SNAPSHOT

Event: 980: Inhibition, Calcineurin Activity

Short Name: Inhibition, Calcineurin Activity

AOPs Including This Key Event

Stressors

Biological Context

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

CN is broadly distributed in T-cells, B‑cells, and throughout the body. The structure of CnA and CnB is highly conserved from yeasts to humans. Also highly conserved are the amino acid sequences of the catalytic and regulatory domains of CnA isoforms from different organisms (Kincaid. 1996).

As for immunophilins, of which complexes inhibit the CN activity, FKBP is found in a wide variety of organisms, from prokaryotes to multicellular organisms (Siekierka et al. 1989a). Multiple subfamilies of FKBP have been reported, with at least eight types having been found in mammals. FKBP12 is reported to be expressed in B-cells, Langerhans cells and mast cells as well as in T-cells of humans, mice and other mammalian species.

Cyclophilins have been found in mammals, plants, insects, fungi and bacteria. They are structurally conserved throughout evolution and all living beings have PPIase activity (Wang P et al. 2005).

However, inhibition of CN phosphatase activity through immunophilin-CNI complex has been reported at least in rodents and humans.

Event: 979: Interference, nuclear localization of NFAT

Short Name: Interference, nuclear localization of NFAT

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

NFAT expresses in B cells, mast cells, neutrophils, granulocytes, dendritic cells, macrophages, and natural killer cells as well as T cells from humans, rodents and other mammalian species (Rao et al. 1997).

Event: 981: Reduction, NFAT/AP-1 complex formation

Short Name: Reduction, NFAT/AP-1 complex formation

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

CN-NFAT system functionality is common among mammalian species, including humans and rodents. It is also possible that FK506-induced interference with NFAT/AP-1 complex formation at the promoter site of the IL-2 gene is common among mammalian T cells, including those of humans and rodents (Flanagan et al. 1991).

Event: 1202: Suppression, IL-2 and IL-4 production

Short Name: Suppression, IL-2 and IL-4 production

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Taxonomic Applicability

Life Stage Applicability

Sex Applicability

CNIs suppress production of IL-2, IL-3, IL-4, IL-5, IFN-γ, Granulocyte Macrophage colony-stimulating Factor (GM-CSF), and other cytokines, as induced by CD2/CD3 or CD3/CD26 stimulation, in human peripheral blood mononuclear cells (PBMC) (Sakuma et al. 2001a). Also, CNIs (FK506 and CsA) suppress production of IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, Tumor Necrosis Factor-α, IFN-γ, and GM-CSF, as induced by CD3/PMA stimulation, in human PBMC (Dumont et al. 1998).

CNIs (FK506 and CsA) exhibit suppression of IL-2 production induced from mixed lymphocyte reactions in mice and humans (Kino, T et al. 1987a).

Treatment with CsA or 2C1.1 resulted in reduction of IL-2, IL-4, IL-5, and IL-17 cytokine production from PMA/ionomycin stimulation of whole blood in the cynomolgus monkey (Kevin, G. et al. 2014).

These facts indicate that Calcineurin-NFAT system-mediated suppression of cytokines is commonly found in humans, monkey and mice.

Event: 984: Impairment, T-cell dependent antibody response

Short Name: Impairment, T-cell dependent antibody response

AOPs Including This Key Event

Stressors

Biological Context

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 antibodies 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 (Kevin, G. et al. 2014). As for humans, in vitro experiments showed that treatment with FK506 or CsA of peripheral blood mononuclear cells from blood-bank donors suppressed the production of IgM and IgG antibodies specific to T-cell–dependent antigens. (Heidt et al, 2009) Also, in SKW6.4 cells (IL-6–dependent, IgM-secreting, human B-cell line) cultures, FK506 or CsA suppressed the production of IgM antibodies in the presence of T-cell activation. (Sakuma et al. 2001b)  Considering that FKF506 and CsA reduce T cell-derived cytokines including IL-2 and IL-4, these findings strongly suggest that impairment of TDAR following reduced production of such cytokines occurs at least in common among humans monkey and rodents.

Relationship: 1508: Inhibition, Calcineurin Activity leads to Interference, nuclear localization of NFAT

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

CN is broadly distributed throughout the body, and the structure of CnA and CnB is highly conserved from yeasts to humans (Kincaid. 1993).

NFAT expresses in B cells, mast cells, neutrophils, granulocytes, dendritic cells, macrophages, and natural killer cells as well as T cells from humans, rodents and other mammalian species (Rao et al. 1997).

FKBP is found in a wide variety of organisms, from prokaryotes to multicellular organisms (Siekierka et al. 1989). Multiple subfamilies of FKBP have been reported, with at least eight types having been found in mammals. FKBP12 is reported to be expressed in B-cells, Langerhans cells, and mast cells as well as in T-cells of humans, mice and other mammalian species.

Cyclophilins have been found in mammals, plants, insects, fungi and bacteria. They are structurally conserved throughout evolution and all have PPIase activity (Wang P et al. 2005).

These facts indicate that CN and immunophilins are conserved among animals and plants although they show multiple physiological functions.

In addition, CNI/immunophilin complex-induced inhibition of CN phosphatase activity resulting in suppression of immune responses is found in humans and mice.

Key Event Relationship Description

The phosphatase activity of calcineurin (CN) is known to be inhibited by CN inhibitors (CNIs) such as FK506 and cyclosporin A (CsA) through the formation of complexes with immunophilins.

Immunophilins of FK506-binding protein (FKBP) and cyclophilin bind with CNIs FK506 and CsA to form complexes, which inhibit CN activity (Barik. 2006).

While FKBP12, FKBP12.6, FKBP13, and FKBP52 are all part of the FK506-binding FKBP family, FKBP12 has a significant involvement in the mechanism of action for FK506-induced immunosuppression (Siekierka et al. 1989, Kang et al. 2008).

FKBP12 is a 12-kDa protein localized in cytoplasm and has been isolated from Jurkat T-cells as a receptor that binds with the FK506 (Bram et al. 1993). FKBP12 has an FK506-binding domain (FKBD) that comprises 108 amino acids, and is expressed in T cells, B cells, Langerhans cells, and mast cells (Siekierka et al. 1990, Panhans-Gross et al. 2001, Hultsch et al. 1991).

Cyclophilin and FKBP both exhibit peptidyl propyl isomerase (PPIase) activity, but the PPlase activity and the inhibition of activity that they indicate are unrelated to CN regulation.

CN is a heterodimer that comprises a catalytic subunit (CnA) and a Ca-binding regulatory subunit (CnB). CnA handles phosphatase activity as well as calmodulin binding, and CnB regulates intracellular calcium and CnA (Klee et al. 1988, Zhang et al. 1996). CnA is a 59kDa protein with a serine-threonine phosphatase domain.

CNI-immunophilin complexes such as FK506/FKBP complexes and cyclophilin/CsA complexes bind directly to CnA in the cell, causing steric hindrance of substrate binding to CN, which in turn inhibits phosphatase activity of CN (Schreiber and Crabtree 1992, Liu et al. 1993, Bierer et al. 1993, Bram et al. 1993, Rao et al. 1997, Liu et al. 1991).

The nuclear factor of activated T cells (NFAT) is a substrate of CN (Rao et al. 1997).

