T-cell Dependent Antibody Response Research Articles

Host Resistance Assays.

The goal of immunotoxicity testing is to obtain data useful for immunotoxicity safety assessment. Guidance in the performance of immunotoxicity safety evaluations is provided in documents from the US EPA for chemicals and the ICH S8 document for pharmaceuticals. The ICH S8 document outlines a tiered approach that includes (1) standard toxicity studies with associated hematology, immune system organ weights, and histopathology data; (2) functional assays, such as cytotoxic T lymphocyte (CTL) assays, natural killer (NK) cell assays, respiratory burst, phagocytosis, and T-cell-dependent antibody response (TDAR) assays; and (3) host resistance assays. Host resistance assays are considered the gold standard in immunotoxicity testing and provide a critical overview of the extent to which innate, adaptive, and homeostatic regulatory immune functions are integrated to protect the host. Both comprehensive and targeted host resistance assays are available, each with distinct advantages. This chapter serves to provide a general overview of the various assays that may be used, as well as a summary of procedures.

Methods for Analysis of Nanoparticle Immunosuppressive Properties In Vitro and In Vivo.

Adverse drug effects on the immune system function represent a significant concern in the pharmaceutical industry, because 10-20% of the drug withdrawal from the market is accounted to immunotoxicity. Immunosuppression is one such adverse effect. The traditional immune function test used to estimate materials' immunosuppression is a T-cell-dependent antibody response (TDAR). This method involves a 28day in vivo study evaluating the animal's antibody titer to a known antigen (KLH) with and without challenge. Due to the limited quantities of novel drug candidates, an in vitro method called human leukocyte activation (HuLa) assay has been developed to substitute the traditional TDAR assay during early preclinical development. In this test, leukocytes isolated from healthy donors vaccinated with the current year's flu vaccine are incubated with Fluzone in the presence or absence of a test material. The antigen-specific leukocyte proliferation is then measured by ELISA analyzing incorporation of BrdU into DNA of the proliferating cells. Here, we describe the experimental procedures for investigating immunosuppressive properties of nanoparticles by both TDAR and HuLa assays, discuss the in vitro-in vivo correlation of these methods, and show a case study using the iron oxide nanoparticle formulation, Feraheme.

Abstract 5005: AGEN1884 and AGEN2041: Two functionally distinct anti-CTLA-4 antagonist antibodies

Abstract CTLA-4 is an important negative regulator of T cell function. Together with CD28, these two co-receptors exemplify a co-inhibitory/stimulatory system that is critical in the regulation of T cell immune responses. Key to this regulatory system are the shared ligands, CD80 and CD86, whose engagement determines whether T cells receive stimulatory (CD28) or inhibitory (CTLA-4) signals. Pre-clinical and clinical studies have shown that anti-CTLA-4 antibodies can enhance tumor-specific immunity through a combination of mechanisms including: 1) blockade and or displacement of CD80/CD86 binding to CTLA-4, leading to CD28 activation; 2) prevention of the trans-endocytosis of CD80/CD86 from the surface of antigen presenting cells by CTLA-4 expressing regulatory T cells; and 3) the selective depletion of CTLA-4 expressing intratumoral regulatory T cells by an Fcγ receptor-mediated mechanism. AGEN1884 and AGEN2041, two fully human anti-CTLA-4 antibodies identified using the Retrocyte Display™ platform, are being developed for the treatment of advanced malignancies. The antibodies share heavy and light chain complementarity determining regions (CDRs), but differ in their IgG Fc region (AGEN1884, an IgG1, and AGEN2041, an IgG2). AGEN1884 and AGEN2041 selectively bind to human and cynomolgus monkey CTLA-4 with low single digit nM affinity. Further, both antibodies bind CTLA-4 expressed on T cells, and potently block engagement of CD80 and CD86, leading to enhanced T cell responsiveness. In a T cell-dependent antibody response (TDAR) study in cynomolgus monkeys, administration of a vaccine in combination with either AGEN1884 or AGEN2041 augmented the antibody response to the vaccine antigen. This finding demonstrates that both antibodies are functional in non-human primates, and exemplifies their utility in promoting immunity to co-administered antigens in patients, such as therapeutic cancer vaccines. Consistent with this, an anti-mouse CTLA-4 antibody produced potent tumor regressions in combination with a heat-shock protein-based vaccine (a surrogate vaccine resembling Prophage™ heat shock protein-based autologous vaccine). The distinct IgG backbones of AGEN1884 and AGEN2041 enable distinct optimal effector functions, such as the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). We anticipate that these differences in effector function may be exploited in certain tumors depending on immune cell composition. Taken together, the biochemical and functional attributes of AGEN1884 and AGEN2041 are ideally suited for clinical development, both as single agents and also in combination with other immune education approaches, such as cancer vaccines and immunomodulatory antibodies or small molecule therapies. Citation Format: Elise E. Drouin, Ana Gonzalez, Hao Tang, David Savitsky, Randi Gombos, Jeremy Waight, Benjamin Duckless, Andrea Schuster, Lili Wang, Shiwen Lin, Cornelia Mundt, Gerd Ritter, Taha Merghoub, Kyle Draleau, Jedd Wolchok, Daniel Levey, Jennifer Buell, Marc van Dijk, John M. Goldberg, Robert Stein, Nicholas S. Wilson. AGEN1884 and AGEN2041: Two functionally distinct anti-CTLA-4 antagonist antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5005.

