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 and takes place via 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 receptors for cytokines require activation step through a Janus-kinase (JAK)/Signal Transducers and Activator of Transcription (STAT) system. When cytokine binds to its specific cytokine receptors, the cytokine receptors form dimers, which more closely resemble the JAK molecules. The JAK then activates to phosphorylate adjacent cytokine receptors. STATs bind to the phosphorylated sites of the receptors and are then phosphorylated by the activated JAK. The phosphorylated STAT is dimerized to be translocated into nucleus and bind to promoter regions of cytokine genes, which starts transcription of cytokine genes in the nucleus.

In mammals, four JAK families of enzymes (JAK1, JAK2, JAK3, TYK2) and seven STATs (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, 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 and have a critical function that evokes itchiness. Because these cytokines also interact with JAK, several JAK-inhibitors have received a lot of attention recently as a therapeutic agent for major inflammatory diseases and pruritic diseases.

This proposed AOP consists of JAK3 inhibition as a MIE, blockade of STAT5 phosphorylation as a KE1, suppression of STAT5 binding to the promoter regions of cytokine genes as a KE2, suppression of IL-4 production as a 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 gamma (γ) chain of the interleukin receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In this proposed AOP, JAK3 selective inhibitors (e.g. PF-06651600 (CAS No：1792180-81-4), RB1) are stressors. Upon the phosphorylation of STAT5 by JAK3, it makes a homo-dimer to translocate to nucleus and induce gene expression such as IL-4. Therefore, JAK3 inhibition leads to the suppression of STAT5 binding to the promoter regions of cytokine genes and the subsequent suppression of IL-4 production. In this way, JAK/STAT regulation plays an important role in TDAR. TDAR is frequently affected under immunosuppressive conditions and is a major endpoint in many preclinical immunotoxicity studies.

Background

Although there are numerous stressors that inhibit JAK3 activity, this AOP is based on immunosuppression caused by recently developed, highly selective JAK3 inhibitors PF-06651600 and RB1, about 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 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 kinase and consists of four members: JAK1, JAK2, JAK3, and Tyk2 (Johnston, et al. 1994). All four members 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 (IBD) symptoms (Miyazaki, et al. 1994). Moreover, abnormal activation of JAK3 was associated with human hematological (Ihle, et al. 1997), indicating that a tight balance of its activity was essential for normal hematopoietic development.

Although JAK1, JAK2, and Tyk2 are each widely expressed, JAK3 is predominantly expressed in hematopoietic cells and is known to associate 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 plays a crucial role in the polarization of naïve T cells to type 2 helper T cells. It plays a major role in the growth and proliferation of many immune cells such NK and T cells (Dhupkar and Gordon 2017). Homozygous mutant mice in which the JAK3 gene had been disrupted were generated by gene targeting. JAK3-deficient mice had profound reductions in thymocytes and severe B cell and T cell lymphopenia similar to severe combined immunodeficiency disease (SCID), and the residual T cells and B cells were functionally deficient. Thus, JAK3 plays a critical role in γ chain signaling and lymphoid development.

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

This proposed AOP involves inhibition of JAK activity leading to suppression of TDAR and is not dependent on life stage, sex, or age. Since JAK3 inhibitors (PF-06651600, RB1) are currently under a phase 2 clinical evaluation to treat rheumatoid arthritis, the AOP appears to be applicable to all life stages. Since JAK3 inhibitor-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 JAK3 activity is considered to occur across a variety of mammalian species. For example, PF-06651600 reduces paw swelling with an unbound EC50 of 169 nM in the rat adjuvant-induced arthritis. Similarly, PF-06651600 significantly reduces disease severity in the experimental autoimmune encephalomyelitis (EAE) mouse model at 30 or 100 mg/kg or prophylactically at 20 and 60 mg/kg. Then, PF-06651600 is going to 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 to identify the JAK family member that was involved in the signaling of a group of cytokines that shared in common the 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 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 the responses to cytokines that use the γ chain (Ihle, et al. 1997). The phenotype of the JAK3 deletion mice was quite striking and consisted of a range of deficiencies which collectively constituted 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 is the dramatic reduction seen 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. In spite of the reduced numbers, the cells that do develop are phenotypically normal. These results are consistent with the hypothesis that activation of JAK3 give it a critical role in the expansion but not the 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). Most frequently rare and monogenic, PIDs present clinically with a broad array of phenotypes including increased susceptibility to infection. One of the most deadly categories of PID is SCID, which is invariably caused by severe developmental and/or functional defects of T lymphocytes, but may also present with variable defects of B and/or Natural Killer (NK) cells. The B6.Cg-Nr1d1tm1Ven/LazJ mouse line harbors a spontaneous mutation in JAK3, which generates an 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 tyrosine residue (Y694 in STAT5) and 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).

