T-cell Dependent Antibody Response Research Articles

Challenges and gaps in immunosafety evaluation of therapeutics: An IQ DruSafe survey

Immunotoxicology/immunosafety science is rapidly evolving, with novel modalities and immuno-oncology among the primary drivers of new tools and technologies. The Immunosafety Working Group of IQ/DruSafe sought to better understand some of the key challenges in immunosafety evaluation, gaps in the science, and current limitations in methods and data interpretation. A survey was developed to provide a baseline understanding of the needs and challenges faced in immunosafety assessments, the tools currently being applied across the industry, and the impact of feedback received from regulatory agencies. This survey also focused on current practices and challenges in conducting the T-cell-dependent antibody response (TDAR) and the cytokine release assay (CRA). Respondents indicated that ICH S8 guidance was insufficient for the current needs of the industry portfolio of immunomodulators and novel modalities and should be updated. Other challenges/gaps identified included translation of nonclinical immunosafety assessments to the clinic, and lack of relevant nonclinical species and models in some cases. Key areas of emerging science that will add future value to immunotoxicity assessments include development of additional in vitro and microphysiological system models, as well as application of humanized mouse models. Efforts are ongoing in individual companies and consortia to address some of these gaps and emerging science.

Preclinical Pharmacokinetics and Pharmacology Study of RC98: A Programmed Cell Death Ligand 1 Monoclonal Antibody in Cynomolgus Monkeys.

RC98 is the monoclonal antibody against Programmed Cell Death Ligand 1 (PD-L1). Relevant reports have confirmed that the influence of PD-L1 expressed by tumor cells on antitumor CD8+ T cell responses is well characterized, but the impact of PD-L1 expressed by immune cells has not been well defined. This study aimed to design a Pharmacokinetics/Pharmacology (PK/PD) study of RC98 in normal cynomolgus monkeys to research the effect on the immune system. RC98 and vehicle were administered to cynomolgus monkeys at 15 mg/kg via intravenous infusion once a week for 4 weeks to evaluate the relationship between PK and PD. The pharmacodynamic activity was measured by the PD-L1 receptor occupancy (RO) in CD3+ T cells, A T-cell-dependent antibody response (TDAR), and the concentration of soluble PD-L1. The pharmacokinetic result showed that the exposure from the last administration was lower than that of the first administration, probably due to immunogenicity production. There was a strong correlation between systemic exposure and RO in CD3+ T cells but decreased RO levels after the last dose, which indirectly reflected the activation of T cells. The keyhole limpet hemocyanin (KLH)-induced TDAR in the RC98 group was higher than in the vehicle group. The concentration of soluble PD-L1 had increased feedback with RC98, and the concentration of soluble PD-L1 was maintained at a higher level after multiple doses than before dosing. These data indicate that the immune system was clearly activated. In addition, the non-clinical data could provide a basis for its efficacy evaluation in clinical trials.

Determination of in vitro immunotoxic potencies of a series of perfluoralkylsubstances (PFASs) in human Namalwa B lymphocyte and human Jurkat T lymphocyte cells.

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment.

Methods for Analysis of Nanoparticle Immunosuppressive Properties.

Adverse drug effects on immune system function represent a significant concern in the pharmaceutical industry, because 10-20% of drug withdrawal from the market is attributed to immunotoxicity. Immunosuppression is one such adverse effect. The traditional immune function test used to estimate materials' immunosuppression is T cell dependent antibody response (TDAR). This method involves a 28-day in vivo study evaluating the animal's antibody titer to a known antigen (Keyhole Limpet Hemocyanin; KLH) with and without challenge. Due to the limited quantities of novel drug candidates, an in vitro method called human lymphocyte 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 nanoparticles. The antigen-specific lymphocyte 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.

