibited somatically acquired SCID (Macchi, et al. 1995, Russell, et al. 1995). One of the most striking components of the phenotype are the dramatic reductions in both the T and B-cell lineages. Comparable reductions are seen in mice that lack IL-7 (von Freeden-Jeffry, et al. 1995), the IL-7 receptor α chain (Peschon, et al. 1994), or the γ chain. Despite the reduced numbers, the cells that do develop are phenotypically normal. These results are consistent with the hypothesis that activation of JAK3 is critical in the expansion, but not differentiation, of early lymphoid lineage-committed cells. In addition to the reduced numbers, the differentiated lymphoid cells that are generated fail to respond to the spectrum of cytokines that utilize the γ chain and activate JAK3 normally.

 

B6.Cg-Nr1d1tm1Ven/LazJ mouse

Primary immunodeficiencies (PIDs) are inborn errors that cause developmental and/or functional defects in the immune system (Picard, et al. 2015). PIDs are usually rare and monogenic. They present clinically with a broad array of phenotypes, including increased susceptibility to infection. One of the most deadly categories of PID is SCID. SCID is invariably caused by severe developmental and/or functional defects of T lymphocytes. However, SCID may also present with variable defects of B and/or NK cells. The B6.Cg-Nr1d1tm1Ven/LazJ mouse line harbors a spontaneous mutation in JAK3, which generates the SCID phenotype (Robinette, et al. 2018).

KE1: STAT5-KO mice

STAT5 plays a major role in regulating vital cellular functions, such as proliferation, differentiation, and apoptosis of hematopoietic and immune cells (Rani and Murphy 2016, Wittig and Groner 2005). STAT5 is activated by phosphorylation of a single constituent tyrosine residue (Y694) and is negatively regulated by dephosphorylation. A wide variety of growth factors and cytokines can activate STAT5 through the JAK-STAT pathway. The activation of STAT5 is transient and tightly regulated in normal cells (Quezada Urban, et al. 2018).

Phenotypes observed in STAT5-KO mice

The transcription factor STAT5 is expressed in all lymphocytes and plays a key role in multiple aspects of lymphocyte development and function (Owen and Farrar 2017). STAT5 was initially identified as a transcription factor activated by prolactin in mammary gland epithelial cells (Schmitt-Ney, et al. 1992, Wakao, et al. 1992). Subsequent studies identified STAT5 binding activity in T cells (Beadling, et al. 1994). Other authors described that the expression of STAT5 in multiple cell types and its’ activation by a number of cytokines, including the common γ-chain-dependent cytokines IL-2, IL-4, IL-7, IL-13, and IL-15 (Lin, et al. 1995).

STAT5 in T cell development

The observation that STAT5 is activated by multiple cytokines in T cells suggests that it might play a critical role in the development and/or function of these cells. Disruption of the Stat5a gene or Stat5b gene reportedly resulted in relatively modest phenotypes. For example, Stat5a-/- mice displayed defects in mammary gland development and lactation, while Stat5b-/- mice displayed defects in response to growth hormone in male mice and NK cell proliferation (Imada, et al. 1998, Liu, et al. 1997). To determine whether combined deletion of Stat5a and Stat5b might result in more profound immunodeficiencies, subsequent studies deleted the first coding exons of both Stat5a and Stat5b. This intervention resulted in the production of truncated forms of STAT5a and STAT5b, which acted as functional hypomorphs. These mice had surprisingly mild defects in lymphocyte development, although T cells were grossly dysfunctional as they could no longer proliferate in response to IL-2 (Moriggl, et al. 1999, Teglund, et al. 1998). Finally, complete deletion of Stat5a and Stat5b using Cre-LoxP approaches demonstrated that STAT5a and STAT5b are absolutely required for lymphocyte development, as Stat5a/b-/- mice had profound blocks in lymphocyte development, which mimicked that observed in Il7r-/- mice (Cui, et al. 2004, Yao, et al. 2006). These studies definitively demonstrated the retention of appreciable STAT5 function in STAT5 hypomorph mice.

Weight of Evidence Summary

T cell development is mainly regulated by the JAK-STAT system. JAK3 deficiency in T cells induces multiple types of immunosuppression, including TDAR.

JAK3-deficient mice reportedly displayed profound reductions in thymocytes and severe B cell and T cell lymphopenia, similar to SCID disease. The residual T cells and B cells were functionally deficient (Peschon, et al. 1994).

Mice lacking JAK3 also showed a severe block in B cell development at the pre-B stage in the bone marrow. In contrast, although the thymuses of these mice were small, T cell maturation progressed relatively normally. In response to mitogenic signals, peripheral T cells in JAK3-deficient mice did not proliferate and secreted small amounts of IL-4. These data demonstrate that JAK3 is critical for the progression of B cell development in the bone marrow and for the functional competence of mature T cells (Nosaka, et al. 1995).

Furthermore, the abnormal architecture of lymphoid organs suggested the involvement of JAK3 in epithelial cells. T cells that developed in the mutant mice did not respond to IL-2, IL-4, or IL-7 (Ito, et al. 2017).