TAK1 deficiency in dendritic cells inhibits adaptive immunity in SRBC-immunized C57BL/6 mice.

Dendritic cells (DCs) are important in the initiation of primary T‐cell responses, while transforming growth factor‐β (TGF‐β)‐activated kinase 1 (TAK1) is a critical regulator of DC survival and homeostasis. This study evaluated the T‐cell dependent antibody response (TDAR) to sheep red blood cells (SRBC) on a DC‐specific TAK1‐deficient mice model. The results showed that TAK1 deficiency in DCs significantly suppressed the humoral and cellular immune response in mice. DC‐specific TAK1 deletion impaired splenic T‐cell population and conventional DCs, abolished the cytokine production of splenic T cells and down‐regulated some functional gene expression in the spleen. Collectively, this study suggests that TAK1 plays an essential role in the development of the humoral immune response.

Abstract B58: Early safety assessment of single and combination therapy using TDAR

Abstract The discovery of targeted therapies against tumors offers promise of therapeutic benefit to patients however, it poses challenges in assessing immunologic and toxicologic risk during drug development. We determined that early safety assessment of combination therapy, prior to later stage toxicology studies, can be used to support Go/No-Go NME decision and thereby reduce time, efficiency, and resources used to choose and develop lead candidates. The rodent T-cell-dependent antibody response (TDAR) assay is used to assess the effect of candidate therapeutic agents on the immune system by measuring primary and secondary IgM and IgG antibody responses to exogenous antigen challenge. TDAR responses require intact function of multiple immune cells including antigen presenting cells and T and B lymphocytes, as well as a cytokine-dependent isotype class switch from IgM to IgG, resulting in production of an antigen-specific antibody response. Alterations in the amount of antibody produced therefore can reflect effects on any or all cell populations involved in TDAR. TDAR is commonly used in preclinical drug development especially where increased cause for concern exists (ICH guideline S8). Development of combination therapy that engages multiple targets impacting the immune system poses unique opportunity for increased efficacy but also unique risk for increased immunotoxicity including immune stimulation. For this work, a mouse TDAR evaluating primary and secondary KLH antibody responses in a KLH-Specific IgM and IgG sandwich enzyme-linked immunosorbent assay (ELISA) was first validated and then used to assess immunotoxicologic potential of multiple single immunosuppressive agents (cyclophosphamide (CTX), abatacept, azathioprine (AZT), etanercept, cyclosporine (CsA), and prednisone) and biological therapies (A, B, C, D, E, and F) and combination biologic therapies ( A+B, C+D, E+F). Doses of 100 mg/kg CsA, 250 mg/kg abatacept, 125 mg/kg etanercept, 100 mg/kg AZT, 20 mg/kg prednisone administered subcutaneously (s.c.) on Days 1 and 3 had mild immunosuppressive effects. 200 mg/kg of CTX administered s.c. had an expected robust immunosuppressive effect that was statistically significant than control. Combination of biologic therapies did not result in enhanced TDAR immunotoxicity compared to single biologic therapy alone for the molecules evaluated. Tier 1 immunotoxicology assessments similar to those in standard toxicity studies were added to the TDAR assessment. Together, these data support the use of the TDAR assay for early safety assessment of potential combination therapies against tumors. Citation Format: Amy L. Volk, Mindi Walker, Kerry Brosnan, Dorie Capaldi, Patricia Rafferty, Daniel Weinstock, Eva Emmell. Early safety assessment of single and combination therapy using TDAR. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B58.