When T-cell activation takes place, T-cell–receptor-mediated stimulus increases the intracellular concentration of calcium and activates CnB, which subsequently induces CnA phosphatase activation, leading to dephosphorylation of NFAT. In that process, . dephosphorylated SP motifs exposes nuclear localization signal (NLS) and covers nuclear export signal (NES), thereby promoting nuclear localization of NFAT (Matsuda and Koyasu 2000, Zhu and McKeon 1999).

When CN activity is inhibited by the binding of immunophilin complexes, dephosphorylation does not occur in NFAT, thereby interfering with nuclear localization.

Evidence Supporting this KER

Biological Plausibility

The molecular structures and functions of CN and NFAT are evident based on sufficient scientific findings as mentioned above The known mechanisms for inhibition of CN phosphatase activity by FK506, CsA, or other CNIs are initiated by the formation of complexes with their respective immunophilin species. Immunophilins are general classes of proteins that exhibit PPlase activity, but modification of these functions is unrelated to inhibition of CN activity and thus thought to arise in the molecular structure of the complexes (Schreiber and Crabtree 1992, Liu et al. 1993, Bierer et al. 1993, Bram et al. 1993, Rao et al. 1997, Liu et al. 1991).

As mentioned above, inhibition of CN phosphatase activity interferes with the dephosphorylation of NFAT, which leads to the suppression of its nuclear localization.

Empirical Evidence

Much experimental data is available that supports the inhibition of CN activity induced by CNI/immunophilin complexes, which subsequently suppress nuclear localization of NFAT. In addition, CN phosphatase activity is inhibited by 24 hours treatment with CNI of FK506 and CsA with IC50 values of 0.5 and 5 nM, respectively (Fruman et al.1992).

Also, concentration-dependent reduction of in vitro nuclear localization of NFAT was evident using imaging flowcytometry at the maximum concentration of 1 µM with minimal concentration of 0.1nM (Jurkat human T cell line) or 10nM (T cells from whole blood) after 2 hours treatment of tacrolimus (Maguire et al. 2013). Interference with translocation of NFAT to the nucleus is also detected using gel mobility shift assay to test nuclear extracts and cytoplasmic extracts, in which the examined concentration of FK506 was 10ng/mL (Flanagan et al. 1991).

These findings show that dose responses and temporality of MIE and KE1 seem to be the same.

Uncertainties and Inconsistencies

CN and NFAT are expressed in T cells and other immune cells including B cells, DC, and NKT cells and related to cytokine productions from these immune cells. Also, expression of IL-2 receptors (IL-2R) in DCs are lowered due to the inhibition of CN phosphatase activity by CNI treatment. Of these, reduced production of IL-2 and IL-4 from T cells plays a major role in suppression of TDAR due to lower proliferation, differentiation, and class switching of B cells. There have been no reports of CNI-induced reduction of cytokines other than IL-2 and IL-4 or reduced expression of IL-2R resulting in TDAR suppression.

FKBP12, a specific immunophilin that binds with FK506, is also an accessory molecule that binds to IP3 and Ryanodine receptors, both of which occur in Ca channels located on the membrane of the endoplasmic reticulum and participate in the regulation of intracellular Ca concentration. When binding with FK506, FKBP12 leaves these receptors to increase the influx of Ca2+ from the endoplasmic reticulum to cytoplasm, which should increase CN activity. Treatment with FK506, however, suppresses NFAT nuclear localization. In addition, FKBP12-knock out mice show no changes in immune function, including T-cell function. These facts suggest that the inhibition of CN-NFAT systems induced by FK506 treatment results from direct inhibition of CN phosphatase activity by FK506/FKBP12 complexes and not by affecting Ryanodine and IP3 receptors associated with FKBP12.

Quantitative Understanding of the Linkage

Response-response relationship

MIE:

Dose-response analysis of the effects of FK506 on CN phosphatase activity in mast cell-derived KiSVMC4W cells transfected with human FKBP12 cDNA showed that increased expression of FKBP12 resulted in a greater than ten-fold increase in sensitivity to FK506-mediated inhibition, as indicated by an IC50 value of roughly 2 nM with linear inverse dose-response curve after 1 hour incuvation (Fruman et al.1995). Another phosphatase assay showed that FK506 inhibition of CN activity was concentration-dependent reverse sigmoidal and that IC50 values for CN inhibition were approximately 0.5 nM for FK 506 and 5 nM for CsA after 1 hour culture (Fruman et al.1992).

KE1:

Dose-dependent interference with nuclear translocation of NFAT1 was observed with increasing CNI concentrations from 0.1 nM (Jurkat human T cells) up to 1 μM (1000 nM) using imaging flowcytometer. Higher concentrations induced cellular toxicity and resulted in cell death. Dose-dependent interference of nuclear NFAT1 translocation per CN inhibition was also observed in CD4+ T cells from healthy donors, again at maximal concentrations of 1 μM with minimum concentration of 10nM (Maguire et al. 2013).

There have been no literature available to compare directly the dose response of inhibition of CN phosphatase activity with that of nuclear translocation of NFAT; however, the concentration ranges of CNIs for inhibition of CN phosphatase activity and nuclear translocation of NFAT seem to be the same range.

Time-scale

Inhibition of CN phosphatase activity was examined after 1 hour culture of T cells (Fruman et al.1995, Fruman et al.1992), and inhibition of nuclear translocation of NFAT was measured by imaging flowcytometry after 2 hour culture of T cells with CNI (Maguire et al. 2013).

Known modulating factors

At present, no evidence is found.

Known Feedforward/Feedback loops influencing this KER

At present, no evidence is found.