PF-06651600 and RB1 specifically inhibit JAK3 with over 100-fold preference over JAK2, JAK1, and TYK2 in kinase assays. Reduced inflammation and associated pathology have been described in collagen-induced arthritis mice. Importantly, the administration of PF-06651600 or RB1 results in decreased pro-inflammatory cytokines and JAK3 and STAT phosphorylation in mice. The findings suggest that the inhibition of JAK3/STAT signaling is closely correlated with the induction of multiple types of immunosuppression, including TDAR.

Quantitative Consideration

KER1 (MIE => KE1)：

Treatment with the highly selective JAK3 inhibitor PF-06651600 or RB1 suppresses the complex formation of STAT5 in the nucleus. IL-2 stimulates STAT5 and induces tyrosine phosphorylation of STAT5 (Wakao, et al. 1995). RB1 inhibits the phosphorylation of STAT5 elicited by IL-2, as evidenced by an IC50 value of 31 nM in the peripheral blood mononuclear cells (PBMCs) of humans. PBMCs isolated from the buffy coats of healthy volunteers by density gradient centrifugation on Lymphoprep were cultured in complete RPMI 1640 medium (containing 10% fetal bovine serum, 100 mg/mL streptomycin and 100 U/mL penicillin) plus 10 μg/mL lectin phytohemagglutinin (PHA) for 3 days. The cells were then treated with recombinant human IL-6 (400 ng/mL), recombinant human IL-2 (100 ng/mL), or recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 ng/mL) at 37°C for 20 min. To terminate the stimulation, the cells were fixed with Lyse/Fix Buffer and then incubated with 100% methanol for 30 min. The cells were incubated with anti-pSTAT3 and anti-CD4 antibodies, or anti-pSTAT5 and anti-CD4 antibodies at 4°C overnight, washed twice with PBS, and analyzed with by flow cytometry (Ju, et al. 2011).

The fluorescence intensity of phospho-STAT5 in CD3-positive lymphocytes was observed to increase upon incubation of peripheral blood with IL-2. Peficitinib inhibited STAT5 phosphorylation in a concentration-dependent manner with a mean IC50 of 124 nM (101 and 147 nM for two rats). Additionally, the effect of peficitinib on IL-2 stimulated STAT5 phosphorylation in human peripheral T cells was evaluated. Parallel with the results in rats, the fluorescence intensity of phospho-STAT5 in CD3-positive lymphocytes increased in human peripheral blood after adding IL-2. Peficitinib inhibited STAT5 phosphorylation in a concentration-dependent manner with a mean IC50 of 127 nM in human lymphocytes (Ito, et al. 2017).

 

KER2 (KE1 => KE2)：

STAT5 can be activated and phosphorylated by cytokines, such as IL-2 and IL-15. Tyrosine phosphorylation of STAT5 is important for the dimerization of STAT5 (Wakao, et al. 1995). The STAT5 dimer has an identical DNA binding specificity and immunoreactivity.

 

KER3 (KE2 => KE3)：

STAT5 is phosphorylated by JAK kinases, allowing its dimerization and translocation into the nucleus where it can bind to its specific DNA binding sites. Electrophoretic mobility shift assay (EMSA) data revealed that IL-2 activation induced STAT5 dimerization and DNA binding to the gamma interferon activated site (GAS) motif in the IL-4 receptor alpha promoter region (John, et al. 1999). Other EMSA data showed that dexamethasone (10^-6 M) inhibited STAT5 DNA binding in mononuclear cells in a dose-dependent fashion at dexamethasone concentrations of 10^-8 to 10^-7 M (Bianchi, et al. 2000).

 

KER4 (KE3 => AO)：

Binding of IL-4 to the T cell 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.

In co-cultured human T and B cells stimulated with anti-CD3 monoclonal antibody, the calcineurin inhibitors (CNIs) FK506 and cyclosporin A (CsA) lowered the levels of T cell cytokines, including IL-2 and IL-4, and inhibited IgM and IgG production in a dose-dependent manner (Heidt, et al. 2010).

The collective results demonstrate the quantitative relationships between the inhibition of IL-4 by specific antibodies or CNI and suppression of antibody production.

References

Appendix 1

List of MIEs in this AOP

Event: 1715: Inhibition of JAK3

Short Name: Inhibition of JAK3

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

JAKs are a family of nonreceptor protein tyrosine kinases that are critical for cytokine-receptor-binding-triggered signal transduction through STAT to the nuclei of cells. In mammals, the JAK1, JAK2, and TYK2 kinases are ubiquitously expressed. In contrast, the expression of JAK3 is more restricted. It is predominantly expressed in hematopoietic cells and is highly regulated by cell development and activation (Gaffen, et al. 1995, Xu, et al. 1996). JAK3 is solely activated by type I cytokine receptors, featuring a common γ-chain subunit that is activated by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-7 (Peschon, et al. 1994). Mutations in either the γ chain or JAK3 have been identified as a cause of SCID in humans, which manifests as a depletion of T, B, and NK cells with no other defects (Darnell 1997, Decker, et al. 1997).