 

The following phenotypes are 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), and it was later established that STAT5 was expressed in multiple cell types and activated 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 suggested that it might play a critical role in the development or function (or both) of these cells. Disruption of Stat5a or Stat5b genes alone resulted in relatively modest phenotypes; for example, Stat5a-/- mice had defects in mammary gland development and lactation while Stat5b-/- mice had 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 that acted as functional hypomorphs. These mice too 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 that the STAT5 hypomorph mice retained significant STAT5 function.

Weight of Evidence Summary

Biological Plausibility

T-cell development is mainly regulated by JAK-STAT system, and JAK3 deficiency in T cells is known to induce multiple types of immunosuppression, including TDAR.

JAK3-deficient mice had profound reductions in thymocytes and severe B cell and T cell lymphopenia similar to SCID disease, and 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 the function of epithelial cells. T cells developed in the mutant mice did not respond to either IL-2, IL-4, or IL-7 (Ito, et al. 2017).

Specific JAK3 inhibitor PF-06651600 or RB1, which selectively inhibited JAK3 with an over 100-fold preference over JAK2, JAK1, and TYK2 in the kinase assay, displayed reduced inflammation and associated pathology in collagen-induced-arthritis mice. Importantly, with PF-06651600 or RB1 administration, pro-inflammatory cytokines and JAK3 and STATs phosphorylation decreased in mice, suggesting that the inhibition of JAK3/STAT signaling was closely correlated with induction of multiple types of immunosuppression, including TDAR.

Quantitative Consideration

KER1 (MIE=>KE1)：

Treatment of highly selective JAK3 inhibitors (PF-06651600 or RB1) clearly suppresses the complex formation of STAT5 in the nucleus. IL-2 have been demonstrated to stimulate STAT5 and induce tyrosine phosphorylation of STAT5 (Wakao, et al. 1995). Highly-selective JAK3 inhibitor RB1 inhibited the phosphorylation of STAT5 elicited by IL-2 at IC50 value of 31 nM in the peripheral blood mononuclear cells (PBMCs) of humans. PBMCs were isolated from the buffy coats of healthy volunteers using density gradient centrifugation on Lymphoprep. Cells were cultured in complete RPMI 1640 medium (containing 10% foetal bovine serum, 100 mg/ml streptomycin and 100 U/ml penicillin) plus 10 μg/ml lectin phytohemagglutinin (PHA) for 3 days and then treated with either recombinant human IL-6 (400 ng/ml), recombinant human IL-2 (100 ng/ml), or recombinant human GM-CSF (50 ng/ml) at 37 °C for 20 min. To terminate the stimulation, cells were fixed with Lyse/Fix Buffer and then incubated with 100% methanol for 30 minutes; cells were incubated with anti-pSTAT3 and anti-CD4 Abs, or anti-pSTAT5 and anti-CD4 Abs at 4 °C overnight, washed twice with PBS, and analyzed with a flow cytometer (Ju, et al. 2011).

Fluorescence intensity for phospho-STAT5 in CD3-positive lymphocytes increased 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. Paralleling results in rats, the fluorescence intensity of phospho-STAT5 in CD3-positive lymphocytes increased in human peripheral blood after adding IL-2, but 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 could be activated and phosphorylated by cytokines such as IL-2 and IL-15. Tyrosine phosphorylation of STAT5 is important for dimerization of STAT5 (Wakao, et al. 1995). Dimer of STAT5 has an identical DNA binding specificity and immunoreactivity.