References

1. Barik, S. (2006). Immunophilins: for the love of proteins. Cellular and Molecular Life Sciences 63(24): 2889-900.
2. Bierer, B.E., Holländer, G., Fruman, D. and Burakoff, S.J. (1993). Cyclosporin A and FK506: molecular mechanisms of immunosuppression and probes for transplantation biology. Current opinion in immunology 5 (5): 763-73.
3. Bram, R.J., Hung, D.T., Martin, P.K., Schreiber, S.L. and Crabtree, G.R. (1993). Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcimeurin binding and cellular location. Molecular and cellular biology 13 (8): 4760-9.
4. Flanagan, W.M., Corthésy, B., Bram, R.J. and Crabtree, G.R. (1991). Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352 (6338): 803-7.
5. Fruman, D. A., Klee, C. B., Bierer, B. E. and Burakoff, S. J. (1992). Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proceedings of the National Academy of Sciences of the United States of America. 89(9):3686-90.
6. Fruman, D. A., Bierer, B. E., Benes, J. E., Burakoff, S. J., Austen, K. F. and Katz, H. R. (1995). The complex of FK506-binding protein 12 and FK506 inhibits calcineurin phosphatase activity and IgE activation-induced cytokine transcripts, but not exocytosis, in mouse mast cells. Journal of Immunology.154(4):1846-51.
7. Hultsch, T., Albers, M. W., Schreiber, S.L. and Hohman, R. J. (1991). Immunophilin ligands demonstrate common features of signal transduction leading to exocytosis or transcription. Proceedings of the national academic science of the United States of America. 14: 6229-6233.
8. Kang, C. B., Hong, Y., Dhe-Paganon, S. and Yoon, H. S. (2008). FKBP family proteins: immunophilins with versatile biological functions. Neurosignals. 16: 318-325.
9. Kincaid, R .L. (1993). Calmodulin-dependent protein phosphatases from microorganisms to man. A study in structural conservatism and biological diversity. Adv Second Messenger Phosphoprotein Res. 27:1-23.
10. Klee, C. B., Draetta, G. F. and Hubbard, M. J. (1988). Calcineurin. Advances in enzymology and related areas of molecular biology. 61:149-200.
11. Liu, J., Farmer, J. D. Jr., Lane, W. S., Friedman, J., Weissman, I. and Schreiber, S. L. (1991). Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 66(4): 807-815.
12. Liu, J., Albers, M. W., Wandless, T. J., Luan, S., Alberg, D. G., Belshaw, P. J., Cohen, P., MacKintosh, C., Klee, C. B. and Schreiber, S.L.. (1992). Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry. 31(16):3896-901.
13. Liu, J. (1993). FK506 and cyclosporin, molecular probes for studying intracellular signal transduction. Immunology today. 14(6): 290-305.
14. Maguire O, Tornatore KM, O'Loughlin KL, Venuto RC and Minderman H. (2013) Nuclear translocation of nuclear factor of activated T cells (NFAT) as a quantitative pharmacodynamic parameter for tacrolimus. Cytometry A. 83(12):1096-104.
15. Matsuda, S., Koyasu, S. (2000). A second target of cyclosporin A and FK506. Tanpakushitsu kakusan koso. 45(11): 1823-1831.
16. Panhans-Gross, A., Novak, N., Kraft, S. and Bieber, T. (2001). Human epidermal Langerhans' cells are targets for the immunosuppressive macrolide tacrolimus (FK506). Journal of Allergy and Clinical Immunology 107(2): 345-52.
17. Rao, A., Luo, C. and Hogan, PG. (1997). Transcription factors of the NFAT family: regulation and function. Annual Review of Immunology 15: 707-47.
18. Schreiber, SL. and Crabtree, GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunology Today 13(4): 136-42. >
19. Siekierka, JJ., Hung, SH., Poe, M., Lin, CS. and Sigal, NH. (1989). A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature 341(6244): 755-57.
20. Siekierka, JJ., Wiederrecht, G., Greulich, H., Boulton, D., Hung, SH., Cryan, J., Hodges, PJ. and Sigal, NH. (1990). The cytosolic-binding protein for the immunosuppressant FK-506 is both a ubiquitous and highly conserved peptidyl-prolyl cis-trans isomerase. Journal of Biological Chemistry 265(34): 21011-5.
21. Wang, P. and Heitman, J. (2005) The cyclophilins. Genome Biology 6 (7):226.
22. Zhang, B.W., Zimmer, G., Chen, J., Ladd, D., Li, E., Alt, F.W., Wiederrecht, G., Cryan, J., O'Neill, E.A., Seidman, C.E., Abbas, A.K. and Seidman, J.G. (1996). T cell responses in calcineurin A alpha-deficient mice. Journal of experimental medicine 183(2): 413-20.
23. Zhu, J. and McKeon, F. (1999). NF-AT activation requires suppression of Crm1-dependent export by calcineurin. Nature. 398(6724): 256-60.

Relationship: 1017: Interference, nuclear localization of NFAT leads to Reduction, NFAT/AP-1 complex formation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

NFAT expresses in B cells, mast cells, neutrophils, granulocytes, dendritic cells, macrophages, and natural killer cells as well as T cells from humans, rodents, and other mammalian species (Rao et al. 1997).

CN-NFAT system functionality is common among mammalian species, including humans and rodents. It is also possible that FK506-induced interference with NFAT/AP-1 complex formation at the promoter site of the IL-2 gene is common among mammalian T cells, including those of humans and rodents (Flanagan et al. 1991).

Key Event Relationship Description

Activated (dephosphorylated) nuclear factor of activated T cells (NFAT) is translocated into the nucleus through the molecular changes of exposing nuclear localization signal (NLS) and concomitant masking of nuclear export signal (NES) due to dephosphorylation of the SP motifs of NFAT. (Matsuda and Koyasu 2000, Zhu and McKeon 1999).

Nuclear localization of NFAT results in the NFAT binding with AP 1 at the IL-2 promoter region, (Schreiber and Crabtree 1992; Jain et al. 1992) and induces transcription of IL-2 (Jain et al. 1993). In addition to IL-2, NFAT localized in the nucleus of T cells also binds to the promoter region of the other classes of cytokines including IL-4 and IL-13.

Once CN phosphatase activity is inhibited, dephosphorylation of NFAT and subsequent nuclear localization of NFAT decreases, which results in a decrease of NFAT/AP-1 complex formation at the cytokine promoter sites (Rao et al. 1997).

Evidence Supporting this KER

Biological Plausibility

As has been mentioned, NFAT has NLS and NES among and adjacent to the N-terminal region rich in SP motifs, and once the SP region is dephosphorylated, the NLS is exposed and the NES is covered, which leads to translocation of NFAT into the nucleus (Matsuda and Koyasu 2000).

It is well known from the experiments using CN inhibitors (CNIs) that interference with the nuclear localization of NFAT in T cells leads to a reduction in the formation of NFAT/AP-1 complexes, thereby suppressing transcription of IL-2, IL-4, and a number of other cytokines (Maguire et al. 2013, Jain et al. 1992, Jain et al. 1993).

In contrast to T cells, B-cell receptor-mediated increases in intracellular concentration of calcium in B cells leads to NFAT nuclear localization, thereby producing some classes of cytokines in the same manner as T-cells (Bhattacharyya et al.2011). However, there has been no report of any evidence that CNI acts directly on B cells to effect antibody production.

Expression of IL-2 receptors in dendritic cells and NKT cells is also reported to be regulated by this CN-NFAT system (Panhans-Gross A et al. 2001; Kim et al. 2010), but there is no report showing that CNIs suppress TDAR through the changes in IL-2R expression in these cells.

Empirical Evidence

The relationship of the interference of nuclear localization of NFAT leading to reduced NFAT/AP-1 complex formation bound at the promoter sites of cytokine genes in the presence of CNIs is well known as mentioned above.

Imaging flowcytometry revealed that concentration-dependent reduction of in vitro nuclear localization of NFAT was evident at the maximum concentration of 1 µM with minimal concentration of 0.1nM (Jurkat human T cell line) or 10nM (CD4⁺T cells from whole blood) after 2 hours treatment of tacrolimus (Maguire et al. 2013).

The experiment of gel mobility shift assay using Ar-5 human T cells stimulated with cross-linked anti-CD3 antibody showed that NFAT/AP-1 (cFos and Jun) complexes were found only in the nuclear extract with preexisting NFAT in the cytoplasm after T cell stimulation and that the NFAT/AP-1 complexes in the nucleus decreased after 2 hours treatment with CsA at 1µM (Jain et al. 1992). Decreased NFAT translocated to the nucleus, induced by FK506 at 100ng/mL (124nM) or CsA at 500ng/mL (416nM) after 2 hours treatment, hinders the formation of the functional NFAT/AP-1 complexes necessary to binding at the site of IL-2 promoters (Flanagan et al. 1991) NFAT/AP-1 complex formation was also reported to be inhibited by CNI (Rao et al. 1997).

Quantitative understanding of NFAT/AP-1 complex formation in the nucleus is insufficient although nuclear NFAT/AP-1 complex formation was shown to be inhibited with FK506 at the concentration within the concentration range of FK506 for the inhibition of nuclear translocation of NFAT.