Laboratory safety evaluation of lokivetmab, a canine anti-interleukin-31 monoclonal antibody, in dogs

Lokivetmab (Cytopoint®, Zoetis) is a canine monoclonal antibody that specifically binds and neutralizes interleukin (IL)-31. Lokivetmab is approved for use in dogs for the treatment of atopic dermatitis (AD) and allergic dermatitis. The laboratory safety of lokivetmab was evaluated in 2 studies by adapting the science-based, case-by-case approach used for preclinical and early clinical safety evaluation of human biopharmaceuticals. The main objectives were to demonstrate the safety of lokivetmab in healthy laboratory Beagle dogs by using integrated clinical, morphologic, and functional evaluations. In Study 1, dogs were treated s.c. with saline or lokivetmab at 3.3 mg/kg (1X, label dose) or 10 mg/kg (3X intended dose) for 7 consecutive monthly doses, with terminal pathology and histology assessments. In Study 2, the functional immune response was demonstrated in naïve dogs using the T-cell dependent antibody response (TDAR) test with 2 different dose levels of unadjuvanted keyhole limpet hemocyanin (KLH) as the model immunogen. The primary endpoint was anti-KLH IgG antibody titer, and secondary endpoints were ex vivo IL-2 enzyme-linked immunospot (ELISpot) and peripheral blood mononuclear cell lymphoproliferation assays. Both studies included monitoring general health, periodic veterinary clinical evaluations, serial clinical pathology and toxicokinetics, and monitoring for anti-drug antibodies.In both studies, the health of dogs receiving lokivetmab was similar to controls, with no treatment-related changes uncovered. Extensive pathology evaluations of immune tissues (Study 1) revealed no lokivetmab-related morphologic changes, and in dogs treated at 10 mg/kg lokivetmab, immunization with the model antigen KLH did not impair the functional antibody or T-cell recall responses. There were no immunogenicity-related or hypersensitivity-related responses observed in either study. These studies in healthy laboratory dogs showed that lokivetmab was well-tolerated, did not produce any treatment-related effects, and had no effect on immune system morphology or its functional response. These studies also demonstrated the utility of a science-based case-by-case approach to the safety evaluation of a veterinary biopharmaceutical product.

Twelve-month in utero safety assessment of gardenia blue, a natural food colorant.

Toxicity assessment of the food colorant Gardenia jasminoides Ellis at dietary exposures of 0.0%, 0.1%, 0.5%, 1.5%, 3.0% and 5.0% included measures of T-cell- dependent antibody response, neurotoxicity, and clinical and anatomic pathology in Sprague Dawley rats during mating, gestation, lactation, postnatal development, and following weaning for up to 12 months including 3- and 6-month interim evaluations. Blue coloration of the gastrointestinal tract, mesenteric lymph nodes and kidneys was present in treated rats only at necropsy with minimal blue coloration at the lowest dose and without histopathological correlates in any of the tissues. There was good survival with no consistent treatment-related changes in hematology, clinical chemistry, enhanced evaluation of lymphoid tissues, or tissue histopathology at interim and final time points. T-cell dependent antibody response and neurotoxicity screening were negative in treated rats. The no-observed-adverse-effect level (NOAEL) was determined to be 5.0% gardenia blue (2,854.5 and 3,465.4 mg/kg/day in parental males and females, respectively, prior to mating; 3,113.5 and 4,049.6 mg/kg/day in male and female offspring, respectively, following up to 12 months of exposure.

A T-cell-dependent antibody response (TDAR) method in BALB/c mice based on a cytometric bead array

Most current methods to assess T-cell-dependent antibody responses (TDAR) are semi-quantitative and based on measures of antibody titer generated against a standard antigen like keyhole limpet hemocyanin (KLH). The precision, sensitivity, and convenience of TDAR assays might be improved by applying rapid, sensitive, specific cytometric bead assays (CBA). In the study here, KLH antigen was covalently coupled onto the surface of cytometric beads using immune microsphere technology, and IgM antibody capture spheres were prepared for use in pretreatment processing of samples. The working parameters associated with this novel TDAR-CBA system were optimized in orthogonal experiments. The optimal concentration of the KLH coating solution in this system was 160 μg/ml, that of the anti-KLH IgG capture spheres 6.0 × 105/ml, and the optimal dilution of fluorescein isothiocyanate (FITC)-conjugated Affini-Pure Goat Anti-Mouse IgG (H + L) was 60 μg/ml. Repeated tests indicated that this approach yielded good linearity (r 2 = 0.9937) method, with a within-run precision of 3.1–4.9%, and a between-run precision of 4.4–4.9%. This new approach had a limit of detection of 113.43 ng/ml (linear range = 390.63–50 000), and an interference rate of just 0.04–3.51%. Based on these findings, it seems that a new mouse TDAR assay based on CBA can be developed that would appear to be more sensitive, accurate, and precise than the current TDAR assay approaches based on traditional ELISA.