Loss-of-function mutations in JAK3 cause autosomal recessive SCID. Defects in this form of SCID are restricted to the immune system, which leads to the development of immunosuppressive JAK inhibitors.

Key Event Description

Janus tyrosine kinase (JAK) 3 is a member of the JAK family that is constitutively associated with the Box-1 region of the cytokine receptor intracellular domain. JAK3 is activated upon ligand-induced receptor dimerization (Stahl, et al. 1994).

The PF-06651600 selective JAK3 inhibitor is undergoing phase 2 clinical evaluation for use in treating rheumatoid arthritis. This compound inhibits JAK3 kinase activity with an IC50 of 33.1 nM (IC50 > 10000 nM). It lacks activity against JAK1, JAK2, or TYK2 (Telliez, et al. 2016, Thorarensen, et al. 2017). The RB1 novel and highly selective JAK3 inhibitor  blocks JAK3 kinase in vitro and abrogates functional activity in various cell types (Pei, et al. 2018). When orally administered to mice, RB1 mediate the JAK-STAT pathway and reduces the clinical and microscopic manifestations of paw damage in collagen-induced arthritis mice.

How it is Measured or Detected

Enzymatic activities against JAK1, JAK2, JAK3, and TYK2 were examined using a Caliper Mobility Shift Assay. In the presence of an ATP concentration at Km for ATP for each JAK isoform, RB1 inhibited JAK3 kinase activity with an IC50 value of 40 nM without inhibiting JAK1, JAK2, or TYK2 (IC50 > 5000 nM) (Gianti and Zauhar 2015). The PF-06651600 JAK3 inhibitor displays potent inhibitory activity with an IC50 of 33.1 nM (IC50>10 000 nM), with no activity against JAK1, JAK2, and TYK2. PF-06651600 inhibits the phosphorylation of STAT5 elicited by IL-2, IL-4, IL-7, and IL-15 with an IC50 of 244, 340, 407, and 266 nM, respectively (Telliez, et al. 2016).

References

Darnell JE, Jr. 1997. STATs and gene regulation. Science 277:1630-1635.

Decker T, Kovarik P, Meinke A. 1997. GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression. J Interferon Cytokine Res 17:121-134. DOI: 10.1089/jir.1997.17.121.

Gaffen SL, Lai SY, Xu W, Gouilleux F, Groner B, Goldsmith MA, Greene WC. 1995. Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity. Proc Natl Acad Sci U S A 92:7192-7196.

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.

Pei H, He L, Shao M, Yang Z, Ran Y, Li D, Zhou Y, Tang M, Wang T, Gong Y, Chen X, Yang S, Xiang M, Chen L. 2018. Discovery of a highly selective JAK3 inhibitor for the treatment of rheumatoid arthritis. Sci Rep 8:5273. DOI: 10.1038/s41598-018-23569-y.

Peschon JJ, Morrissey PJ, Grabstein KH, Ramsdell FJ, Maraskovsky E, Gliniak BC, Park LS, Ziegler SF, Williams DE, Ware CB, Meyer JD, Davison BL. 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med 180:1955-1960.

Stahl N, Boulton TG, Farruggella T, Ip NY, Davis S, Witthuhn BA, Quelle FW, Silvennoinen O, Barbieri G, Pellegrini S, et al. 1994. Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Science 263:92-95.

Telliez JB, Dowty ME, Wang L, Jussif J, Lin T, Li L, Moy E, Balbo P, Li W, Zhao Y, Crouse K, Dickinson C, Symanowicz P, Hegen M, Banker ME, Vincent F, Unwalla R, Liang S, Gilbert AM, Brown MF, Hayward M, Montgomery J, Yang X, Bauman J, Trujillo JI, Casimiro-Garcia A, Vajdos FF, Leung L, Geoghegan KF, Quazi A, Xuan D, Jones L, Hett E, Wright K, Clark JD, Thorarensen A. 2016. Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition. ACS Chem Biol 11:3442-3451. DOI: 10.1021/acschembio.6b00677.

Thorarensen A, Dowty ME, Banker ME, Juba B, Jussif J, Lin T, Vincent F, Czerwinski RM, Casimiro-Garcia A, Unwalla R, Trujillo JI, Liang S, Balbo P, Che Y, Gilbert AM, Brown MF, Hayward M, Montgomery J, Leung L, Yang X, Soucy S, Hegen M, Coe J, Langille J, Vajdos F, Chrencik J, Telliez JB. 2017. Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1-((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1-yl)prop -2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans. J Med Chem 60:1971-1993. DOI: 10.1021/acs.jmedchem.6b01694.

Xu BC, Wang X, Darus CJ, Kopchick JJ. 1996. Growth hormone promotes the association of transcription factor STAT5 with the growth hormone receptor. J Biol Chem 271:19768-19773.

List of Key Events in the AOP

Event: 1716: Blockade of STAT5 phosphorylation

Short Name: STAT5 inhibition

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

STAT5 is expressed in hematopoietic cells, including T cells and B cells from humans, rodents, and other mammalian species (Thibault, et al. 2016).

