Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Homo sapiens||Homo sapiens||High||NCBI|
|Mus musculus||Mus musculus||High||NCBI|
|Rattus norvegicus||Rattus norvegicus||High||NCBI|
|All life stages||High|
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 stimulates B cells to proliferate through surface IL-2 receptors. 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 impaire TDAR, as seen in the result of FK506 treatment (Heidt et al, 2009).
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 T cell dependent antibody production. IL-2 promotes differentiation of B cells through IL-2 stimulates differentiation of the activated T cell into T cell called Th2 cell. Therefore, suppressed production of IL-2 and IL-4 impairs TDAR (Alberts et al. 2008).
After a 9-day culture of B cells and non-pre-activated T cell stimulation with FK506 or CsA, the levels of IgM and IgG in the culture supernatant were reduced at 0.3 and 1.0 ng/mL (0.37 and 1.24 nM) of FK506 or 50 and 100 ng/mL (41.6 and 83.2 nM) of CsA (Heidt et al, 2009).
After a 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 concentrations of 0.01 to 100 ng/mL (0.012 to 124 nM) of FK506 or 0.1 to 1000 ng/mL (0.083 to 83.2 nM) of CsA (Sakuma et al. 2001b).
Rats were treated with FK506 for over four weeks and immunized with KLH, after which serum concentration of anti-KLH IgM and IgG was reduced at the dose level 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 were reduced at 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).
As immunosuppression-derived adverse outcomes by CN inhibition, FK506 and CsA increase the frequency and/or severity of infections； impaired TDAR deems to be one of the causative factors for increased infection. Some clinical trials of FK506 and CsA revealed these adverse effects as follows.
- In clinical trials of renal transplantation using FK506 or CsA, opportunistic infections such as candida, cytomegalovirus and herpes simplex virus were reported (Ekberg et al. 2007).
- In recipients of liver transplants treated with FK506 or CsA, opportunistic infections such as cytomegalovirus, hepatitis C virus, hepatitis B and herpes simplex virus were reported (Fung et al. 1991).
- Cardiac transplant patients treated with cyclosporin developed pulmonary infections within the first year after surgery (Luster, M.I. et al. 1993).
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 or with a plaque-forming cell (PFC) assay.
- Rats were repeatedly administered FK506 orally for 4 weeks and immunized with Keyhole limpet hemocyanin (KLH), after which the serum was examined for T-cell–dependent, antigen-specific, IgM and IgG levels using a Sandwich ELISA kit (Ulrich et al. 2004).
- Mice were repeatedly administered CNI including FK506 and CsA orally for 4 days and immunized with sheep red blood cells (SRBC), after which spleen cells were examined using a PFC assay (Kino et al. 1987).
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 peripheral blood mononuclear cells (PBMC) were co-cultured with a CNI for nine days in the presence of polyclonal–T-cell stimulation, after which supernatants were tested for immunoglobulin IgM and IgG levels using a Sandwich ELISA kit. Treatment with FK506 or CsA reduced the levels of IgM and IgG at the concentrations of 0.3 and 1.0 ng/mL or 50 and 100 ng/mL (Heidt et al, 2009).
- 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 four days, IgM produced in the culture supernatants was measured using an ELISA kit. 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. 2001b).
- In order to examine class switching, T cells derived from human PBMCs were cultured with CNI, and cytokine mRNA levels of IFN-gamma, IL-2, IL-4, IL-5, IL-10, IL-13, and other B-cell–stimulatory cytokines produced in T cells were measured by quantitative PCR (Dumont et al. 1998).
Domain of Applicability
CNI 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 plaque-forming cells (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(keyhole limpet hemocyanin)-IgG and IgM antibodies after subcutaneous immunization with KLH (Ulrich et al. 2004). 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 cyclosporin 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 and rodents.
Evidence for Perturbation by Stressor
Regulatory Significance of the Adverse Outcome
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 be evaluated whenever the target cells of immunotoxicity are not clear based on pharmacology and findings in standard toxicity studies.
- 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
- 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.
- 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.
- Kino, T., Hatanaka, H., Hashimoto, M., Nishiyama, M., Goto, T., Okuhara, M., Kohsaka, M., Aoki, H. and Imanaka, H. (1987). 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.
- 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.
- 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.
- Ekberg, H., Tedesco-Silva, H., Demirbas, A., Vítko, S., Nashan, B., Gürkan, A., Margreiter, R., Hugo, C., Grinyó, J.M., Frei, U., Vanrenterghem, Y., Daloze, P. and Halloran, P.F.; ELITE-Symphony Study. (2007). Reduced exposure to calcineurin inhibitors in renal transplantation. The New England journal of medicine 357 (25): 2562-75.
- Fung, J., Abu-Elmagd, K., Jain, A., Gordon, R., Tzakis, A., Todo, S., Takaya, S., Alessiani, M., Demetris, A., Bronster, O., Martin, M., Mieles, L., Selby, R., Reyes, J., Doyle, H., Stieber, A., Casavilla, A. and Starzl, T. (1991). A randomized trial of primary liver transplantation under immunosuppression with FK 506 vs cyclosporine. Transplantation proceedings 23 (6): 2977-83.
- Luster, M.I., and Rosenthal, G.J. (1993). Environmental Health Perspectives. 100: 219-36.