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

ORAI1 is the pore-forming component of calcium release-activated calcium (CRAC) channels. CRAC channels are the primary route for calcium ion (Ca2+) entry into T-cells following antigen stimulation. This Ca2+ entry induces proliferation and cytokine production through activation of calcineurin and the nuclear factor of activated T-cells (NFAT) transcription factor along with subsequent cytokine-related genes. It was hypothesized that the in vivo inhibition of T-cell function by blocking ORAI1 or calcineurin would lead to similar functional consequences. To test this hypothesis the activity of 2C1.1, a fully human anti-ORAI1 monoclonal antibody, and cyclosporin A (CsA) were tested in vivo for their suppressive effect on T-cell-derived cytokine production and a T-cell-dependent antibody response (TDAR) using sheep red blood cells (SRBC) in cynomolgus monkeys. Despite showing similar inhibition of ex vivo interleukin (IL)-2 production by stimulated T-cells, both molecules exhibited different pharmacologic effects on the SRBC antibody response. CsA blocked the development of SRBC-specific antibodies, while 2C1.1 failed to inhibit the antigen-specific antibody response. These surprising observations suggest that full inhibition of the CRAC channel is required to inhibit a functional immune response, consistent with findings from human patients with loss of function mutations in ORAI1.

The T-cell-dependent antibody response assay in nonclinical studies of pharmaceuticals and chemicals: Study design, data analysis, interpretation

The T-cell-dependent antibody response (TDAR) assay is a measure of immune function that is dependent upon the effectiveness of multiple immune processes, including antigen uptake and presentation, T cell help, B cell activation, and antibody production. It is used for risk and safety assessments, in conjunction with other toxicologic assessments, by the chemical and pharmaceutical industries, and research and regulatory agencies. It is also employed to evaluate investigational drug efficacy in animal pharmacology studies, provide evidence of biological impact in clinical trials, and evaluate immune function in patients with primary or secondary immunodeficiency diseases. Various immunization schemes, analytical methods, approaches to data analysis, and data interpretations are in use. This manuscript summarizes some recommended practices for the conduct and interpretation of the assay in animal studies.

Immunotoxicity of silver nanoparticles in an intravenous 28-day repeated-dose toxicity study in rats