Key Event Description

The STAT family of proteins regulate gene transcription upon activation. The proteins rely on cytokine signaling and a number of growth factors through the JAK/STAT  pathway (Kisseleva, et al. 2002). STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization (Gianti and Zauhar 2015). STAT5 has been implicated in cell growth and differentiation. STAT5 was originally purified and cloned from mammary epithelial cells in sheep and identified as a signal transducer that confers the specific biological responses of prolactin (Wakao, et al. 1992, Xu, et al. 1996).Thus, STAT5 proteins function as signal transduction molecules in the cytoplasm and as transcription factors upon translocation to the nucleus.

How it is Measured or Detected

Phosphorylation of STAT5 tyrosine can be detected by specific antibodies using several detection systems, including flow cytometry. In one study, phosphorylated STAT5 expression was measured in T lymphocytes, and MFIs were reported for each subset (Osinalde, et al. 2017). A cell-permeable non-peptidic nicotinoyl hydrazone compound selectively targets the SH2 domain of STAT5 (IC50 = 47 µM against STAT5b SH2 domain EPO peptide binding activity), with markedly less recognition of the SH2 domain of STAT1, STAT3, or Lck (IC50 >500 µM). The compound was reported to block STAT5/STAT5 DNA binding activity in K562 nuclear extract and inhibit IFN-α-stimulated STAT5 tyrosine phosphorylation in Daudi cells, with no effect on STAT1 or STAT3 (Muller, et al. 2008).

Tyrosine phosphorylation of STAT5 induced by IL-2 has been analyzed using an anti-STAT5 antibody. In the study, this antibody immunoprecipitated STAT5 (p94 kDa). Peripheral blood lymphocytes were untreated (control) or treated with IL-2, IL-4, or IL-15 for 15 min. The extracts were incubated with biotinylated oligonucleotide bound to streptavidin-coated agarose. The agarose beads were washed and the eluted protein was immunoblotted with an antibody to STAT5 (Stahl, et al. 1994).

Other authors described the inhibition of JAK3 kinase activity by PF-06651600, followed by inhibition of the phosphorylation of STAT5 elicited by IL-2, IL-4, IL-7, and IL-15 with IC50 values of 244, 340, 407, and 266 nM, respectively (Telliez, et al. 2016).

Pimozide is a specific inhibitor of STAT5 phosphorylation. Pimozide decreased the survival of chronic myelogenous leukemia cells resistant to kinase inhibitors (Nelson, et al. 2011). IL-2 markedly stimulated STAT5 phosphorylation in PBMCs from patients with chronic kidney disease (CKD). Pretreatment with pimozide (3 µM) dramatically suppressed IL-2-induced STAT5 phosphorylation, indicating that it is a potent blocker of IL-2-stimulated STAT5 phosphorylation in PBMCs from CKD patients.

References

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.

Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW. 2002. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 285:1-24.

Muller J, Sperl B, Reindl W, Kiessling A, Berg T. 2008. Discovery of chromone-based inhibitors of the transcription factor STAT5. Chembiochem 9:723-727. DOI: 10.1002/cbic.200700701.

Nelson EA, Walker SR, Weisberg E, Bar-Natan M, Barrett R, Gashin LB, Terrell S, Klitgaard JL, Santo L, Addorio MR, Ebert BL, Griffin JD, Frank DA. 2011. The STAT5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors. Blood 117:3421-3429. DOI: 10.1182/blood-2009-11-255232

blood-2009-11-255232 [pii].

Osinalde N, Sanchez-Quiles V, Blagoev B, Kratchmarova I. 2017. Data on interleukin (IL)-2- and IL-15-dependent changes in IL-2Rbeta and IL-2Rgamma complexes. Data Brief 11:499-506. DOI: 10.1016/j.dib.2017.02.030.

Stahl N, Boulton TG, Farruggella T, Ip NY, Davis S, Witthuhn BA, Quelle FW, Silvennoinen O, Barbieri G, Pellegrini S, et al. 1994. Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Science 263:92-95.

Telliez JB, Dowty ME, Wang L, Jussif J, Lin T, Li L, Moy E, Balbo P, Li W, Zhao Y, Crouse K, Dickinson C, Symanowicz P, Hegen M, Banker ME, Vincent F, Unwalla R, Liang S, Gilbert AM, Brown MF, Hayward M, Montgomery J, Yang X, Bauman J, Trujillo JI, Casimiro-Garcia A, Vajdos FF, Leung L, Geoghegan KF, Quazi A, Xuan D, Jones L, Hett E, Wright K, Clark JD, Thorarensen A. 2016. Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition. ACS Chem Biol 11:3442-3451. DOI: 10.1021/acschembio.6b00677.

Thibault G, Paintaud G, Legendre C, Merville P, Coulon M, Chasseuil E, Ternant D, Rostaing L, Durrbach A, Di Giambattista F, Buchler M, Lebranchu Y. 2016. CD25 blockade in kidney transplant patients randomized to standard-dose or high-dose basiliximab with cyclosporine, or high-dose basiliximab in a calcineurin inhibitor-free regimen. Transpl Int 29:184-195. DOI: 10.1111/tri.12688.