P12-04 Development of a hematopoietic stem cell (murine system) based system as an alternative for the in vivo T-cell-dependent antibody response (TDAR) assay within the EOGRTS: Case-study with Parabens

P12-04 Development of a hematopoietic stem cell (murine system) based system as an alternative for the in vivo T-cell-dependent antibody response (TDAR) assay within the EOGRTS: Case-study with Parabens

Evaluation of immunotoxicitiy in biomedical research and development

Immunotoxicology aims at studying toxic effects of any substance on the immune system and its functions. In its various fields of application, this science is dependent on regulatory texts and guidelines. Studies are based on in vitro, ex vivo and in vivo techniques and are observational or functional allowing the identification of a toxic effect and its underlying mechanisms, respectively. Here, we review the various tests to perform in biomedical research and development, with a particular interest for the T-cell Dependent Antibody Response (TDAR) assay. We also briefly discuss the upcoming evolutions in this domain within a more ethically sound framework such as limiting the use of laboratory animals. These evolutions are represented by the development of relevant cell models.

Seroconversion after coronavirus disease 2019 vaccination in patients with immune deficiency

Safety and efficacy are 2 major drivers for any vaccination strategy and have come to the forefront in the setting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. Patients with underlying primary or secondary immunodeficiencies have variable response to vaccination, but inactivated or nonviable vaccines are generally considered safe in patients with immune deficiency.1 The efficacy of inactivated vaccines is variable in patients with humoral immune deficiencies, in which many can mount a protective T-cell–dependent antibody response to protein-conjugated vaccines, except for patients with congenital agammaglobulinemia, such as X-linked agammaglobulinemia (XLA), who may lack the ability to mount any antibody response.

A Comprehensive Overview of the Clinical Relevance and Treatment Options for Antibody-mediated Rejection Associated With Non-HLA Antibodies.

Although solid organ transplant results have improved significantly in recent decades, a pivotal cause of impaired long-term outcome is the development of antibody-mediated rejection (AMR), a condition characterized by the presence of donor-specific antibodies to HLA or non-HLA antigens. Highly HLA-sensitized recipients are treated with desensitization protocols to rescue the transplantation. These and other therapies are also applied for the treatment of AMR. Therapeutic protocols include removal of antibodies, depletion of plasma and B cells, inhibition of the complement cascade, and suppression of the T-cell–dependent antibody response. As mounting evidence illustrates the importance of non-HLA antibodies in transplant outcome, there is a need to evaluate the efficacy of treatment protocols on non-HLA antibody levels and graft function. Many reviews have been recently published that provide an overview of the literature describing the association of non-HLA antibodies with rejection in transplantation, whereas an overview of the treatment options for non-HLA AMR is still lacking. In this review, we will therefore provide such an overview. Most reports showed positive effects of non-HLA antibody clearance on graft function. However, monitoring non-HLA antibody levels after treatment along with standardization of therapies is needed to optimally treat solid organ transplant recipients.

Decreased immune response in monkeys administered a human T-effector cell agonist (OX40) antibody

