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Key Event Title
Impairment of T-cell dependent antibody response
|Level of Biological Organization|
Key Event Components
|T cell activation involved in immune response||decreased|
Key Event Overview
AOPs Including This Key Event
|All life stages||High|
Key Event Description
The production of antibodies to T cell-dependent antigens is a coordinated process involving B cells, antigen-presenting cells, and T cell derived cytokines. The B cells are stimulated to proliferate and differentiate. The TDAR might be altered if any of these cell populations are affected.
IL-2 and IL-4 are produced and secreted by helper T cells. Both are important in the development of TDAR. IL-4 affects maturation and class switching of B cells as well as proliferation. Both events induce and enhance TDAR. IL-2 promotes differentiation of B cells, which stimulates differentiation of activated T cells to Th2 cells. The suppressed production of IL-2 and IL-4 impairs TDAR (Justiz Vaillant and Qurie 2020).
A mutant form of human IL-4, in which the tyrosine residue at position 124 is replaced by aspartic acid (hIL-4Y124D), was reported to specifically block IL-4 and IL-13-induced proliferation of B cells. In addition, hIL-4Y124D also strongly inhibited both IL-4- or IL-13-induced IgG4 and IgE synthesis in cultures of PBMCs, or highly purified sIgD+ B cells cultured in the presence of anti-CD40 monoclonal antibodies. IL-4 may be necessary to produce antibodies and to proliferate in B cells. The mutation of IL-4 may impair TDAR (Aversa, et al. 1993).
IL-4 stimulates B cells to proliferate, switch immunoglobulin classes, and differentiate to plasma and memory cells. Suppressing the production of these B cell related cytokines appears to impair TDAR, as evident from the results of FK506 treatment (Heidt, et al. 2010).
STAT5 is able to inhibit peroxisome proliferator activated receptor (PPAR)-regulated gene transcription. Conversely, ligand-activated PPAR can inhibit STAT5-regulated transcription. As a peroxisome proliferator, perfluorooctanoic acid (PFOA) induces PPARs. The suppression of TDAR has been observed with a no observable effect level (NOEL) of 1.88 mg/kg/d and lowest observed adverse effect level (LOEL) of 3.75 mg/kg/d for PFOA administered in drinking water over 15 days (Dewitt, et al. 2008). The increase in PPAR expression induced by PFOA may inhibit STAT5-regulated transcription, which is important for IL-4 production in TDAR.
How It Is Measured or Detected
TDAR can be examined in vivo and in vitro. In vivo studies of antigen-specific antibodies are usually performed by measuring serum antibody levels with ELISA (Onda, et al. 2014) or with a plaque-forming cell (PFC) assay.
To assess keyhole limpet hemocyanin (KLH) antigen-specific T cell proliferation, 1 × 105 CD4+ T cells were co-cultured with 2 × 105 autologous PBMCs in 96-well plates in the presence of KLH. Cells were cultured for 5 or 7 days before being pulsed with 0.5 μCi 3[H]-thymidine (PerkinElmer) for 18 h. The cells were harvested using a 96-well cell FilterMate harvester. 3[H]-thymidine incorporation in CD4+ T cell response to biopharmaceuticals was measured by liquid scintillation counting using a TopCount NXT (Schultz, et al. 2017).
In another in vivo study, rats were repeatedly administered FK506 orally for 4 weeks and immunized with KLH. Rat serum was examined for T cell dependent, antigen-specific IgM and IgG levels by ELISA (Ulrich, et al. 2004).
Other authors repeatedly administered CNIs, including FK506 and CsA, to mice orally for 4 days and immunized with sheep red blood cells (SRBCs). Spleen cells were examined using a PFC assay (Kino, et al. 1987). Antigen-specific plaque-forming splenocytes were reduced at doses of 3.2, 10, 32, and 100 mg/kg of FK506 or 32 and 100 mg/kg CsA.