BackgroundNanosilver is used in a variety of medical and consumer products because of its antibacterial activity. This wide application results in an increased human exposure. Knowledge on the systemic toxicity of nanosilver is, however, relatively scarce. In a previous study, the systemic toxicity of 20 nm silver nanoparticles (Ag-NP) was studied in a 28-day repeated-dose toxicity study in rats. Ag-NP were intravenously administered with a maximum dose of 6 mg/kg body weight (bw)/day. Several immune parameters were affected: reduced thymus weight, increased spleen weight and spleen cell number, a strongly reduced NK cell activity, and reduced IFN-γ production were observed.MethodsPrompted by these affected immune parameters, we wished to assess exposure effects on the functional immune system. Therefore, in the present study the T-cell dependent antibody response (TDAR) to keyhole limpet hemocyanin (KLH) was measured in a similar 28-day intravenous repeated-dose toxicity study. In addition, a range of immunological parameters was measured. Data obtained using the benchmark dose (BMD) approach were analyzed by fitting dose-response models to the parameters measured.ResultsA reduction in KLH-specific IgG was seen, with a lowest 5% lower confidence bound of the BMD (BMDL) of 0.40 mg/kg bw/day. This suggests that Ag-NP induce suppression of the functional immune system. Other parameters sensitive to Ag-NP exposure were in line with our previous study: a reduced thymus weight with a BMDL of 0.76 mg/kg bw/day, and an increased spleen weight, spleen cell number, and spleen cell subsets, with BMDLs between 0.36 and 1.11 mg/kg bw/day. Because the effects on the spleen are not reflected by increased KLH-specific IgG, they, however, do not suggest immune stimulation.ConclusionsIntravenous Ag-NP administration in a 28-day repeated-dose toxicity study induces suppression of the functional immune system. This finding underscores the importance to study the TDAR to evaluate immunotoxicity and not to rely solely on measuring immune cell subsets.

T-cell-dependent antibody responses in the rat: Forms and sources of keyhole limpet hemocyanin matter

The T-cell-dependent antibody response (TDAR) is a functional assay used in immunopharmacology and immunotoxicology to assess ability to mount an antibody response to immunization. Keyhole limpet hemocyanin (KLH) is extensively used as the immunogen of choice in non-clinical and clinical settings. Native KLH is comprised of high molecular weight (HMW; 4–8 MDa) assemblies of KLH subunit dimers (> 600–800 kDa). It is not known how the different forms (HMW vs subunit) and manufacturing processes (commercial sources) may impact the nature of anti-KLH immune responses (e.g. magnitude and inter-animal variability). Anti-KLH IgM and IgG responses were studied in Sprague-Dawley rats immunized with different forms and commercial sources of KLH: 100 µg of HMW KLH from two different sources or subunit KLH from three different sources. Biophysical and biochemical analyses were conducted to characterize the KLH formulations. Anti-KLH IgM and IgG responses were measured using a proprietary indirect quantitative electrochemiluminescence immunoassay. The HMW KLH preparations showed a greater number of sub-visible particles (2–150 µm size range) than the subunit KLH preparations. All HMW KLH and all subunit KLH were equivalent on SEC (hydrodynamic volume), PAGE (size and charge), and SDS-PAGE (molecular radius). Robust primary and secondary anti-KLH responses were detected for both sources of HMW KLH. The subunit KLH immunizations resulted in lower IgG and IgM responses compared to the HMW KLH, with the exception of Stellar Biotechnologies subunit KLH that produced both robust primary and secondary responses, which approached the HMW KLH responses. Inter-animal variability for IgM and IgG responses was lower with HMW KLH than with subunit KLH. In conclusion, different forms and commercial sources of KLH were associated with different magnitudes and inter-animal variability in IgM and IgG responses, a critical finding to take into consideration when designing TDAR studies for robust immunotoxicology or immunopharmacology testing.

Evaluation of canine T-cell dependent antibody response to the primary and secondary immunization with keyhole limpet hemocyanin