The OX40 receptor plays a crucial co-stimulatory role in T effector cell survival, expansion, cytokine production, and cytotoxicity to tumor cells; therefore, OX40 agonists are being evaluated as anti-cancer immunotherapies, especially in combination with checkpoint inhibitors. To support clinical development of BMS-986178 (an OX40 agonist antibody), two repeat-dose toxicity studies were conducted in cynomolgus monkeys. In the first study, BMS-986178 was administered intravenously (IV) once weekly for one month at doses from 30 to 120 mg/kg. BMS-986178 was well tolerated; surprisingly, immune function was suppressed rather than increased based on pharmacodynamic (PD) and flow cytometry readouts (e.g. T-cell dependent antibody response [TDAR]). To determine whether immune suppression was due to a bi-phasic response, a follow-up study was conducted at lower doses (1 and 10 mg/kg). Although receptor engagement was confirmed, immune function was still suppressed at both doses. In addition, treatment-emergent anti-drug antibodies (ADAs) at 1 mg/kg resulted in hypersensitivity reactions and reduced BMS-986178 exposure after repeated dosing, which precluded a full PD assessment at this dose. In conclusion, BMS-986178 was clinically well-tolerated by monkeys at weekly IV doses from 10 to 120 mg/kg (AUC[0–168] ≤ 712,000 μg●h/mL). However, despite target engagement, PD assays and other immune endpoints demonstrated immune suppression, not stimulation. Due to the inverted immune response at higher doses and the onset of ADAs, additional repeat-dose toxicity studies of BMS-986178 in monkeys (that would typically be required to support Phase 3 clinical trials and registration) would not add value for human safety assessment.

OP0316 EMERGING BEST-IN-CLASS IL-2 VARIANT HIGHLIGHTS TREG-DIRECTED THERAPY FOR AUTOIMMUNE DISEASE

Background:Impairment or deficiency of regulatory T cells (Treg) is associated with chronic inflammation and autoimmune diseases. Interleukin 2 (IL-2) is a cytokine indispensable for Treg expansion and immunosuppressive function. However, expansion of cytotoxic effector T (Teff) and NK cells and the associated vascular leakage side effect limit the use of IL-2 in autoimmune diseases [1].Objectives:Cugene developed a long-acting IL-2 variant with high Treg specificity and low toxicity to restore immune homeostasis and self-tolerance, and potentially cure autoimmune and inflammatory diseases.Methods:IL-2 variants were generated based on the quaternary structure of IL-2 and IL-2Rαβγ (alpha, beta, gamma) complex. Biological activity was determined by examining differential signaling activity in induction of STAT5 phosphorylation in defined lymphocyte populations of human PBMC using flow cytometry. Binding activity was evaluated by ELISA. Pharmacokinetics, pharmacodynamics, safety and tolerability were assessed in mice and cynomolgus monkeys. Treg suppressive function was determinedin vivo/ex vivo,and anti-inflammatory and anti-antibody production efficacy were determined in delayed-type hypersensitivity (DTH) and T-cell-dependent antibody response (TDAR) models.Results:Structure-based rational design and activity-guided fine-tuning generated an optimized IL-2 variant, CUG252. It demonstrated a strong and near wild-type IL-2 ability to stimulate STAT5 phosphorylation in IL-2Rαβγ dominant Treg cells but abolished activities in IL-2Rβγ dominant effector CD4, CD8 and NK cells. This was a result of biased binding activity to IL-2Rα while dramatically attenuated binding to IL-2Rβγ complex. In mice and monkeys, administration of CUG252 resulted in dose-dependent increases in Treg proliferation and expansion by more than 10- and 30-fold, respectively, with largely abolished activities in CD4+ T conventional, cytotoxic CD8+ Teff and NK cells. The ratio of Treg/Teff cells achieved was as high as 0.4 in mice and 1.2 in monkeys. Both CD4+ and CD8+ Tregs were expanded with preferential increases in memory over naïve subsets. A substantial increase in Treg-suppressive capacity over T effector cells was corroborated by enhanced expression of functional and inhibitory markers, including CD25, Foxp3, PD-1, CTLA-4, Tim3 and ICOS. In DTH and TDAR models, CUG252 strongly inhibited antigen-driven inflammation, B cell maturation, and antibody production. The sustained PK/PD profile supports monthly dosing or better in humans. CUG252 was well-tolerated and no changes in body weight, body temperature, clinical pathology or signs of vascular leakage were observed. Moreover, CUG252 demonstrated superior manufacturability.Conclusion:CUG252 demonstrates an emerging best-in-class profile among IL-2 variants. It displayed exquisite Treg-selectivity while retaining potency comparable to wild-type IL-2. It showed strong anti-inflammatory and anti-antibody production efficacy with significantly improved therapeutic index and manufacturability. Its favorable drug-like property and robust preclinical efficacy warrant further evaluation in patients with a variety of inflammation and autoimmune diseases.