 

KER3：(KE2 =>KE3)：

STAT5 is phosphorylated by the 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) showed that IL-2 activation induced STAT5 dimerization and DNA binding to gamma interferon-activated site (GAS) motif in IL-4 receptor alpha promoter region (John, et al. 1999). Furthermore, EMSAs showed that dexamethazone (Dex, 10^-6 M) inhibited STAT5 DNA binding in mononuclear cells with a dose-dependent fashion, including concentrations of dex (10^-8 M ～ 10^-7 M) (Bianchi, et al. 2000).

 

KER4：(KE3 =>AO)：

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

In the human T-B cell co-culture stimulated with anti-CD3 monoclonal antibody, calcineurin inhibitors (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, et al. 2010).

These results show 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. The JAK family consists of four members: JAK1, JAK2, JAK3, and TYK. In mammals JAK1, JAK2, and TYK2 are ubiquitously expressed. In contrast, the expression of JAK3 is more restricted: it is predominantly expressed in hematopoietic cells and is highly regulated with 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 are 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 severe combined immunodeficiency disease (SCID) in humans, which manifests as a depletion of T, B, and natural killer (NK) cells with no other defects (Darnell 1997, Decker, et al. 1997).

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

Key Event Description

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

To date, PF-06651600 is a selective JAK3 inhibitor, which is undergoing to phase 2 clinical evaluation for rheumatoid arthritis. This compound inhibits JAK3 kinase activity with an IC50 of 33.1 nM (IC50 > 10000 nM) but without activity against JAK1, JAK2, or Tyrosine kinase (TYK)2 (Telliez, et al. 2016, Thorarensen, et al. 2017). RB1 has been also identified as a novel and highly selective JAK3 inhibitor, which blocks in vitro JAK3 kinase and functional activity in various cell types (Pei, et al. 2018). When administered to mice orally, RB1 has been proven to mediate the JAK-Signal Transducer and Activator of Transcription (STAT) (JAK-STAT) pathway and reduce the clinical and microscopic manifestations of paw damage in collagen induced arthritis mice.

How it is Measured or Detected

The enzymatic activities against JAK1, JAK2, JAK3, and TYK2 are tested by 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). PF-06651600 is also a potent JAK3-selective inhibitor which can inhibit the JAK3 kinase activity with an IC50 of 33.1 nM (IC50>10 000 nM) but without 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 IC50 values 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

Signal transducer and activator of transcription (STAT) is a family of proteins that regulate gene transcription upon activation, operated by cytokine signaling and a number of growth factors through the Janus-Associated-Kinase (JAK)/STAT  pathway (Kisseleva, et al. 2002). The STAT5 is a member of the STAT family of proteins, implicated in cell growth and differentiation. 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 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 play a double role as signal transduction molecules in the cytoplasm and as transcription factors upon translocation to the nuclear compartment.

How it is Measured or Detected

Phosphorylation of tyrosine of STAT5 was detected by specific antibodies using several detection systems, including a flow cytometer. Phosphprylated-STAT5 expression was measured in T lymphocytes, and MFIs were reported for each subset (Osinalde, et al. 2017). A cell-permeable nonpeptidic nicotinoyl hydrazone compound that selectively targets the SH2 domain of STAT5 (IC50 = 47 µM against STAT5b SH2 domain EPO peptide binding activity), while exhibiting much less effect towards the SH2 domain of STAT1, STAT3, or Lck (IC50 >500 µM). Shown to block STAT5/STAT5 DNA binding activity in K562 nuclear extract and inhibit IFN-α-stimulated STAT5, but not STAT1 or STAT3, tyrosine phosphorylation in Daudi cells (Muller, et al. 2008).

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

JAK3 selective inhibitor PF-06651600 can inhibit the JAK3 kinase activity 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. Nelson et al., showed the STAT5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors (Nelson, et al. 2011). IL-2 remarkably stimulated STAT5 phosphorylation in peripheral blood mononuclear cells (PBMCs) from Chronic kidney disease (CKD) patients. Pimozide (3 µM) pretreatment 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, B cells from humans, rodents and other mammalian species (Gilmour, et al. 1995).

Key Event Description

Interleukin-2 (IL-2) and other cytokines rapidly activate the Janus-Associated-Kinase (JAK)1 and JAK3 (Beadling, et al. 1994) in peripheral blood lymphocytes (PBLs). The activation of JAK kinases and signal transducer and activator of transcription (STAT) proteins by IL-2 and interferon (IFN)-α does not include the T cell antigen receptor in human T lymphocytes (EMBO J. 13:5606–5615). After activation of JAKs, latent STAT transcription factors make dimeric STAT proteins (Gaffen, et al. 1995). The STAT 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, which can additionally bind to GAS motifs where m is 4 (Darnell 1997, Decker, et al. 1997, Ihle 1996, Leonard and O'Shea 1998).