T-cell dependent antibody response (TDAR) incorporating both primary and secondary responses to keyhole limpet hemocyanin (KLH) in canine models have not yet been fully understood. To develop a practical dog TDAR model, we characterized primary and secondary antibody responses by intravenous or intramuscular immunization of KLH twice at intervals of 8 days during a 28-day course of study. Primary immunization with KLH by both routes induced a maximum IgM response on 6 to 8 days after the treatment, whereas the IgG response started 6 to 8 days after the treatment with relatively low levels. Remarkable increases in anti-KLH IgG levels (about 10-times compared with the primary response) were produced 5 to 7 days after the secondary KLH immunization by both routes. These results indicate that IgM-predominant and IgG-predominant responses were respectively induced by the primary and secondary immunization. Furthermore, the intravenous route showed higher baseline titers of primary and secondary anti-KLH IgM responses, suggesting that intravenous immunization of KLH might be a more suitable method for immunotoxicity evaluation. No remarkable inter-individual variability was noted in our canine models. Treatment with cyclophosphamide at 2 mg/kg/day for a consecutive 28 days significantly suppressed primary and secondary anti-KLH IgM and IgG responses induced by KLH injection on Days 15 and 23 of CPA treatment. These results demonstrate that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in dogs when exposed to potential immunomodulatory drugs.

Evaluation of primary and secondary responses to a T-cell-dependent antigen, keyhole limpet hemocyanin, in rats

To develop a rat T-cell-dependent antibody response (TDAR) model evaluating both primary and secondary antibody responses, keyhole limpet hemocyanin (KLH) was used to immunize rats twice during a 14-day course of study, a pattern closely linked to that of a short-term general toxicity study. Female rats of four representative strains (e.g., Sprague-Dawley, Wistar, Fischer, and Lewis) were immunized twice with intravenous administrations of KLH (300 µg/rat) on Days 5 and 9 during a 14-day treatment regimen with cyclophosphamide (CPA) at 1, 3, or 6 mg/kg/day. The primary and secondary immunizations of KLH markedly elevated serum anti-KLH IgM and IgG levels in all strains on Days 9 and 15. Remarkable higher levels of anti-KLH IgG (≈ 1000 µg/ml) were noted in all strains, which were more than 4-times compared with those of anti-KLH IgM levels at Day 9, indicating that predominant IgG reactions were induced by the dual immunizations. A large inter-individual variability in KLH-specific IgM and IgG production was observed in all strains. However, levels of the KLH-specific antibodies were considered sufficient for the evaluation, even in Sprague-Dawley and Wistar rats reported as strains with a wide range of variability since immunosuppression of CPA on responses in both anti-KLH IgM and IgG were observed in all strains to the same extent. In addition, the sensitivity of the KLH-ELISA assay system detecting the immunosuppressive effects of CPA was comparable to other assay systems with PFC assay or ELISA using SRBC. The results here demonstrated that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in rats when exposed to potential immunomodulatory drugs. Furthermore, the design of the presented TDAR study would support comprehensive evaluation together with the outcome of the conventional general toxicity study.

Assessing a Theoretical Risk of Dolutegravir-Induced Developmental Immunotoxicity in Juvenile Rats

HIV-1 integrase inhibitors (INIs) are a promising class of antiretrovirals for the treatment of HIV in adults; there is interest in expanding their use into pediatric populations. A theoretical concern for developmental immunotoxicity was raised after a publication suggested that two HIV INI tool compounds inhibited in vitro cleavage activity of recombination activating genes 1 and 2 (RAG1/2) through the inhibition of their binding to recombination signal sequences. RAG1/2 are required for the development of mature B and T lymphocyte populations. The potential effects of the investigational INI dolutegravir on RAG1/2 were addressed by developing assays in juvenile rats to measure T cell receptor (TCR) Vβ usage by flow cytometry as an indicator of TCR repertoire diversity and a T cell dependent antibody response (TDAR) as an indicator of immunosuppression. These endpoints were incorporated into a juvenile rat toxicity study, along with immunophenotyping, hematology, and histopathology of immunologic organs. Dose levels of 0, 0.5, 2, or 75mg/kg/day dolutegravir were given via oral gavage from postnatal day 4 through 66. At the highest dose, there was decreased body weight gain and two preweanling deaths; however, there were no treatment-related effects on developmental parameters. There were no effects on immunologic competence, as measured by TDAR, and no effects on lymphocyte subsets or CD4 and CD8 TCR Vβ usage in peripheral blood. Histopathology of immunologic organs (spleen, thymus, lymph nodes) and hematology evaluation revealed no effects. The no observed adverse effect level for immunotoxicity endpoints was 75mg/kg/day.

