Upstream eventSuppression, IL-2 and IL-4 production
Impairment, T-cell dependent antibody response
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Inhibition of Calcineurin Activity Leading to Impaired T-Cell Dependent Antibody Response||adjacent||High||High|
|Homo sapiens||Homo sapiens||High||NCBI|
|Mus musculus||Mus musculus||High||NCBI|
Life Stage Applicability
|All life stages||High|
Key Event Relationship Description
IL-2 and IL-4 are produced and secreted by helper T cells and play important roles in the development of 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, after treatment with FK506, production of IL-2, IL-4, and other cytokines decreases in T cells (Dumont et al. 1998), reducing stimulation of B cells including proliferation, activation, and class switching, and leading to impairment of TDAR. Therefore, FK506 and cyclosporin A (CsA) are potent inhibitors of T-cell–dependent-antibody production and suppressing the production of these B-cell-related cytokines appears to be the main factor in impairment of TDAR by FK506 (Heidt, S. et al. 2009).
Evidence Supporting this KER
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 antibody. Dupilumab (Dupixent) reduces productions of IgE 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, 2009)..
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, PBMC treated with FK506 and CsA suppressed the production of immunoglobulin (Ig) M and G 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 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, 2009).
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 support of the suppression, IL-2 and IL-4 production leads to impairment, T-cell dependent antibody response is strong.
- In the allergen-induced pneumonia model in mice, dupilumab (anti-IL-4/13 receptor 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/PMA-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 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 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).
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
In the blocking of IL-4 receptor in mice by dupilumab (anti-IL-4/13 receptor 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 (an inhibitor of the production of cytokines on Th2 cell) 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 addition, cynomolgus monkeys treated wth CsA showed suppression of IL-2 and TDAR using sheep red blood cells with a dose dependent manner (Gaidal K. 2015).
In the human T-B cell co-culture stimulated with anti-CD3 monoclonal antibody, CNIs of FK506 and CsA lowered the 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).
In human T cell culture, suplatast tosilate (an inhibitor of the production of cytokines on Th2 cell) 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
(To be described)
Known Feedforward/Feedback loops influencing this KER
(To be described)
Domain of Applicability
Suppressed IgE and antigen specific IgG1 productions by the blocking of IL-4 receptor were reported in mice using dupilumab (anti-IL-4/13 receptor antibody) (Sanofi K.K. 2018).
Suppressed antigen specific IgE production by the inhibition of IL-4 production was reported in mice using suplatast tosilate (an inhibitor of the production of cytokines on Th2 cell) (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 and rodents in inhibitions of IL-2 and IL-4 production and TDAR induction.
- 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
- Sanofi K.K. (2018) Drug interview form Dupixent subcutaneous injection 300 mg syringe. 2nd edition.
- Taiho Pharmaceutical Co.,Ltd. (2013) Drug interview form IPD capsule 50 and 100. Revised 5th edition.
- Novartis Pharma K.K. (2016). Drug interview form Simulect i.v. injection 20 mg. 10th edition.
- 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.
- Heidt, S., Roelen, D. L., Eijsink, C., Eikmans, M., van Kooten, C., Claas, F. H. and Mulder, A. (2010). Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help. Clinical and experimental immunology. 159(2): 199-207.
- Gaida K., Salimi-Moosavi H., Subramanian R., Almon V., Knize A., Zhang M., Lin F.F., Nguyen H.Q., Zhou L., Sullivan J.K., Wong M., McBride H.J. (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.
- Heidt S., Roelen D.L., Eijsink C., Eikmans M., van Kooten C., Claas F.H., Mulder A.(2010). Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help.
- Kino, T., Hatanaka, H., Miyata, S., Inamura, N., Nishiyama, M., Yajima, T., Goto, T., Okuhara, M., Kohsaka, M. and Aoki, H. (1987a). FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. Journal of antibiotics. 40(9): 1256-1265.
- Kino, T., Hatanaka, H., Hashimoto, M., Nishiyama, M., Goto, T., Okuhara, M., Kohsaka, M., Aoki, H. and Imanaka, H. (1987b). FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. Journal of antibiotics. 40(9): 1249-1255.
- Owens T.(1991). Requirement for noncognate interaction with T cells for the activation of B cell immunoglobulin secretion by IL-2. Cell Immunol 133:352-366.
- Sakuma, S., Kato, Y., Nishigaki, F., Magari, K., Miyata, S., Ohkubo, Y., and Goto, T. (2001b). Effects of FK506 and other immunosuppressive anti-rheumatic agents on T cell activation mediated IL-6 and IgM production in vitro. International Immunopharmacology 1(4): 749-57.
- Ulrich, P., Paul, G., Perentes, E., Mahl, A., and Roman D. (2004). Validation of immune function testing during a 4-week oral toxicity study with FK506. Toxicology Letters 149(1-3): 123-31.