Uncertainties and Inconsistencies

Nothing especially

Quantitative Understanding of the Linkage

Response-response relationship

The relationship of the interference of nuclear localization of NFAT leading to reduced NFAT/AP-1 complex formation bound at the promoter sites of cytokine genes in the presence of CNIs is well known as mentioned above.

KE1:

Dose-dependent interference with nuclear translocation of NFAT1 was observed with increasing FK506 concentrations from 0.01nM (Jarkat T cells) up to 1 μM (1000 nM). Higher concentrations induced cellular toxicity and resulted in cell death. Dose-dependent interference of nuclear NFAT1 translocation per CN inhibition was also observed in CD4+ T cells from healthy donors, again from 10nM to maximal concentrations of 1 μM (Maguire et al. 2013). Both parameters were measured after 2 hour culture of T cells with FK506.

KE2:

Reduction in generation of NFAT/AP-1 complexes can be detected using a gel shift assay (Rao et al. 1997, Jain et al. 1992, Jain et al. 1993).

Decreased NFAT translocated to the nucleus, induced by FK506 at 100ng/mL (124nM) or CsA at 500ng/mL (416nM) after 2 hours treatment, hinders the formation of the functional NFAT/AP-1 complexes necessary to binding at the site of IL-2 promoters (Flanagan et al. 1991). As mentioned above, the gel mobility shift assay also showed that NFAT/AP-1 complexes were formed only in the nucleus after T cell activation with unchanged preexisting NFAT in the cytoplasm and that treatment of T cells with 1µM FK506 led to decease the levels of NFAT/AP-1 complex (Jain et al. 1992).

These findings suggest that nuclear translocation of NFAT after T cell stimulation is strongly related to the complex formation with AP-1 in the nucleus, and FK506 was shown to inhibit NFAT/AP-1 complex formation in the nucleus at the concentrations within the concentration range of FK506 for suppressing nuclear translocation of NFAT (Maguire et al. 2013).

Time-scale

Nuclear translocation of NFAT was shown to be inhibited in vitro using imaging flowcytometry after 2 hours culture of T cells with FK506 (Maguire et al. 2013), and gel mobility shift assay revealed the inhibition of nuclear translocation of NFAT and following complex formation with AP-1 within the nucleus after 2 hours culture of T cells with FK506 (Jain et al. 1992, Flanagan et al. 1991).

Known modulating factors

At present, no evidence is found.

Known Feedforward/Feedback loops influencing this KER

At present, no evidence is found.

References

1. Bhattacharyya, S., Deb, J., Patra, A.K., Thuy Pham, D.A., Chen, W., Vaeth, M., Berberich-Siebelt, F., Klein-Hessling, S., Lamperti, E.D., Reifenberg, K., Jellusova, J., Schweizer, A., Nitschke, L., Leich, E., Rosenwald, A., Brunner, C., Engelmann, S., Bommhardt, U., Avots, A., Müller, M.R., Kondo, E. and Serfling, E. (2011). NFATc1 affects mouse splenic B cell function by controlling the calcineurin-NFAT signaling network. The Journal of experimental medicine 208 (4): 823-39.
2. Flanagan, W.M., Corthésy, B., Bram, R.J. and Crabtree, G.R. (1991). Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352 (6338): 803-7.
3. Jain, J., McCaffrey, P. G., Valge-Archer, V. E. and Rao, A. (1992). Nuclear factor of activated T cells contains Fos and Jun. Nature. 356(6372): 801-4.
4. Jain, J., Miner, Z. and Rao, A. (1993). Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells. Journal of immunology. 151(2): 837-48.
5. Kim, T., Kim, N. and Kang, H. J. (2010). FK506 causes cellular and functional defects in human natural killer cells. Journal of leukocyte biology. 88:1089-1097.
6. Maguire O, Tornatore KM, O'Loughlin KL, Venuto RC and Minderman H. (2013) Nuclear translocation of nuclear factor of activated T cells (NFAT) as a quantitative pharmacodynamic parameter for tacrolimus. Cytometry A. 83(12):1096-104.
7. Matsuda, S., Koyasu, S. (2000). A second target of cyclosporin A and FK506. Tanpakushitsu kakusan koso. 45(11): 1823-31.
8. Panhans-Gross, A., Novak, N., Kraft, S., and Bieber, T. (2001). Human epidermal Langerhans’ cells are targets for the immunosuppressive macrolide tacrolimus (FK506). Journal of Allergy and Clinical Immunology 107(2): 345-52.
9. Rao, A., Luo, C., and Hogan, PG. (1997). Transcription factors of the NFAT family: regulation and function. Annual Review of Immunology 15: 707-47.
10. Schreiber, SL., and Crabtree, GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunology Today 13(4): 136-42.
11. Zhu, J. and McKeon, F. (1999). NF-AT activation requires suppression of Crm1-dependent export by calcineurin. Nature. 398(6724): 256-60.

Relationship: 1509: Reduction, NFAT/AP-1 complex formation leads to Suppression, IL-2 and IL-4 production

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

In purified T cell from male C57BL/6J mice, T-5224 (a selective c-Fos/AP-1 inhibitor) inhibits the DNA-binding activity of AP-1, IL-2 production and CD25 (IL-2R) up-regulation (Yoshida et al. 2015).

In splenic lymphocytes and/or CD4+ T cells, ursolic acid suppressed products of NF-kB, NFAT and AP-1, and inhibits secretion of IL-2 and IL-4, mRNA level of IL-2 and CD25 expression (Checker et al. 2012).

NFATp- and NFAT4-deficient mice indicate decreased production of IL-2 (Ranger et al. 1998).

NFAT/AP-1 complex formation in the nucleus was shown using murine and human T cells lines (Jain J et al. 1992). In addition to data on suppression of cytokine production by CNI in rodents, FK506 is reported to inhibit expression of both IL-2 and mRNA in human anti-CD3/PMA-activated cells (Dumont et al. 1998).

Key Event Relationship Description

Localized nuclear factor of activated T cells (NFAT) in the nucleus of T cells binds to form complexes with activator protein-1 (AP-1) at the Interleukin (IL)-2 promoter region (Schreiber and Crabtree 1992; Jain et al. 1992), which induces transcription of IL-2 (Jain et al. 1993). In addition to IL-2, NFAT localized in the nucleus of T cells also binds to the promoter region of the other classes of cytokines including IL-4 and IL-13.

For IL-2, NFAT proteins are necessary for IL-2 gene expression and cooperation of NFAT with AP-1 is required for IL-2 gene transcription. For IL-4, At least five different NFAT sites have been described in the IL-4 promoter with at least three of them being composite sites binding NFAT and AP-1 (Macián et al. 2001).

Decreased formation of NFAT/AP-1 complex at the promoter region of IL-2 genes in the nucleus of T cells following lowed nuclear localization of NFAT by calcineurin inhibitor (CNI) treatment reduces the transcription of IL-2 (Dumont et al. 1998). Production in T cells of IL-4 and other classes of cytokines is also suppressed in the same manner as IL-2 (Dumont et al. 1998).

Evidence Supporting this KER

Biological Plausibility

T-5224, a selective c-Fos/AP-1 inhibitor, inhibits the DNA-binding activity of AP-1 in primary murine T cells. T-5224 also inhibits CD25 (one of IL-2 receptors) up-regulation, IL-2 production, and c-Fos DNA-binding activity in mice (Yoshida et al. 2015).