Germinal Center Texture Entropy as Possible Indicator of Humoral Immune Response: Immunophysiology Viewpoint

In this article, we present the results indicating that spleen germinal center (GC) texture entropy determined by gray-level co-occurrence matrix (GLCM) method is related to humoral immune response. Spleen tissue was obtained from eight outbred male short-haired guinea pigs previously immunized by sheep red blood cells (SRBC). A total of 312 images from 39 germinal centers (156 GC light zone images and 156 GC dark zone images) were acquired and analyzed by GLCM method. Angular second moment, contrast, correlation, entropy, and inverse difference moment were calculated for each image. Humoral immune response to SRBC was measured using T cell-dependent antibody response (TDAR) assay. Statistically highly significant negative correlation was detected between light zone entropy and the number of TDAR plaque-forming cells (r (s) = -0.86, p < 0.01). The entropy decreased as the plaque-forming cells increased and vice versa. A statistically significant negative correlation was also detected between dark zone entropy values and the number of plaque-forming cells (r (s) = -0.69, p < 0.05). Germinal center texture entropy may be a powerful indicator of humoral immune response. This study is one of the first to point out the potential scientific value of GLCM image texture analysis in lymphoid tissue cytoarchitecture evaluation. Lymphoid tissue texture analysis could become an important and affordable addition to the conventional immunophysiology techniques.

Developmental immunotoxicity in male rats after juvenile exposure to di- n-octyltin dichloride (DOTC)

To determine relevant endpoints for evaluating developmental immunotoxicity due to juvenile exposure and optimal age of the animals at assessment, a wide range of immunological parameters were assessed in a juvenile toxicity study. Rats were exposed to di- n-octyltin dichloride (DOTC) by gavage from postnatal day (PND) 10 through PND 21 and via the diet after weaning using a benchmark dose (BMD) approach. Immune assessments were performed in male rats on PNDs 21, 42, and 70 and a subset of animals was used to evaluate the T-cell dependent antibody response (TDAR) to Keyhole limpet hemocyanin. Immune effects were more pronounced on PND 21 and 42 and observed at lower doses than developmental effects. The most sensitive immune parameters affected included TDAR parameters and thymocyte subpopulations with lower confidence limits of the benchmark doses (BMDLs) below the overall no-observed-adverse-effect-level (NOAEL) for DOTC reported so far in literature. These findings illustrate the relative sensitivity of the developing immune system for DOTC, the additional value of assessing functional immune parameters, and underscore the relevance of juvenile immunotoxicity testing in view of the risk assessment of chemicals.

Antibody-mediated rejection: treatment alternatives and outcomes

Over the past 10 years, thanks to the development of sensitive methods of antibody detection and markers of antibody injury such as C4d staining, the role of anti-human leukocyte antigen (HLA) and non-HLA alloantibodies as effectors of acute and chronic immune allograft injury has been revisited. Antibody-mediated rejection (AMR) defines all allograft rejection caused by antibodies directed against donor-specific HLA molecules, blood group antigen (ABO)-isoagglutinins, or endothelial cell antigens. Antibody-mediated rejection can be a recalcitrant process, resistant to therapy and carries an ominous prognosis to the graft. In concordance with these views, treatment protocols for AMR use permutations of a multiple-prong approach that include (1) the suppression of the T-cell dependent antibody response, (2) the removal of donor reactive antibody, (3) the blockade of the residual alloantibody, and (4) the depletion of naive and memory B-cells. Although all published protocols report a variable rate of success, a major weakness of all current protocols is the lack of effective anti-plasma cell agents. In comparison to acute AMR, the treatment for chronic AMR (CAMR) is not well characterized. Although in acute AMR large titers of pre-existent alloantibodies result in massive activation of the complement system and lytic injury of the graft endothelium, thereby requiring aggressive and fast removal of the offending agents, in CAMR, complement activation results in sublytic endothelial cell injury and activation. Although this type of injury results in chronic graft failure, its slow progression likely renders it amenable of suppression by heightening of maintenance immunosuppression and anti-idiotypic blockade of the circulating alloantibody without the need of plasma exchange. In this review, we will discuss the rationale behind the design of treatment protocols for acute AMR and CAMR as well as their reported results and complications.