In another study, cynomolgus monkeys received 50 mg/kg CsA twice a day via oral gavage (10 h apart) for 23 days and were immunized with SRBCs. Serum was examined for anti-SRBC IgM and IgG levels using an ELISA specific for SRBC antigen (Gaida, et al. 2015).
In the final in vivo study cited here, mice were exposed to a single pharyngeal aspiration of 1,2:5,6-Dibenzanthracene, after which the supernatants of splenocytes were cultured for 24 h in the presence of lipopolysaccharide and assayed using a mouse IgM or IgG matched pairs antibody kit (Smith, et al. 2010).
For in vitro studies, total IgM and IgG levels in culture supernatants are often measured after polyclonal T cell activation rather than after antigen stimulation in immune cell cultures.
In one study, T and B cells isolated from human PBMCs were co-cultured with CNIs for 9 days in the presence of polyclonal T cell stimulation. The supernatants were examined for IgM and IgG levels by ELISA. Treatment with FK506 or CsA reduced the levels of IgM and IgG at concentrations of 0.3 and 1.0 ng/mL (0.37 and 1.24 nM) or 50 and 100 ng/mL (41.6 and 83.2 nM), respectively (Heidt, et al. 2010).
In another study, SKW6.4 IL-6-dependent IgM-secreting human B cells were cultured for 4 days with anti-CD3/CD28 antibody-stimulated PBMC culture supernatant. IgM produced in the culture supernatants was measured by ELISA. FK506 or CsA reduced the levels of IgM at concentrations of 0.01 to 100 ng/mL or 0.1 to 1000 ng/mL (Sakuma, et al. 2001).
Domain of Applicability
CNI-induced impairment of TDAR has been demonstrated in rodent studies. In one study, 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 4-week period reduced the production of both anti-KLH-IgG and IgM after subcutaneous immunization with KLH (Ulrich, et al. 2004). Other authors described that treatment with CsA at 50 mg/kg BID via oral gavage in cynomolgus monkeys resulted in reduction of serum SRBC-specific IgM and IgG (Gaida, et al. 2015). As for humans, in vitro experiments showed that treatment with FK506 or CsA of PBMCs from blood bank donors suppressed the production of IgM and IgG specific to T cell dependent antigens (Heidt, et al. 2010). In SKW6.4 cells (IL-6 dependent, IgM-secreting, human B cell line) cultures, FK506 or CsA suppressed the production of IgM in the presence of T cell activation (Sakuma, et al. 2001). Considering that FK506 and CsA reduce T cell derived IL-2, these findings strongly suggest that impairment of TDAR following reduced production of IL-2 occurs at least in common among humans, monkeys, and rodents.
Yang et al. (2002b) exposed male C57BL/6 mice to a single concentration (0.02%) of PFOA in the diet for 16 days. TDAR was measured after inoculating PFOA-treated mice with horse red blood cells intravenously on day 10; serum levels of horse red blood cell-specific IgM and IgG in response to the immunization were significantly decreased (Yang, et al. 2002).
The suppression of TDAR in adult C57BL/6 female mice has been observed in several studies. NOEL of 1.88 mg/kg/d and LOEL of 3.75 mg/kg/d were identified for PFOA administered in drinking water for over 15 days (Dewitt, et al. 2008).
The suppression of TDAR in adrenalectomized or sham-operated C57BL/6N female mice was observed when PFOA was provided in drinking water for 10 days at doses of 0, 3.75, 7.5, or 15 mg/kg/d. TDAR was determined as the primary antibody response to the T cell dependent antigen in SRBCs. The day after exposure ended, SRBCs were introduced intravenously and SRBC-specific IgM was measured 5 days later (DeWitt, et al. 2009).
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 TDAR. Thus, changes in any of these immune cell populations can influence TDAR.