Wakao H, Schmitt-Ney M, Groner B. 1992. Mammary gland-specific nuclear factor is present in lactating rodent and bovine mammary tissue and composed of a single polypeptide of 89 kDa. J Biol Chem 267:16365-16370.

Xu BC, Wang X, Darus CJ, Kopchick JJ. 1996. Growth hormone promotes the association of transcription factor STAT5 with the growth hormone receptor. J Biol Chem 271:19768-19773.

Event: 1717: Suppression of STAT5 binding to cytokine gene promoters

Short Name: Suppression of STAT5 binding to cytokine gene promoters

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

STAT5 is expressed in hematopoietic cells, such as T and B cells from humans, rodents, and other mammalian species (Gilmour, et al. 1995).

Key Event Description

IL-2 and other cytokines rapidly activate JAK1 and JAK3 (Beadling, et al. 1994) in peripheral blood lymphocytes. The activation of JAK kinases and STAT proteins by IL-2 and IFN-α does not include the T cell antigen receptor in human T lymphocytes (Beadling, et al. 1994). After activation of JAKs, latent STAT transcription factors induce dimeric STAT proteins (Gaffen, et al. 1995). These proteins then translocate to the nucleus, where they bind to and regulate the transcriptional activation of the promoters of target genes. Dimeric STAT proteins can bind to the palindromic gamma interferon-activated (GAS) sequence TTCNmGAA, where m is 3 for all the STATs, except STAT6. The latter can additionally bind to GAS motifs. The m for STAT6 denotes 4 (Darnell 1997, Decker, et al. 1997, Ihle 1996, Leonard and O'Shea 1998).

How it is Measured or Detected

EMSA using nuclear extracts and specific oligonucleotides, including transcription factor binding sites, such as cytokine-inducible SH2-containing protein (CIS) gene promoters, are useful to evaluate DNA binding activity (Johnston, et al. 1995). Activated STAT5 binds to specific DNA-probes in splenocytes (Liu, et al. 2010). A cell-permeable non-peptidic nicotinoyl hydrazone compound selectively targets the SH2 domain of STAT5 (IC50 = 47 µM against STAT5b SH2 domain EPO peptide binding activity), with markedly less recognition of the SH2 domain of STAT1, STAT3, or Lck (IC50 > 500 µM). This compound inhibited  STAT5/STAT5 DNA binding activity in K562 nuclear extract and inhibited IFN-α-stimulated STAT5 tyrosine phosphorylation in Daudi cells, but not STAT1 or STAT3 (Muller, et al. 2008).

 

Nuclear extracts were prepared from untreated YT cells or cells treated with recombinant IL-2 (2 nM) for 30 min at 37°C. EMSA was performed using glycerol-containing 5% polyacrylamide gels (29:1) containing 0.5× Tris-borate-EDTA buffer. For supershift assays, nuclear extracts were preincubated for 10 min with antibodies against STAT5. Oligonucleotide sequences from PRRIFV have been used as probes (Maeshima, et al. 2012). Other authors described a supershift ESMA that involved preincubating whole-cell extract with 3 μL of pan-STAT5 antiserum that recognizes both STAT5a and STAT5b. Electrophoresis was carried out at room temperature using 5% or 6% polyacrylamide gels (Heidt, et al. 2010).

References

Event: 1718: Suppression of IL-4 production

Short Name: Suppression of IL-4 production

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

In one study, only 1% of CD4 T cells from STAT5a-/- mice primed with soluble anti-CD3 and anti-CD28 with IL-2 produced IL-4, whereas 10.5% of control C57BL/6 CD4 T cells produced IL-4 (Cote-Sierra, et al. 2004).

Cells from STAT5A-deficient mice or cells treated with phospho-STAT5 peptide are defective in Th2 differentiation. STAT5A single-deficient mice showed impaired Th2 differentiation. Reconstituting STAT5A by retroviral infection restored the capacity of cells to induce IL-4 (Kagami, et al. 2001). 

IL-2 directly activates STAT5A and STAT5B. T cells from mice deficient in either STAT5A or STAT5B did not show a dramatic change in T cell proliferation, but cells from mice in which both had been knocked out proliferated poorly in response to IL-4 (Moriggl, et al. 1999).

Key Event Description

IL-4 is a mammalian protein found in Homo sapiens. IL-4 is pivotal in shaping the nature of immune responses. Upon activation, naïve peripheral CD4+ T cells begin to synthesize and secrete cytokines. Type 2 helper cells (Th2 cells) produce IL-4, IL-5, IL-6, and IL-13. IL-4 is a 15-kD polypeptide with pleiotropic effects on many cell types. In T cells, binding of IL-4 to its receptor induces proliferation and differentiation into Th2 cells. Th2 cells assist B cells in promoting class switching from IgM to IgG1 and IgE (Choi and Reiser 1998).