T cell tolerance to AChR, the autoantigen in myasthenia gravis, is mediated primarily by peripheral mechanisms (128.25)

Abstract In myasthenia gravis (MG), a T-cell dependent antibody response is directed against acetylcholine receptor (AChR) at the neuromuscular junction. To study T cell tolerance, we generated a transgenic mouse in which the TAChRα chain is expressed as a self protein. This transgenic was bred to nude mice and thymus transplants were performed to produce progeny expressing TAChRα in the thymus and/or the periphery (muscle). These mice were immunized with the TAChRα dominant epitope, p146–162, and in vitro proliferation of responding T cells was measured. Since AChR is expressed at low levels in the thymus, it was presumed that central tolerance would be important. However, this was not the case; only mice with peripheral expression of TAChRα showed tolerance (a lack of proliferation). Furthermore, CFSE-labeled TAChR specific T cells from a TCR transgenic were adoptively transferred into TAChR transgenic (tolerizing) and non-transgenic recipients. Following immunization, proliferation of the transferred T cells was lower in the TAChR transgenic host. Moreover, when the TAChR transgene was bred to scurfy mice, progeny lacking Tregs (foxp3−/Y) exhibited an increase in serum anti-TAChR Ig. Autoantibodies were not seen in the foxp3+/Y mice or in mice lacking Aire function (which have defective central tolerance). These results are consistent with a primary role for peripheral tolerance in regulating the response to AChR. Supported by an APC Subcontract, a UTHSCSA ERC grant and NIH (T32AG021890, T32DE11438).

Primary Immune Response to Sheep Red Blood Cells (SRBC) as the Conventional T-Cell Dependent Antibody Response (TDAR) Test

The production of antigen-specific antibodies represents a major defense mechanism of humoral immune responses. Several assays have been developed to assess T-cell-dependent antibody responses (TDAR). Of these assays, the antibody forming cell assay (AFC) or plaque forming cell (PFC) assay and ELISA are the two most often used tests to assess immunotoxicity. Historically, the T-cell-dependent antigen of choice has been sheep red blood cells (SRBC). The SRBC AFC assay is considered the “gold standard” for TDAR based on extensive intra- and inter-laboratory validation in mice and has been utilized for over 35 years. The quantification of the primary AFC response (i.e., the specific IgM antibody-forming cell response) was found to provide one of the best predictors of immunotoxicity in mice. The SRBC-specific ELISA is relatively new, with the first publication of the method appearing in 1993. Data from the application of using both the SRBC specific AFC and ELISA for evaluation of potential immunotoxicity of chemicals in rodents and the pros and cons and associated issues of each method were presented. Specifically, the following was discussed: (1) studies investigating the incorporation of the SRBC-specific IgM ELISA in rats on standard toxicology study; (2) characterization of an approach to developmental immunotoxicology assessment in the rat using SRBC as the antigen; and, (3) data from an inter-laboratory study comparing the AFC assay and ELISA in outbred rodents using both cyclophosphamide and dexamethasone.