Dexamethasone represses the IL-2 mRNA induction. glucocorticoid-induced leucine zipper (GILZ) is one of the most prominent glucocorticoid-induced genes, and inhibited the induction of the NFAT reporter and interferes with the AP-1 component of the NFAT/AP-1 complex. GILZ also inhibits the IL-2 promoter (Mittelstadt et al. 2001).

Ursolic acid suppressed activation of three immunoregulatory transcription factors NF-kB, NFAT and AP-1. Treatment of lymphocytes and CD4+ T cells with ursolic acid inhibited secretion of IL-2 and IL-4 cytokines. Treatment of CD4+ T cells with ursolic acid suppressed mRNA level of IL-2. Treatment of lymphocytes with ursolic acid inhibited the upregulation of CD25 expression on T cells (Checker et al. 2012).

NFATp- and NFAT4-deficient mice indicate decreased production of Th1 cytokine including IL-2 (Ranger et al. 1998).

It is generally accepted that NFAT, translocated to the nucleus after T-cell stimulation, binds with AP-1 to the promoter regions of the cytokine genes to mount transcription, which follows production of these T-cell–derived cytokines. Of these cytokines, IL-2 and IL-4 promote proliferation, maturation, and class-switching of B cells to enhance TDAR.

There is also sufficient evidence to support the hypothesis that CNI-induced decreases in T-cell–derived cytokine production is mediated through suppressed nuclear localization of NFAT, with a resultant decrease in the amount of NFAT/AP-1 complex binding to the promoter regions of T-cell-derived cytokines.

When stimulated with ovalbumin, calcineurin A (CnA)-knockout (KO) mice produce less Interferon (IFN)- γ, IL-2, and IL-4 than wild-type mice. However, primary antibody response in CnA-KO mice is normal in response to trinitrophenol-ovalbumin (Zhang et al. 1996).

The following phenotypes are observed in NFAT-KO mice: moderate hyperproliferation with splenomegaly; moderately enhanced B- and T-cell responses, with bias towards Th2- cell responses; decreased IFN-γ production in response to TCR ligation; reduced proliferative responses by T cells; impaired repopulation of the thymus and lymphoid organs; impaired Th2-cell responses and IL-4 production; grossly impaired T-cell effector functions, with profound defects in cytokine production and cytolytic activity; B-cell hyperactivity; impaired development of CD4 and CD8 single-positive cells, with increased apoptosis of double-positive thymocytes; and mild hyperactivation of peripheral T cells (Macian, 2005).

Therefore, the study of NFAT-KO mice shows that NFAT is involved in a wide range of immune responses, and some of these phenomenon are known to be regulated by calcineurin (CN). Suppression of T-cell-derived cytokines is noted both in CnA-KO and NFAT-KO mice, which indicates that the production of T-cell derived cytokines such as IL-2 and IL-4 is regulated by the CN-NFAT system.

FK506-FKBP12 complex decreased CN phosphatase activity, which leads to inhibit translocation of NFAT to the nucleus. Because NF-ATp is an essential transcription factor regulating the IL-2 gene, FK506 ultimately blocks the T-cell response by inhibiting IL-2 transcription (Panhans-Gross A et al. 2001). FK506 inhibited IL-2 mRNA expression in anti-CD3/phorbol 12-myristate-13-acetate (PMA)-activated cells (Dumont et al. 1998).

These facts indicate that although NFAT is widely involved in the function of T cells, the effect of CNIs is to suppress production of some classes of T‑cell–derived cytokines through reducing the formation of NFAT/AP-1 complexes induced by inhibition of CN phosphatase activity.

Empirical Evidence

Empirical support of Reduction, NFAT/AP-1 complex formation leading to Suppression, IL-2 and IL-4 production is strong.

Rationale：

• In purified T cell from male C57BL/6J mice, T-5224 (a selective c-Fos/AP-1 inhibitor) inhibits the DNA-binding activity of AP-1 and CD25 (one of IL-2 receptors) up-regulation at 80 μg/mL, and IL-2 production in a dose-dependent manner from 40 to 80 μg/mL (Yoshida et al. 2015).
• In splenic lymphocytes stimulated with concanavalin A for 24 h in C57BL/6 mice, ursolic acid suppressed products of NF-kB, NFAT and AP-1 at 5 μM for 4 h. Secretion of IL-2 and IL-4 was inhibited in lymphocytes stimulated with concanavalin A for 24 h at concentrations of 0.5, 1 and 5 μM of ursolic acid, and lymphocytes and CD4+ T cells stimulated with anti-CD3/anti-CD28 mAb for 24 h at concentration of 5 μM of ursolic acid. In CD4+ T cells stimulated with anti-CD3/anti-CD28 mAb for 24 h, ursolic acid suppressed mRNA level of IL-2 at 5 μM for 4 h. In lymphocytes stimulated with concanavalin A for 24 h, ursolic acid inhibited CD25 expression at 5 μM for 4 h (Checker et al. 2012).
• In NFATp- and NFAT4-deficient mice, cultured splenocytes bound anti-CD3 for 48 h indicates decreased production of Th1 cytokine including IL-2 (Ranger et al. 1998).

It is well established that inhibition of NFAT/AP-1 complex formation at the promoter sites reduces the production of T-cell–derived cytokines including IL-2 and IL-4, which are mainly involved in T-cell–dependent antibody response.

• NFAT/AP-1 complex formation is inhibited by CNI shown by gel shit mobility assay using human T cell line or CD4+ T cells from heathy donners after 2 hours treatment with cyclosporin A (CsA) at 1µM.. Preceding NFAT nuclear localization after T cell activation is suppressed with FK506 at the dose range of 0.01nM (Jarkat T cells) or 10nM (CD4+ T cells) to 1µM (Maguire et al. 2013), and NFAT nuclear localization and NFAT/AP-1 complex formation is shown to be strongly related (Jain et al. 1992, Jain et al. 1993).
• In CD3/PMA-activated human T cells, FK506 suppressed production of IL-2, IL-4, and IFN-γ at the concentrations of 1.2 to 12.5 nM after 22 to 24 hours culture as well as inhibited expression of IL-2, IL-4, and IFN-γ mRNA in a dose-dependent (10 nM) manner after 3 day culture (Dumont et al. 1998).
• Treatment with CsA completely eliminated detectable IL-2 release from 3A9 T cells co-cultured with antigen-bearing Ch27 B cells with an IC25 and IC50 for IL-2 production of 1.19 nM and 1.99 nM. Treatment with other immunosuppressant compounds (dexamethasone, azathioprine, methotrexate, benzo(a)pyrene and urethane) also resulted in decreased IL-2 release from stimulated 3A9 T cells at non-cytotoxic concentrations. Urethane, a weakly immunosuppressive chemical, was least potent in the assay, with an IC25 and IC50 for IL-2 secretion of 4.24 mM and 13.26 mM (D.M. Lehmann. et al. 2018).
• In female B6C3F1 mice, 1,2:5,6-dibenzanthracene exposure reduced production of IL-2 in spleen cell culture supernatants after in vitro stimulation with Concanavalin A or lipopolysaccharide (Donna, C. et al. 2010).
• Treatment with CsA at 50 mg/kg BID via oral gavage or 2C1.1 (a fully human anti-ORAI1 monoclonal antibody) at 25 mg/kg single IV resulted in reduction of IL-2, IL-4, IL-5, and IL-17 cytokine production from PMA/ionomycin stimulation of whole blood in the cynomolgus monkey (Kevin, G. et al. 2014).
• In male CD-1 mice, chronic psychosocial stress (types of social outcome occurred: residents becoming subordinates) reduced IL-2 release in response to keyhole limpet hemocyanine (Alessandro, B. et al. 2003).