STAT5 phosphorylation facilitates the dimerization of STAT5, transport to the nucleus, and gene regulation (Levy and Darnell 2002). DNaseI hypersensitivity sites II (HSⅡ) and III (HSⅢ) in intron 2 have been identified in several regions of the Il4/Il13 locus. STAT5A binding to sites near HSⅡ and HSⅢ could provide a mechanism through which STAT5A mediates IL-4 gene accessibility and participates in the induction of IL-4 production (Zhu, et al. 2003). The CD3 antibody-induced phosphorylation of STAT5 can be downregulated by tofacitinib, suggesting that JAK3 inhibition by tofacitinib can downregulate STAT5-dependent cytokine signaling. Tofacitinib was shown to abrogate anti-CD3-induced STAT5 activation in CD4+ T cells and inhibit IL-4 production from CD4+ T cells (Migita, et al. 2011).

How it is Measured or Detected

In one study, CD4+ T cells were stimulated with CD3 monoclonal antibodies in the presence or absence of tofacitinib (CP-690550) for 48 h. Supernatants were collected and the levels of IL-4 production were measured by ELISA (Migita, et al. 2011). The authors also extract total RNA after 8 h or 24 h of stimulation and measured IL-4 mRNA expression was measured by real-time PCR (Migita, et al. 2011).

 

In another study, flow cytometry analysis involving intracellular staining was used to measure cytosolic IL-4 content in stimulated cells (Zhu, et al. 2001). Relative gene expression levels were determined by quantitative RT-PCR using Taqman Gene Expression primer probe sets and ABI PRISM 7700 or 7900 Taqman systems (Applied Biosystems). The comparative threshold cycle method and internal controls (cyclophillin or β-actin) were used to normalize the expression of target gene (IL-4) (Ghoreschi, et al. 2011).

Cytokine content was quantified in appropriately diluted samples in duplicate using ELISA kits to test matched antibody pairs with biotin-horseradish peroxidase-streptavidin detection and 3,3',5,5'-tetramethylbenzidine substrate. ELISA plates were scanned using the UVmax plate reader (Molecular Devices) using SOFT max software (Dumont, et al. 1998).

References

Choi P, Reiser H. 1998. IL-4: role in disease and regulation of production. Clin Exp Immunol 113:317-319. DOI: 10.1046/j.1365-2249.1998.00690.x.

Cote-Sierra J, Foucras G, Guo L, Chiodetti L, Young HA, Hu-Li J, Zhu J, Paul WE. 2004. Interleukin 2 plays a central role in Th2 differentiation. Proc Natl Acad Sci U S A 101:3880-3885. DOI: 10.1073/pnas.0400339101.

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.

Ghoreschi K, Jesson MI, Li X, Lee JL, Ghosh S, Alsup JW, Warner JD, Tanaka M, Steward-Tharp SM, Gadina M, Thomas CJ, Minnerly JC, Storer CE, LaBranche TP, Radi ZA, Dowty ME, Head RD, Meyer DM, Kishore N, O'Shea JJ. 2011. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol 186:4234-4243. DOI: 10.4049/jimmunol.1003668.

Kagami S, Nakajima H, Suto A, Hirose K, Suzuki K, Morita S, Kato I, Saito Y, Kitamura T, Iwamoto I. 2001. Stat5a regulates T helper cell differentiation by several distinct mechanisms. Blood 97:2358-2365. DOI: 10.1182/blood.v97.8.2358.

Levy DE, Darnell JE, Jr. 2002. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3:651-662. DOI: 10.1038/nrm909.

Migita K, Miyashita T, Izumi Y, Koga T, Komori A, Maeda Y, Jiuchi Y, Aiba Y, Yamasaki S, Kawakami A, Nakamura M, Ishibashi H. 2011. Inhibitory effects of the JAK inhibitor CP690,550 on human CD4(+) T lymphocyte cytokine production. BMC Immunol 12:51. DOI: 10.1186/1471-2172-12-51.

Moriggl R, Topham DJ, Teglund S, Sexl V, McKay C, Wang D, Hoffmeyer A, van Deursen J, Sangster MY, Bunting KD, Grosveld GC, Ihle JN. 1999. Stat5 is required for IL-2-induced cell cycle progression of peripheral T cells. Immunity 10:249-259.

Zhu J, Cote-Sierra J, Guo L, Paul WE. 2003. Stat5 activation plays a critical role in Th2 differentiation. Immunity 19:739-748. DOI: 10.1016/s1074-7613(03)00292-9.

Zhu J, Guo L, Watson CJ, Hu-Li J, Paul WE. 2001. Stat6 is necessary and sufficient for IL-4's role in Th2 differentiation and cell expansion. J Immunol 166:7276-7281. DOI: 10.4049/jimmunol.166.12.7276.

List of Adverse Outcomes in this AOP

Event: 1719: Impairment of T-cell dependent antibody response

Short Name: Impairment, TDAR

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

CNI-induced impairment of TDAR has been demonstrated in rodent studies. In one study, oral administration of FK506 or CsA to mice for 4 days impaired the response of PFC in splenocytes after intravenous immunization with sheep erythrocytes (Kino, et al. 1987). Likewise, oral administration of FK506 to rats over a 4-week period reduced the production of both anti-KLH-IgG and IgM after subcutaneous immunization with KLH (Ulrich, et al. 2004). Other authors described that treatment with CsA at 50 mg/kg BID via oral gavage in cynomolgus monkeys resulted in reduction of serum SRBC-specific IgM and IgG (Gaida, et al. 2015). As for humans, in vitro experiments showed that treatment with FK506 or CsA of PBMCs from blood bank donors suppressed the production of IgM and IgG specific to T cell dependent antigens (Heidt, et al. 2010). In SKW6.4 cells (IL-6 dependent, IgM-secreting, human B cell line) cultures, FK506 or CsA suppressed the production of IgM in the presence of T cell activation (Sakuma, et al. 2001). Considering that FK506 and CsA reduce T cell derived IL-2, these findings strongly suggest that impairment of TDAR following reduced production of IL-2 occurs at least in common among humans, monkeys, and rodents.

