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Key Event Title
Impairment, T-cell dependent antibody response
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Key Event Components
Key Event Overview
AOPs Including This Key Event
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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.
Interleukin (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).
In male CD-1 mice, chronic psychosocial stress (types of social outcome occurred: residents becoming subordinates) decrease in anti- keyhole limpet hemocyanine (KLH) immunoglobulin (Ig)G. (Alessandro, B. et al. 2003).
In female B6C3F1 mice, 1,2:5,6-dibenzanthracene (DBA) exposure reduced total IgG antibody in spleen cell culture supernatants after in vitro stimulation with lipopolysaccharide (LPS) (Donna, C. et al. 2010).
Treatment with cyclosporin A (CsA) at 50 mg/kg BID via oral gavage in cynomolgus monkey resulted in reduction of serum sheep red blood cells (SRBC)-specific IgM and IgG (Kevin, G. et al. 2014).
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 peripheral blood mononuclear cells (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 calcineurin inhibition, FK506 and CsA increase the frequency and/or severity of infections and allergic reactions impaired TDAR deems to be one of the causative factors for these side effects . 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).
- In patients of X-linked autoimmune enteropathy treated with CsA or FK506, serum levels of IgE developed extremely high during the immunosuppressive therapy (Kawamura et al. 1997).
- Renal transplant recipients treated with belatacepy/mycophenolate (MMF)/predonisone or FK506/MMF/prednisone showed significantly lower the geometric mean hemagglutination inhibition titer against influenza vaccine, hemagglutination-specific IgG and isotype IgG1 antibodies, and IgG-antibody secreting cells response (Gangappa et al. 2019).
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 Enzyme-Linked ImmunoSorbent Assay (ELISA) or with a plaque-forming cell (PFC) assay.
- 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 using a Sandwich ELISA kit (Ulrich et al. 2004).
- Mice were repeatedly administered calcineurin inhibitors (CNIs) including FK506 and CsA orally for 4 days and immunized with SRBC, after which spleen cells were examined using a PFC assay (Kino et al. 1987).
- 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 (Kevin, G. et al. 2014).
- 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 (Bethyl Laboratories, Montgomery, TX) (Donna, C. 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 peripheral blood mononuclear cells (PBMC) were co-cultured with a CNIs 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 CNIs, and cytokine mRNA levels of Interferon-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
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.
Regulatory Significance of the Adverse Outcome
The ICH S8 guideline, which covers immunosuppression of small molecule drugs, determines the need for immunotoxicity studies by comprehensively evaluating the findings of pharmacology, changes in the immune system in repeated-dose toxicity studies, and other factors using a Weight of Evidence approach. If there is concern about immunotoxicity, the presence or absence of immunotoxicity should be determined using an in vivo test system capable of assessing the functional changes of predicted immunotoxic target cells. If immunotoxicity is observed, additional studies including in vitro assays or clinical evaluation should be considered to assess the risk of immunotoxicity in humans. Because TDAR involves many immune cell populations, including T cells, B cells, and antigen-presenting cells, evaluation of TDAR is recommended when there is concern about immunotoxicity but the immunotoxic target cells are unclear. The S8 guidelines list KLH, SRBC, and tetanus toxin as antigens for TDAR.
The draft FDA immunotoxicity testing guidance (2020) covers immunosuppressive and immunostimulatory drugs and biologics; evaluating immunosuppressive drugs in the draft FDA guidance is similar to that in the S8 guideline, with in vivo TDAR assays recommended when toxic target cells are unknown. The draft guidance states that TDAR assays using KLH as an antigen have been established in mice, rats, dogs, minipigs, and cynomolgus monkeys, but the use of SRBC and tetanus toxin as antigens is also acceptable.
For the assessment for pesticides, US EPA OPPTS 870.7800 immunotoxicity testing guideline recommends TDAR using SRBC. The REACH guideline does not provide for immunotoxicity testing, but it provides triggers for conducting immunotoxicity testing.
The WHO/IPSS Immunotoxicity Risk assessment Guidance (2012) describes a strategy for assessing five categories of immunotoxicity risks, including immunosuppression. For risk assessment of immunosuppression, it calls for identification of immunosuppression risks, prediction of pathogenesis that may occur, and consideration of safety margins based on the WoE approach from human findings, infection resistance tests, immune function tests, general immune system assays, histopathological findings and organ weights in general toxicity studies, and hematological data.
The evaluation of immunotoxicity in F1 animals in the OECD Guidelines for Extended First Generation Reproductive and Developmental Toxicity Studies (TG443) requires that PFC and ELSA assays to measure primary IgM antibody production by TDAR using T-cell dependent antigens (SRBC, KLH, etc.) be performed. Furthermore, if changes are observed, the significance of the changes should be examined by comprehensively evaluating other data.
The outcomes of immunosuppression are susceptibility to infection and tumorigenesis, and the FDA guidance requires that immunosuppressive drugs be evaluated for carcinogenic risk using WoE approach based on the results of carcinogenicity and immunotoxicity studies. Meanwhile, the ICH S1B(R1) Draft Step 2 Guidelines for Carcinogenicity Testing calls for evaluation of carcinogenicity by WoE approach instead of rat carcinogenicity testing, because rodent carcinogenicity test models are less capable of detecting carcinogenicity. On the other hand, it is difficult to define susceptibility to infection as a measurable AO with a clear mechanism, because immune responses vary among pathogens. In fact, many immunotoxicity guidelines require that the risk of immunotoxicity be identified and assessed by evaluating immune functions.
It was difficult to define susceptibility to infection as an AO, so TDAR, which is recommended as an indicator of immunosuppresoin in many guidelines, was used as an AO. It is expected that several AOPs with TDARs as AOs will be developed, and based on these AOPs, it may be possible to develop an IATA to assess the risk of immunotoxicity characterized by TDARs.
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