Reduced nuclear translocation of NFAT followed by NFAT/AP-1 complex formation and suppression of IL-2/IL-4 productions are shown to occur under similar dose ranges and treatment duration.

Uncertainties and Inconsistencies

CNIs are reported to suppress IL-17 release from Th17 cells and development of Th17 cells from naïve T cells (Tsuda et al, 2012).  On the other hand, Yadav reported that Th17 cells increased and Treg cells decreased in number and that the levels of RORC mRNA increased and those of FOXP3 decreased in renal transplanted patients with chronic calcineurin inhibitor toxicity (Yadav, 2015). From these findings, CNIs suppress the functions of Th17 and Treg cells，which enhance Th17 cells to develop chronic CNI toxicity.

FK506 suppresses expression of IL-2 receptor (IL-2R: CD25) and costimulatory molecules CD80 (B7.1)/CD40 in Langerhans cells (Panhans-Gross A et al. 2001).

In human NK cells, FK506 suppresses IL-2 responsive proliferation and cytokine production as well as lowers cytotoxicity directed toward K562 tumor cells (Kim et al. 2010). FK506 suppresses IL-2 production of NKT cell line DN32.D3 induced by stimulus from PMA/calcium -ionophore (van Dieren et al. 2010).

The relationship between these FK506-induced mechanisms and NFAT and contribution of those to TDAR are unclear.

In addition to NFAT/AP-1 complexes, NFAT forms complexes at the site of IL-3 and IL-4 enhancers with avian musculoaponeurotic fibrosarcoma oncogene homolog, early growth response 1, early growth response 4, interferon-regulatory factor 4, octamer-binding transcription factor, and other transcriptional partners to induce transcription of a variety of cytokines (Macian 2005). The production of cytokine induced by these transcriptional partners also suppressed by CNI; however, contribution of these additional transcription factors to TDAR is also unclear.

Quantitative Understanding of the Linkage

Response-response relationship

In purified T cells from male C57BL/6J mice, T-5224 (a selective c-Fos/AP-1 inhibitor) inhibits the DNA-binding activity of AP-1 at 80 μg/mL. On the other hand, T-5224 inhibits IL-2 production in a dose-dependent manner from 40, 60 and 80 μg/mL after 48 hours culture. T-5224 also inhibits CD25 (IL-2R) up-regulation at 80 μg/mL (Yoshida et al. 2015).

In splenic lymphocytes stimulated with concanavalin A for 24 h in C57BL/6 mice, ursolic acid suppressed products of NF-kB, NFAT and AP-1 at 5 μM. In lymphocytes stimulated with concanavalin A for 24 h, ursolic acid inhibits secretion of IL-2 and IL-4 at 0.5, 1 and 5 μM. In lymphocytes and CD4+ T cells stimulated with anti-CD3/anti-CD28 mAb for 24 h, ursolic acid also inhibits secretion of IL-2 and IL-4 at 5 μM. In CD4+ T cells stimulated with anti-CD3/anti-CD28 mAb for 24 h, ursolic acid suppressed mRNA level of IL-2 at 5 μM. In lymphocytes stimulated with concanavalin A for 24 h, ursolic acid inhibited CD25 expression at 5 μM (Checker et al. 2012).

These findings showed that T-5244 and ursolic acid treated for 24 hours inhibit NFAT/AP-1 complex formation at a single concentration each and that these compounds suppress IL-2 and IL-4 production with dose dependent manner including the doses for inhibition of NFAT/AP-1 complex formation.

FK506 suppressed proliferation in human T cells induced by anti-CD3 mAb in the presence of adherent autologous peripheral blood mononuclear cells (mean IC50 = 0.06 nM). FK506 suppressed, in a dose-dependent (1.2 to 12.5 nM) manner after 22-24 hours culture, production of IL-2, IL-4, and IFN-γ by human T cells stimulated with anti-CD3 mAb in the presence of PMA, as well as inhibited, also in a dose-dependent (10 nM) manner, expression of IL-2, IL-4, and IFN-γ mRNA in anti-CD3/PMA- activated cells (Dumont et al. 1998). On the other hand, the quantitative data for the decreased formation of NFAT/AP-1 complexes by CNI is insufficient, although the formation was suppressed by FK506 at the concentration within the range needed for suppressed production of IL2/IL-4 by FK506 after 2 hours culture.

Time-scale

Inhibition of NFAT/AP-1 complex is detected by gel mobility shit assay after 2 hours culture with CNI; however, suppression of IL2/IL-4 could be measured after 22-48 hours in vitro culture.

Known modulating factors

At present, no evidence is found.

Known Feedforward/Feedback loops influencing this KER

At present, no evidence is found.

References

1. Schreiber, SL., and Crabtree, GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunology Today 13(4): 136-42.
2. Jain J., McCaffrey P.G., Valge-Archer V.E., Roa A.(1992). Nuclear factor of activated T cells contains Fos and Jun. Nature 356(6372):801-804.
3. Jain J., Miner Z., Rao A. (1993). Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells. Journal of Immunology 151(2): 837-848.
4. Macián, F., López-Rodríguez, C. and Rao, A. (2001). Partners in transcription: NFAT and AP-1. Oncogene. 20(19): 2476-89.
5. Yoshida, T., Yamashita, K., Watanabe, M., Koshizuka, Y., Kuraya, D., Ogura, M., Asahi, Y., Ono, H., Emoto, S., Mizukami, T., Kobayashi, N., Shibasaki, S., Tomaru, U., Kamachi, H., Matsushita, M., Shiozawa, S., Hirono, S. and Todo, S. (2015). The Impact of c-Fos/Activator Protein-1 Inhibition on Allogeneic Pancreatic Islet Transplantation. Am J Transplant. 15(10): 2565-75.
6. Mittelstadt, PR. and Ashwell, JD. (2001). Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ. J Biol Chem. 276(31):29603-10.
7. Checker, R., Sandur, SK., Sharma, D., Patwardhan, RS., Jayakumar, S., Kohli, V., Sethi, G., Aggarwal, BB. and Sainis, KB. (2012). Potent Anti-Inflammatory Activity of Ursolic Acid, a Triterpenoid Antioxidant, Is Mediated through Suppression of NF-κB, AP-1 and NF-AT PLoS One. 7(2): e31318.
8. Ranger, AM., Oukka, M., Rengarajan, J. and Glimcher, LH. (1998). Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development. Immunity. 9(5):627-35.
9. Dumont, F.J., Staruch, M.J., Fischer, P., DaSilva, C. and 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. Journal of immunology 160 (6): 2579-89.
10. Macian, F. (2005) NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol. 5(6): 472-84.
11. Panhans-Gross, A., Novak, N., Kraft, S., and Bieber, T. (2001). Human epidermal Langerhans’ cells are targets for the immunosuppressive macrolide tacrolimus (FK506). Journal of Allergy and Clinical Immunology 107(2): 345-52.
12. van Dieren, J.M., Lambers, M.E.H., Kuipers, E.J., Samsom, J.N., van der Woude, C.J. and Nieuwenhuis, E.E.S. (2010). Local immune regulation of mucosal inflammation by tacrolimus. Digestive diseases and sciences 55(9): 2514-19.
13. Zhang, BW., Zimmer, G., Chen, J., Ladd, D., Li, E., Alt, FW., Wiederrecht, G., Cryan, J., O'Neill, EA., Seidman, CE., Abbas, AK., Seidman, JG. (1996). T cell responses in calcineurin A alpha-deficient mice. J Exp Med. 183(2): 413-20.
14. Alessandro B, Paola S, Alberto E. Paneraic, Tiziana P,Paola Palanzaa and Stefano P(2003). Chronic psychosocial stress-induced down-regulation of immunity depends upon individual factors Journal of Neuroimmunology 141: 58–64
15. Donna C. S, Matthew J. S and Kimber L. W Jr. (2010) Systemic immunosuppression following a single pharyngeal aspiration of 1,2:5,6-dibenzanthracene in female B6C3F1 mice, Journal of Immunotoxicology, 7:3, 219-231
16. Kevin G, Hossein S, Raju S, Valerie A, Anna K, Ming Z, Fen-Fen L, Hung Q. N, Lei Z, John K. S, Min W and Helen J. M(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, Journal of Immunotoxicology, 12:2, 164-173,
17. D.M. Lehmann, W.C. Williams.(2018) Development and Utilization of a Unique In Vitro Antigen Presentation Co-culture Model for Detection of Immunomodulating Substances. Toxicol In Vitro.53: 20–28.