 

Yang et al. (2002b) exposed male C57BL/6 mice to a single concentration (0.02%) of PFOA in the diet for 16 days. TDAR was measured after inoculating PFOA-treated mice with horse red blood cells intravenously on day 10; serum levels of horse red blood cell-specific IgM and IgG in response to the immunization were significantly decreased (Yang, et al. 2002).

 

The suppression of TDAR in adult C57BL/6 female mice has been observed in several studies. NOEL of 1.88 mg/kg/d and LOEL of 3.75 mg/kg/d were identified for PFOA administered in drinking water for over 15 days (Dewitt, et al. 2008).

The suppression of TDAR in adrenalectomized or sham-operated C57BL/6N female mice was observed when PFOA was provided in drinking water for 10 days at doses of 0, 3.75, 7.5, or 15 mg/kg/d. TDAR was determined as the primary antibody response to the T cell dependent antigen in SRBCs. The day after exposure ended, SRBCs were introduced intravenously and SRBC-specific IgM was measured 5 days later (DeWitt, et al. 2009).

Key Event Description

The production of antibodies to T cell-dependent antigens is a coordinated process involving B cells, antigen-presenting cells, and T cell derived cytokines. The B cells are stimulated to proliferate and differentiate. The TDAR might be altered if any of these cell populations are affected.

 

IL-2 and IL-4 are produced and secreted by helper T cells. Both are important in the development of TDAR. IL-4 affects maturation and class switching of B cells as well as proliferation. Both events induce and enhance TDAR. IL-2 promotes differentiation of B cells, which stimulates differentiation of activated T cells to Th2 cells. The suppressed production of IL-2 and IL-4 impairs TDAR (Justiz Vaillant and Qurie 2020).

 

A mutant form of human IL-4, in which the tyrosine residue at position 124 is replaced by aspartic acid (hIL-4Y124D), was reported to specifically block IL-4 and IL-13-induced proliferation of B cells. In addition, hIL-4Y124D also strongly inhibited both IL-4- or IL-13-induced IgG4 and IgE synthesis in cultures of PBMCs, or highly purified sIgD+ B cells cultured in the presence of anti-CD40 monoclonal antibodies. IL-4 may be necessary to produce antibodies and to proliferate in B cells. The mutation of IL-4 may impair TDAR (Aversa, et al. 1993).

 

IL-4 stimulates B cells to proliferate, switch immunoglobulin classes, and differentiate to plasma and memory cells. Suppressing the production of these B cell related cytokines appears to impair TDAR, as evident from the results of FK506 treatment (Heidt, et al. 2010).

STAT5 is able to inhibit peroxisome proliferator activated receptor (PPAR)-regulated gene transcription. Conversely, ligand-activated PPAR can inhibit STAT5-regulated transcription. As a peroxisome proliferator, perfluorooctanoic acid (PFOA) induces PPARs. The suppression of TDAR has been observed with a no observable effect level (NOEL) of 1.88 mg/kg/d and lowest observed adverse effect level (LOEL) of 3.75 mg/kg/d for PFOA administered in drinking water over 15 days (Dewitt, et al. 2008). The increase in PPAR expression induced by PFOA may inhibit STAT5-regulated transcription, which is important for IL-4 production in TDAR.

How it is Measured or Detected

TDAR can be examined in vivo and in vitro. In vivo studies of antigen-specific antibodies are usually performed by measuring serum antibody levels with ELISA (Onda, et al. 2014) or with a plaque-forming cell (PFC) assay.

To assess keyhole limpet hemocyanin (KLH) antigen-specific T cell proliferation, 1 × 10⁵ CD4+ T cells were co-cultured with 2 × 10⁵ autologous PBMCs in 96-well plates in the presence of KLH. Cells were cultured for 5 or 7 days before being pulsed with 0.5 μCi ³[H]-thymidine (PerkinElmer) for 18 h. The cells were harvested using a 96-well cell FilterMate harvester. ³[H]-thymidine incorporation in CD4+ T cell response to biopharmaceuticals was measured by liquid scintillation counting using a TopCount NXT (Schultz, et al. 2017).

 

In another in vivo study, rats were repeatedly administered FK506 orally for 4 weeks and immunized with KLH. Rat serum was examined for T cell dependent, antigen-specific IgM and IgG levels by ELISA (Ulrich, et al. 2004).