How it is Measured or Detected

Electrophoretic mobility shift assays (EMSAs) using nuclear extracts and specific oligo including transcription factor binding sites such as cytokine-inducible SH2-containing proteins (CIS) gene promoters is useful for evaluation of DNA binding activity (Johnston, et al. 1995). Liu et al. demonstrated that activated STAT5 binds specific DNA-probe in splenocytes (Liu, et al. 2010). A cell-permeable nonpeptidic nicotinoyl hydrazone compound that selectively targets the SH2 domain of STAT5 (IC50 = 47 µM against STAT5b SH2 domain EPO peptide binding activity), while exhibiting much less effect towards the SH2 domain of STAT1, STAT3, or Lck (IC50 > 500 µM). It shows blockage STAT5/STAT5 DNA binding activity in K562 nuclear extract and inhibit IFN-α-stimulated STAT5, but not STAT1 or STAT3, tyrosine phosphorylation in Daudi cells (Muller, et al. 2008).

Nuclear extracts were prepared from untreated YT cells or cells that had been treated with recombinant IL-2 (2 nM) for 30 min at 37°C. EMSAs were performed by using glycerol-containing 5% polyacrylamide gels (29:1) containing 0.5X Tris-borate-EDTA buffer. For supershifting assays, nuclear extracts were preincubated for 10 min with antibodies to STAT5. Oligonucleotide sequences from PRRIFV were used as probes (Maeshima, et al. 2012). Heidt et al. also showed supershift in EMSA. Supershifting was performed by preincubating the whole-cell extract with 3 μl of a 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

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Taxonomic Applicability Life Stage Applicability Sex Applicability

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, and reconstituting STAT5A by retroviral infection restored the capacity of cells to make 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 have been knocked out proliferate poorly in response to IL-4 (Moriggl, et al. 1999).

Key Event Description

IL-4 is mammalian protein found in Homo sapiens. IL-4 plays a pivotal role in shaping the nature of immune responses. Upon activation, naive 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 provide help for B cells to promote class switching from IgM to IgG1 and IgE (Choi and Reiser 1998).

STAT5 phosphorylation facilitates STAT5 dimerization, transport to the nucleus and gene regulation (Levy and Darnell 2002). DNaseI hypersensitivity sites II (HSⅡ) and III (HSⅢ) in intron 2 were included in several regions of the Il4/Il13 locus. STAT5A binds to the sites near HSⅡ and HSⅢ, which 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 was down regulated by Tofacitinib, suggesting that JAK3 inhibition by Tofacitinib down regulated STAT5-dependent cytokine signaling. Tofacitinib abrogated anti-CD3-induced STAT5 activation in CD4+ T cells and inhibited IL-4 production from CD4+ T cells (Migita, et al. 2011).

How it is Measured or Detected

CD4+ T cells were stimulated with CD3 monoclonal antibodies in the presence or absence of tofacitinib (CP690,550) for 48 hr. Supernatants were collected and the levels of IL-4 production was measured by ELISA (Migita, et al. 2011). Additionally, after 8 hr or 24 hr stimulation, total RNA was extracted and IL-4 mRNA expression was measured by real-time PCR (Migita, et al. 2011).

Flow cytometry analysis (intracellular staining) were used for measurement of 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 expression of target gene (IL-4) (Ghoreschi, et al. 2011).

Quantitation of cytokine content was done on appropriately diluted samples, run 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 in a Molecular Devices UVmax plate reader, 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

CNIs induced impairment of TDAR is demonstrated with rodent studies. That is, 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 four-week period reduced production of both anti-KLH-IgG and IgM after subcutaneous immunization with KLH (Ulrich, et al. 2004). Moreover, Treatment with CsA at 50 mg/kg BID via oral gavage in cynomolgus monkey 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). Also, 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, monkey, 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 (HRBC) intravenously on day 10; serum levels of HRBC-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 and a NOEL of 1.88 mg/kg/d and LOEL of 3.75 mg/kg/d identified for PFOA administered in drinking water over 15 days (Dewitt, et al. 2008).