 

Relationship: 1510: Suppression, IL-2 and IL-4 production leads to Impairment, T-cell dependent antibody response

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

In cynomolgus monkeys, the effects of CsA on production of IL-2 and IL-4, and antigen-specific IgM and IgG in TDAR were demonstrated (Gaida K. 2015).

Suppressed IgE and antigen specific IgG1 productions by the blocking of IL-4 receptor were reported in mice using dupilumab (anti-IL-4/13R antibody) (Sanofi K.K. 2018).

Suppressed antigen specific IgE production by the inhibition of IL-4 production was reported in mice using suplatast tosilate (Taiho Pharmaceutical 2013).

Suppressed antigen specific IgE and IL-4 productions by the inhibition of IL-4 production were reported in human cell culture using suplatast tosilate(Taiho Pharmaceutical 2013).

The effects of FK506 on serum concentration of anti-KLH antibodies IgM and IgG have been demonstrated in rats treated with FK506 for over four weeks and immunized with KLH (Ulrich et al. 2004). The effects of FK506 and CsA on antigen-specific plaque-forming splenocytes have been demonstrated in mice treated with FK506 or CsA for 4 days and immunized with SRBC (Kino et al. 1987b).

The effects of FK506 and CsA on the levels of IgM and IgG in the culture supernatant have been demonstrated in human cells (Heidt et al, 2009, Sakuma et al, 2001).

The effects of FK506 and CsA on production of IL-2 and IL-4 have been demonstrated using mice and human cells (Kino et al. 1987a, Dumont et al. 1998).

These facts suggest that there are no species differences between humans, monkeys and rodents in inhibitions of IL-2 and IL-4 production and TDAR induction.

Key Event Relationship Description

Interleukin (IL)-2 and IL-4 are produced and secreted by helper T cells and play important roles in the development of T-cell dependent antibody response (TDAR), both of which induces/enhances T cell dependent antibody production. IL-4 affects maturation and class switching of B cells as well as proliferation, IL-2 promotes differentiation of B cells through IL-2 receptors and stimulates the activated T cell into T cell called Th2 cell. Therefore, suppressed production of IL-2 and IL-4 impairs T cell dependent antibody production (Alberts et al. 2008).

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

T cell-derived cytokines play important roles in the development of TDAR. Among them, IL-2 promotes proliferation of B cells, and IL-4 affects maturation and class switching of B cells as well as proliferation, both of which induces/enhances T cell dependent antibody production.

Thus, suppressing the production of IL-2, IL-4, and other cytokines in T cells reduces stimulation of B cells including proliferation, activation, and class switching, and leading to impairment of TDAR. Therefore, suppressing the production of these B-cell-related cytokines appears to be the main factor in impairment of TDAR by inhibitors of T-cell–dependent-antibody production.

Evidence Supporting this KER

Biological Plausibility

Cyclosporin A (CsA) is known one of the calcineurin inhibitiors. CsA-treatment is reported to suppresses the productions of IL-2 and IL-4 and result in the reduction of the productions of antigen-specific IgM and IgG in cynomolgus monkeys (Gaida K. 2015).

It is established that IL-2 stimulates B cells to proliferate through the surface IL-2 receptors and that IL-4 stimulates B cells to proliferate, to induce class switch, and to differentiate into plasma and memory cells.

Dupilumab is known as anti-IL-4/13 receptor (IL-4/13R) antibody. Dupilumab (Dupixent) reduces productions of immunoglobulin (Ig) E and antigen specific IgG1 in mice (Sanofi K.K. 2018). It suggests that the blocking of IL-4 signaling by anti-IL-4/13R antibody results in the decrease in T cell dependent antibody production.

Th2 cell produces cytokines including IL-4. Suplatast tosilate (IPD) is known as an inhibitor of the production of IL-4 and IL-5 from Th2 cells and reduces the production of antigen specific IgE in human cell culture and mice (Taiho Pharmaceutical 2013). These findings suggests that the reduction of IL-4 production by the inhibitor of Th2 cell cytokines results in reduced production of IgE and/or IgG1 through inhibitions of maturation, proliferation and class switching of B cells.

IL-2 binds to IL-2 receptor (IL-2R) and acts on T cell. CD25 is one of IL-2R. Basiliximab (Simulect) is known as anti-CD25 antibody. Basiliximab binds to IL-2R and blocks IL-2 signaling. Clinical transplantation study of basiliximab reveals decreases in rejections. On the other hand, basiliximab inhibits the activation of antigen specific T cells (Novartis Pharma 2016). They suggest that the blocking of IL-2 signaling by anti-IL-2R antibody results in decreased rejection through the inhibition of the activation of antigen specific T cell with reduced antibody production.

FK506 and CsA suppress mRNA expression levels of cytokines in T cells including IL-2 and IL-4 that stimulate proliferation of B cells as well as B cell activation and class switching (Heidt et al, 2010).

Several in vivo studies in rodents showed decreased TDAR by the treatment of FK506 (Kino et al. 1987b, Ulrich et al. 2004). In in vitro tests examining antibody production in blood samples obtained from blood-bank donors, peripheral blood mononuclear cells (PBMC) treated with FK506 and CsA suppressed the production of IgM and IgG antibodies to T-cell dependent antigens (Heidt et al, 2009).

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.

However, as for the suppression of humoral immunity induced by the inhibition of calcineurin (CN) phosphatase activity, calcineurin inhibitors (CNIs) do not affect B cells directly but rather indirectly through T cells. That is, FK506 and CsA are capable of inhibiting immunoglobulin production when B cells are cultured with non-pre-activated T cells, but FK506 and CsA fail to inhibit immunoglobulin levels when pre-activated T cells are used to stimulate B cells. Hence, the inhibition of B cell response by FK506 and CsA appears due solely to inhibition of T helper cells (Heidt et al, 2010).

Therefore, it is concluded that decreased amounts of IL-2 and IL-4 secreted from helper T cells is the main factor for suppression of TDAR induced by CN phosphatase inhibition.