 

Other authors repeatedly administered CNIs, including FK506 and CsA, to mice orally for 4 days and immunized with sheep red blood cells (SRBCs). Spleen cells were examined using a PFC assay (Kino, et al. 1987). Antigen-specific plaque-forming splenocytes were reduced at doses of 3.2, 10, 32, and 100 mg/kg of FK506 or 32 and 100 mg/kg CsA.

 

In another study, cynomolgus monkeys received 50 mg/kg CsA twice a day via oral gavage (10 h apart) for 23 days and were immunized with SRBCs. Serum was examined for anti-SRBC IgM and IgG levels using an ELISA specific for SRBC antigen (Gaida, et al. 2015).

 

In the final in vivo study cited here, mice were exposed to a single pharyngeal aspiration of 1,2:5,6-Dibenzanthracene, after which the supernatants of splenocytes were cultured for 24 h in the presence of lipopolysaccharide and assayed using a mouse IgM or IgG matched pairs antibody kit (Smith, et al. 2010).

 

For in vitro studies, total IgM and IgG levels in culture supernatants are often measured after polyclonal T cell activation rather than after antigen stimulation in immune cell cultures.

 

In one study, T and B cells isolated from human PBMCs were co-cultured with CNIs for 9 days in the presence of polyclonal T cell stimulation. The supernatants were examined for IgM and IgG levels by ELISA. Treatment with FK506 or CsA reduced the levels of IgM and IgG at concentrations of 0.3 and 1.0 ng/mL (0.37 and 1.24 nM) or 50 and 100 ng/mL (41.6 and 83.2 nM), respectively (Heidt, et al. 2010).

In another study, SKW6.4 IL-6-dependent IgM-secreting human B cells were cultured for 4 days with anti-CD3/CD28 antibody-stimulated PBMC culture supernatant. IgM produced in the culture supernatants was measured by ELISA. FK506 or CsA reduced the levels of IgM at concentrations of 0.01 to 100 ng/mL or 0.1 to 1000 ng/mL (Sakuma, et al. 2001).

Regulatory Significance of the AO

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 TDAR. Thus, changes in any of these immune cell populations can influence TDAR.

The ICH S8 immunotoxicity testing guideline on pharmaceuticals recommends that TDAR can be evaluated whenever the target cells of immunotoxicity are not clear based on pharmacology and findings in standard toxicity studies. For the assessment of pesticides, the United States Environmental Protection Agency Office of Prevention, Pesticides and Toxic Substances  870.7800 immunotoxicity testing guideline recommends TDAR using SRBC.

Finally, the draft Food and Drug Administration guidance of nonclinical safety evaluation for immunotoxicology recommends the TDAR assay.

References

Aversa G, Punnonen J, Cocks BG, de Waal Malefyt R, Vega F, Jr., Zurawski SM, Zurawski G, de Vries JE. 1993. An interleukin 4 (IL-4) mutant protein inhibits both IL-4 or IL-13-induced human immunoglobulin G4 (IgG4) and IgE synthesis and B cell proliferation: support for a common component shared by IL-4 and IL-13 receptors. J Exp Med 178:2213-2218. DOI: 10.1084/jem.178.6.2213.

Dewitt JC, Copeland CB, Strynar MJ, Luebke RW. 2008. Perfluorooctanoic acid-induced immunomodulation in adult C57BL/6J or C57BL/6N female mice. Environ Health Perspect 116:644-650. DOI: 10.1289/ehp.10896.

DeWitt JC, Shnyra A, Badr MZ, Loveless SE, Hoban D, Frame SR, Cunard R, Anderson SE, Meade BJ, Peden-Adams MM, Luebke RW, Luster MI. 2009. Immunotoxicity of perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator-activated receptor alpha. Crit Rev Toxicol 39:76-94. DOI: 10.1080/10408440802209804.

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.

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.

Justiz Vaillant AA, Qurie A. 2020. Interleukin. In StatPearls: Treasure Island (FL)

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.

Onda M, Ghoreschi K, Steward-Tharp S, Thomas C, O'Shea JJ, Pastan IH, FitzGerald DJ. 2014. Tofacitinib suppresses antibody responses to protein therapeutics in murine hosts. J Immunol 193:48-55. DOI: 10.4049/jimmunol.1400063.

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.

Schultz HS, Reedtz-Runge SL, Backstrom BT, Lamberth K, Pedersen CR, Kvarnhammar AM, consortium A. 2017. Quantitative analysis of the CD4+ T cell response to therapeutic antibodies in healthy donors using a novel T cell:PBMC assay. PLoS One 12:e0178544. DOI: 10.1371/journal.pone.0178544.

Smith DC, Smith MJ, White KL. 2010. Systemic immunosuppression following a single pharyngeal aspiration of 1,2:5,6-dibenzanthracene in female B6C3F1 mice. J Immunotoxicol 7:219-231. DOI: 10.3109/1547691X.2010.487193.

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.

Yang Q, Abedi-Valugerdi M, Xie Y, Zhao XY, Moller G, Nelson BD, DePierre JW. 2002. Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferator, perfluorooctanoic acid. Int Immunopharmacol 2:389-397. DOI: 10.1016/s1567-5769(01)00164-3.

Appendix 2

List of Key Event Relationships in the AOP