The suppression of TDAR in adrenalectomised (adx) or sham-operated C57BL/6N female mice has been observed for PFOA administration (0, 3.75, 7.5, or 15 mg/kg/d in drinking water for 10 days). Immune tests: TDAR, ie. primary antibody response to T-cell dependent antigen (SRBC). Day after exposure ended, i.v. SRBC with SRBC-specific IgM measurement 5 days later (DeWitt, et al. 2009).

Key Event Description

Antibody production to T-cell–dependent antigens is established through the coordination of B cells, antigen-presenting cells as well as T-cell–derived cytokines, which stimulate B cells to proliferate and differentiate. T-cell–dependent antibody response (TDAR) might be altered if any of these cell populations is affected.

IL-2 and IL-4 are produced and secreted by helper T cells and play important roles in the development of TDAR. IL-4 affects maturation and class switching of B cells as well as proliferation, both of which induces/enhances TDAR. IL-2 promotes differentiation of B cells through IL-2 stimulates differentiation of the activated T cell into Th2 cell. Therefore, 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), specifically blocks 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 peripheral blood mononuclear cells, or highly purified sIgD+ B cells cultured in the presence of anti-CD40 monoclonal Abs. It was suggested that IL-4 is necessary to produce antibodies and to proliferate in B cells, and the mutation of IL-4 may cause the impairment of TDAR (Aversa, et al. 1993).

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

STAT5 is able to inhibit PPAR (peroxisome proliferator activated receptors)-regulated gene transcription and conversely, ligand-activated PPAR able to inhibit STAT5-regulated transcription. As a peroxisome proliferator, PFOA is able to induce PPARs. The suppression of TDAR has been observed in a NOEL of 1.88 mg/kg/d and LOEL of 3.75 mg/kg/d identified for PFOA administered in drinking water over 15 days (Dewitt, et al. 2008). It was suggested that the increase of PPAR expression induced by PFOA inhibited STAT5-regulated transcription which is important for IL-4 production in TDAR.

How it is Measured or Detected

TDAR could 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 x 10⁵ CD4+ T cells were co-cultured with 2 x 10⁵ autologous peripheral blood mononuclear cell (PBMCs) in 96-well plates in the presence of KLH. Cells were cultured for five or seven days before being pulsed with 0.5 μCi ³[H]-thymidine (Perkin Elmer, Groningen; Netherlands) for 18 hr. 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).

Rats were repeatedly administered FK506 orally for 4 weeks and immunized with KLH, after which the serum was examined for T-cell–dependent, antigen-specific, IgM and IgG levels by ELISA (Ulrich, et al. 2004).

Mice were repeatedly administered calcineurin inhibitors (CNIs) including FK506 and cyclosporin A (CsA) orally for 4 days and immunized with SRBC, after which spleen cells were examined using a PFC assay (Kino, et al. 1987). 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.

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

Mice were exposed a single pharyngeal aspiration of DBA, after which supernatants of splenocytes cultured for 24 h in the presence of LPS 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 supernatant are often measured after polyclonal T-cell activation rather than measuring antigen stimulation in immune cell cultures.

T cells and B cells isolated from human PBMC were co-cultured with a CNIs for 9 days in the presence of polyclonal–T-cell stimulation, after which supernatants were tested for IgM and IgG levels by ELISA. Treatment with FK506 or CsA reduced the levels of IgM and IgG at the 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).

SKW6.4 cells (IL-6-dependent IgM-secreting human B-cell line) were cultured with anti-CD3/CD28 antibody-stimulated PBMC culture supernatant. After culturing for 4 days, IgM produced in the culture supernatants was measured by ELISA. FK506 or CsA reduced the levels of IgM at the concentrations of 0.01 to 100 ng/mL or 0.1 to 1000 ng/mL (Sakuma, et al. 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 of TDAR. Thus, changes in any of these immune cell populations can influence TDAR.

Moreover, 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 for pesticides, US EPA OPPTS 870.7800 immunotoxicity testing guideline recommends TDAR using SRBC.

The draft FDA guidance of nonclinical safety evaluation for immunotoxicology recommends 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