Empirical Evidence

Empirical support of the suppression, IL-2 and IL-4 production leads to impairment, T-cell dependent antibody response is strong.

Rationale：

• Cynomolgus monkeys treated wth CsA at 50 mg/kg BID for 24 days suppression of IL-2, IL-4 and sheep red blood cell (SRBC)-specific IgM and IgG (Gaida K. 2015).
• In the allergen-induced pneumonia model in mice, dupilumab (anti-IL-4/13R antibody) reduced productions of IgE and antigen specific IgG1 at 25 mg/kg of twice weekly subcutaneous administration for 4weeks (Sanofi K.K. 2018).
• In mice immunized with dinitrophenyl antigen by i.p. injection, suplatast tosilate (an inhibitor of the production of cytokines on Th2 cell) reduced productions of antigen specific IgE at 10, 20, 50 and 100 mg/kg of oral administration for 5 days (Taiho Pharmaceutical 2013). In human cell culture immunized with Japanese cedar antigen, suplatast tosilate reduced productions of antigen specific IgE at the concentration of 10 μg/mL for 10 days (Taiho Pharmaceutical 2013).
• In the clinical study of renal transplantation, basiliximab decreased incidence of acute rejection at 20 mg/kg (Novartis Pharma 2016). In human T cell culture immunized with PPD, basiliximab reduced activation of antigen specific T cell at the concentration of 300 ng/mL (Novartis Pharma 2016).
• In CD3/phorbol 12-myristate-13-acetate-activated human T cells, FK506 suppressed production of IL-2, IL-4 and Interferon (IFN)-γ at the concentrations of 1.2 to 12.5 nM as well as inhibited expression of IL-2, IL-4 and IFN-γ mRNA at the concentrations of 10 nM. (Dumont et al. 1998).
• FK506 or CsA suppressed production of IL-2 in mouse mixed lymphocyte reaction (MLR) at 0.1 to 10 nM of FK506 and 10 to 100 nM of CsA as well as in human MLR at 0.1 to 10 nM of FK506 and 10 to 100 nM of CsA (Kino et al. 1987a).
• After 9-day culture of B cells and non-pre-activated T cell stimulationwith FK506 or CsA, the levels of IgM and IgG in the culture supernatant were reduced at 0.3 and 1.0 ng/mL (0.37 and 1.24 nM) of FK506 or 50 and 100 ng/mL (41 and 83nM) of CsA (Heidt et al, 2009).
• After 4-day culture of SKW6.4 cells (IL-6-dependent IgM-secreting human B-cell line) and anti-CD3/CD28 stimulated PBMC culture supernatant with FK506 or CsA, the level of IgM in the culture supernatant was reduced at the concentrations of 0.01 to 100 ng/mL (0.01 to 124 nM) of FK506 or 0.1 to 1000 ng/mL (0.08 to 832 nM) of CsA (Sakuma et al, 2001).
• Rats were treated with FK506 for over four weeks and immunized with keyhole limpet hemocyanine (KLH), after which serum concentration of anti-KLH IgM and IgG reduced at the dose levels of 3 mg/kg/day (Ulrich et al. 2004).
• Mice were treated with FK506 or CsA for 4 days, and immunized with sheep red blood cells (SRBC), after which antigen-specific plaque-forming splenocytes reduced at the dose levels of 3.2, 10, 32 and 100 mg/kg of FK506 or 32 and 100 mg/kg of CsA (Kino et al. 1987b).
• 1,2:5,6-dibenzanthracene single adoministration suppressed production of IL-2 and total IgG antibody in mice at the dose levels of 3 and 30 mg/kg(Donna, C. et al. 2010).
• In male CD-1 mice, chronic psychosocial stress (types of social outcome occurred: residents becoming subordinates) for 21 days reduced IL-2 release in response to KLH and decrease in anti-KLH IgG(Alessandro, B. et al. 2003).

In vitro suppression of T-cell–derived cytokines and T-cell-dependent antibody production or antibody production after polyclonal T-cell stimulation showed similar dose responses to CNIs. Time gaps were found, however, between these two KEs, which showed earlier onset of cytokine production and delayed onset of antibody production.

Uncertainties and Inconsistencies

IL-2 affects multiple populations of immune cells expressing IL-2 receptors, while IL-4 mainly acts on B cells. Therefore, reduced production of both IL-2 and IL-4 might certainly induce suppression of TDAR; however, there remains some possibility of additional suppression of other immune functions.

Quantitative Understanding of the Linkage

Response-response relationship

Cynomolgus monkeys treated wth CsA at 50 mg/kg BID showed suppression of IL-2 and IL-4 production and inhibition of SRBC-specific IgM and IgG in TDAR (Gaida K. 2015).

In the blocking of IL-4 receptor in mice by dupilumab (anti-IL-4/13R antibody) at 25 mg/kg of twice weekly subcutaneous administration for 4weeks, IgE production was suppressed to about 1/100 and antigen specific IgG1 production was suppressed to about 1/200 (Sanofi K.K. 2018).

In the inhibition of IL-4 production in mice by suplatast tosilate at 10, 20, 50 and 100 mg/kg of oral administration for 5 days, antigen specific IgE production was suppressed from about 1/10 to 1/100 (Taiho Pharmaceutical 2013). In human T cell culture by suplatast tosilate at the concentration of 10 μg/mL, antigen specific IgE production after 10 days was suppressed from 56 to 72% and IL-4 production after 3 days was suppressed from 58 to 76% (Taiho Pharmaceutical 2013).

As for IL-2 and antibody production, in vitro T-cell-induced polyclonal B cell activation to produce antibody was inhibited with anti-IL-2 and anti-IL-2R antibodies. That is, murine small resting B cells, cultured with irradiated hapten-specific TH1 clone, were induced to enter cell cycle at 2 days and to secret antibody at 5 days. An anti-IL-2 and anti-IL-2R antibodies completely inhibited this T-cell dependent antibody production (Owens T, 1991).

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

Time-scale

In CsA-treatment for 24 days at 50 mg/kg BID, cynomolgus monkeys showed suppression of IL-2 and IL-4 production and inhibition of SRBC-specific IgM and IgG in TDAR (Gaida K. 2015).

In human T cell culture, suplatast tosilate inhibits IL-4 production after 3 days and antigen specific IgE production after 10 days (Taiho Pharmaceutical 2013).

In the human T-B cell co-culture, CNIs of FK506 and CsA lowered the m-RNA levels of IL-2 and IL-4 at 8h post-stimulation and inhibited IgM and IgG productions after 9 days (Heidt S. 2010).

Known modulating factors

At present, no evidence is found.

Known Feedforward/Feedback loops influencing this KER

At present, no evidence is found.

References

1. Alberts, B., Johnson, A., Lewis, L., Raff, M., Roberts, K. and Walter, P. (2008). Molecular Biology of the Cell. 5th ed., Garland Science, New York. 1539-1601
2. Sanofi K.K. (2018) Drug interview form Dupixent subcutaneous injection 300 mg syringe. 2nd edition.
3. Taiho Pharmaceutical Co.,Ltd. (2013) Drug interview form IPD capsule 50 and 100. Revised 5th edition.
4. Novartis Pharma K.K. (2016). Drug interview form Simulect i.v. injection 20 mg. 10th edition.
5. Dumont, F.J., Staruch, M.J., Fischer, P., DaSilva, C. and 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. Journal of immunology 160 (6): 2579-89.
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