The ICH S8 immunotoxicity testing guideline on pharmaceuticals recommends that TDAR can be evaluated whenever the target cells of immunotoxicity are not clear based on pharmacology and findings in standard toxicity studies. For the assessment of pesticides, the United States Environmental Protection Agency Office of Prevention, Pesticides and Toxic Substances 870.7800 immunotoxicity testing guideline recommends TDAR using SRBC.
Finally, the draft Food and Drug Administration guidance of nonclinical safety evaluation for immunotoxicology recommends the TDAR assay.
Aversa G, Punnonen J, Cocks BG, de Waal Malefyt R, Vega F, Jr., Zurawski SM, Zurawski G, de Vries JE. 1993. An interleukin 4 (IL-4) mutant protein inhibits both IL-4 or IL-13-induced human immunoglobulin G4 (IgG4) and IgE synthesis and B cell proliferation: support for a common component shared by IL-4 and IL-13 receptors. J Exp Med 178:2213-2218. DOI: 10.1084/jem.178.6.2213.
Dewitt JC, Copeland CB, Strynar MJ, Luebke RW. 2008. Perfluorooctanoic acid-induced immunomodulation in adult C57BL/6J or C57BL/6N female mice. Environ Health Perspect 116:644-650. DOI: 10.1289/ehp.10896.
DeWitt JC, Shnyra A, Badr MZ, Loveless SE, Hoban D, Frame SR, Cunard R, Anderson SE, Meade BJ, Peden-Adams MM, Luebke RW, Luster MI. 2009. Immunotoxicity of perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator-activated receptor alpha. Crit Rev Toxicol 39:76-94. DOI: 10.1080/10408440802209804.
Gaida K, Salimi-Moosavi H, Subramanian R, Almon V, Knize A, Zhang M, Lin FF, Nguyen HQ, Zhou L, Sullivan JK, Wong M, McBride HJ. 2015. Inhibition of CRAC with a human anti-ORAI1 monoclonal antibody inhibits T-cell-derived cytokine production but fails to inhibit a T-cell-dependent antibody response in the cynomolgus monkey. J Immunotoxicol 12:164-173. DOI: 10.3109/1547691X.2014.915897.
Heidt S, Roelen DL, Eijsink C, Eikmans M, van Kooten C, Claas FH, Mulder A. 2010. Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help. Clin Exp Immunol 159:199-207. DOI: 10.1111/j.1365-2249.2009.04051.x.
Justiz Vaillant AA, Qurie A. 2020. Interleukin. In StatPearls: Treasure Island (FL)
Kino T, Hatanaka H, Hashimoto M, Nishiyama M, Goto T, Okuhara M, Kohsaka M, Aoki H, Imanaka H. 1987. FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. J Antibiot (Tokyo) 40:1249-1255. DOI: 10.7164/antibiotics.40.1249.
Onda M, Ghoreschi K, Steward-Tharp S, Thomas C, O'Shea JJ, Pastan IH, FitzGerald DJ. 2014. Tofacitinib suppresses antibody responses to protein therapeutics in murine hosts. J Immunol 193:48-55. DOI: 10.4049/jimmunol.1400063.
Sakuma S, Kato Y, Nishigaki F, Magari K, Miyata S, Ohkubo Y, Goto T. 2001. Effects of FK506 and other immunosuppressive anti-rheumatic agents on T cell activation mediated IL-6 and IgM production in vitro. Int Immunopharmacol 1:749-757.
Schultz HS, Reedtz-Runge SL, Backstrom BT, Lamberth K, Pedersen CR, Kvarnhammar AM, consortium A. 2017. Quantitative analysis of the CD4+ T cell response to therapeutic antibodies in healthy donors using a novel T cell:PBMC assay. PLoS One 12:e0178544. DOI: 10.1371/journal.pone.0178544.
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Yang Q, Abedi-Valugerdi M, Xie Y, Zhao XY, Moller G, Nelson BD, DePierre JW. 2002. Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferator, perfluorooctanoic acid. Int Immunopharmacol 2:389-397. DOI: 10.1016/s1567-5769(01)00164-3.