The enhanced immunopharmacology of VIB4920, a novel Tn3 fusion protein and CD40L antagonist, and assessment of its safety profile in cynomolgus monkeys.

Inhibition of the T- and B-cell interaction through the CD40/CD40 ligand (L) axis is a favourable approach for inflammatory disease treatment. Clinical studies of anti-CD40L molecules in autoimmune diseases have met challenges because of thromboembolic events and adverse haemostasis. VIB4920 (formerly MEDI4920) is a novel CD40L antagonist and Tn3 fusion protein designed to prevent adverse haemostasis and immunopharmacology. We evaluated the pharmacokinetics, activity and toxicity of VIB4920 in monkeys. Cynomolgus monkeys received i.v. or s.c. 5-300 mg·kg-1 VIB4920 or vehicle, once weekly for 1 month (Studies 1 and 2) or 28 weeks (Study 3). VIB4920 exposure and bioavailability were determined using pharmacokinetic analyses, and immune cell population changes via flow cytometry. Pharmacological activity was evaluated by measuring the animals' capacity to elicit an immune response to keyhole limpet haemocyanin (KLH) and tetanus toxoid (TT). VIB4920 demonstrated linear pharmacokinetics at multiple doses. Lymphocyte, monocyte, cytotoxic T-cell and NK cell counts were not significantly different between treatment groups. B-cell counts reduced dose-dependently and the T-cell dependent antibody response to KLH was suppressed by VIB4920 dose-dependently. The recall response to TT was similar across treatment groups. No thromboembolic events or symptoms of immune system dysfunctionality were observed. VIB4920 demonstrated an acceptable safety profile in monkeys. VIB4920 showed favourable pharmacokinetics, dose-dependent inhibition of a neoantigen-specific immune response and no adverse effects on immune function following long-term use. Our data support the use of VIB4920 in clinical trials.

Nonclinical Immunotoxicity Testing in the Pharmaceutical World: The Past, Present, and Future.

An examination for potential direct or indirect adverse effects on the immune system (immunotoxicity) is an established component of nonclinical testing to support safe use of new drugs. Testing recommendations occur in various regulatory guidance documents, especially ICH S8, and these will be presented. Key evaluation usually occurs in toxicology studies with further investigative work a consideration if a positive signal is seen. Expectations around whether findings may occur are related to the type of compound being developed, including a chemically synthesized small molecule, a small molecule oncology drug, a biopharmaceutical, an oligonucleotide, a gene therapy/stem cell product, a vaccine, or reformulation of drugs in liposomes or depots. Examples of immunotoxicity/immunogenicity findings will be discussed for all of these types of compound. Overall, it can be concluded that our main tool for evaluation of potential immunotoxicity/immunogenicity for a new drug still remains standard toxicology study testing with key assessment for effects on clinical pathology and lymphoid organs/tissues (weights and cellularity). Additional evaluation from studies using a T cell-dependent antibody response (TDAR) and lymphocyte phenotyping is also valuable, if needed. Thus, using the tools from the past, it is the role of toxicologists to work with clinical teams now and in the future, to interpret findings from nonclinical testing to possible adverse findings in humans.