Immune Function Tests for Hazard Identification: A Paradigm Shift in Drug Development

Routine immune function testing in preclinical drug development was established as a regulatory requirement in June of 2000 under the Committee of Proprietary Medicinal Products (CPMP) Note for Guidance on Repeated Dose Toxicity (CPMP/SWP/1042/99). The purpose of the more stringent approach to immunotoxicology testing was to better identify unintended immunosuppression; however, the requirement was met with much discussion and debate. At the center of the discussion was an attempt to reconcile opposing regulatory directives from agencies outside of Europe that adhere to a more selective, weight-of-evidence approach to functional evaluations. Uncertainty over the predictive value of the recommended immune function tests relative to conventional toxicology parameters prompted an investigation by the International Committee on Harmonization (ICH). The results of a preliminary, industry-wide survey indicated that only a low percentage of pharmaceuticals adversely affect immune function without alterations to standard toxicology parameters. Expected ICH guidelines will ultimately determine to what extent and for what purpose immune function tests will be conducted. In the meantime, optimization of the recommended immune function tests is ongoing. The T-cell dependent antibody response (TDAR) by either conventional Sheep Red Blood Cell (SRBC) plaque assay or by the modified ELISA method using either SRBC or keyhole limpet hemocyanin (KLH) as antigen is being extensively evaluated to determine best practices and procedures for preclinical immunotoxicity evaluations. This review addresses some aspects of the debate concerning the appropriateness of immune function tests for hazard identification, along with recommendations for optimizing TDAR methodology to ensure adequate sensitivity and predictability in risk assessments for immunotoxicity.

Development and Validation of a Canine T-Cell-Dependent Antibody Response Model for Immunotoxicity Evaluation

A T-cell dependent antibody response (TDAR) model to evaluate compounds for potential immunotoxicity in dogs has not been reported. The objective of these studies was to develop and validate a dog TDAR model using the T-cell dependent antigen, keyhole limpet hemocyanin (KLH). Studies were conducted to determine the appropriate dose of KLH, immunization route and kinetics of the antibody response to KLH in the dog. To validate the sensitivity of this method, we investigated the TDAR to KLH in the dog with a known immunosuppressive drug, cyclosporine (Neoral). The results of this study demonstrate that a robust primary IgM and IgG response to KLH can be generated in dogs and the IgG response was sensitive to cyclosporine treatment.

T-Cell-Dependent Antibody Response: Assay Development in Cynomolgus Monkeys

The current study was designed to develop and test a T-cell dependent antibody response to keyhole limpet hemocyanin (KLH) in cynomolgus monkeys. In an optimization experiment, monkeys (3/sex) were given a single intramuscular injection of KLH at 10 mg/animal to evaluate the kinetics of the antibody response. Serum samples were collected pretest, and on Days 4, 6, 8, 11, 15 and 22 for measurement of anti-KLH IgM and IgG endpoint titers. In a subsequent experiment, female monkeys (3/group) were treated once daily by gavage with the immunosuppressive agent cyclosporine (Neoral) at 0, 10 and 50 mg/kg for 21 days, and the effects of drug treatment on anti-KLH IgM and IgG responses were determined. The effects of cyclosporine on hematology, biochemistry, bone marrow, organ weights, gross and histopathology, and peripheral lymphocyte subsets also were evaluated. Robust anti-KLH IgM and IgG responses were seen in monkeys given a single intramuscular injection of KLH at 10 mg/animal, with peak antibody responses at approximately 10–14 days post-immunization for anti-KLH IgM, and 14–21 days for anti-KLH IgG. Decreases in anti-KLH IgG endpoint titers were seen in 1 monkey given cyclosporine at 10 mg/kg, and 1 monkey dosed at 50 mg/kg. Relative to vehicle control animals, mild lymphoid depletion was evident in lymph nodes and tonsil of monkeys with suppressed anti-KLH IgG titers. Collectively, these findings in individual animals provided evidence of cyclosporine-induced immunosuppression. Cyclosporine at 10 and 50 mg/kg did not alter anti-KLH IgM production, hematology, biochemistry, bone marrow, organ weights, or peripheral lymphocyte subsets. Lastly, the results of this study demonstrated that KLH immunization at 10 mg/animal did not alter the standard toxicity endpoints evaluated in control animals.