Cross-company evaluation of the human lymphocyte activation assay

Nonclinical immunotoxicity evaluation is an important component of safety assessment for pharmaceuticals. One in vitro assay that can be applied in a weight of evidence assessment is the human lymphocyte activation (HuLA) assay, an antigen recall assay, similar in many respects to the in vivo T-cell-dependent antibody response (TDAR) in that cooperation of multiple immune cell types are needed to produce responses. This assay uses human cells and is more amenable than the TDAR to compound ranking and mechanistic studies. The HuLA assay requires less time and drug than TDAR assays, uses a relevant antigen (influenza), reflects a human immune response, and applies principles of the 3Rs to non-clinical safety assessment. Peripheral blood mononuclear cells (PBMC) from flu-immunized donors are re-stimulated with flu-vaccine in the presence of test articles, and proliferation is measured. Published data demonstrate the applicability of the HuLA assay, but it has not been evaluated for reproducibility across testing sites. To evaluate assay reproducibility, scientists from a consortium of institutions conducted the assay in parallel, using a common pool of donor PBMC, influenza vaccine, and known immunosuppressant compounds (cyclosporine A and mycophenolic acid). The HuLA assay was highly reproducible in identification of inhibition of antigen-specific responses, and there was significant agreement across testing sites in the half maximal inhibitory concentration (IC50) values. Intra-site variability was the largest contributor to the variability observed within the assay. The HuLA assay was demonstrated to be ideally suited to comparing multiple compounds (i.e. compound ranking or benchmarking) within the same assay. Overall, the data reported herein support the HuLA assay as a useful tool in mechanistic evaluations of antigen-specific immune responses.

Nonclinical Immunotoxicity Testing in the Pharmaceutical World: The Past, Present, and Future.

An examination for potential direct or indirect adverse effects on the immune system (immunotoxicity) is an established component of nonclinical testing to support safe use of new drugs. Testing recommendations occur in various regulatory guidance documents, especially ICH S8, and these will be presented. Key evaluation usually occurs in toxicology studies with further investigative work a consideration if a positive signal is seen. Expectations around whether findings may occur are related to the type of compound being developed, including a chemically synthesized small molecule, a small molecule oncology drug, a biopharmaceutical, an oligonucleotide, a gene therapy/stem cell product, a vaccine, or reformulation of drugs in liposomes or depots. Examples of immunotoxicity/immunogenicity findings will be discussed for all of these types of compound. Overall, it can be concluded that our main tool for evaluation of potential immunotoxicity/immunogenicity for a new drug still remains standard toxicology study testing with key assessment for effects on clinical pathology and lymphoid organs/tissues (weights and cellularity). Additional evaluation from studies using a T cell-dependent antibody response (TDAR) and lymphocyte phenotyping is also valuable, if needed. Thus, using the tools from the past, it is the role of toxicologists to work with clinical teams now and in the future, to interpret findings from nonclinical testing to possible adverse findings in humans.

Improvement and optimization of a T-cell-dependent antibody response (TDAR) method for BALB/c mice using keyhole limpet hemocyanin (KLH) as specific antigen

Although T-cell-dependent antibody response (TDAR) assays with keyhole limpet hemocyanin (KLH) as specific antigen have many advantages, most experiments produce qualitative results based on antibody titers. It was hypothesized that if experimental conditions (like antigen coating concentration, serum dilution, and detecting [here, horseradish peroxidase-goat anti-mouse IgG] antibody dilution) could be optimized, the resulting measured value (here, optical density) could be used to directly analyze and evaluate the experimental results. This means specifically that the assay OD values could be used for approximate quantitative statistical analysis; it does not require a further conversion of the data into qualitative forms or require obtaining further titer data from additional experiments. As such, the use of this “improved” assay would: greatly reduce the complexity of experimental operations; improve overall sensitivity and practicality of traditional TDAR assays; and, allow for direct assessing of any immunosuppression caused by a test drug in a host. Here, KLH-immunized serum was obtained from BALB/c mice, and the means to detect serum anti-KLH antibodies by an indirect ELISA were optimized. The results indicated that in this system, the optimal KLH coating concentration was 80 μg/ml, the optimal dilution range of the serum (at immunization dose of 5 mg KLH/kg) was 1:200–1:800, and the optimal dilution of HRP-goat anti-mouse IgG antibody was 1:16,000. Methodology verification was performed and a regression model of y = 144.16x + 0.9815 (R2 = 0.9571, indicating very good linearity) was obtained. Intragroup precision was 7.51–9.40%; the intergroup coefficient of variation was 9.83–14.22%. The lower limit of detection was 0.1385. The present results showed this indirect ELISA exhibited very good linearity, accuracy, and precision.

Strain differences in histopathological features of lymphoid tissues of SD and F344 rats in a T cell-dependent antibody response assay of cyclophosphamide.

When conducting histopathological evaluation of lymphoid tissues, it is necessary to know the variability and strain differences in histological features of different sites of lymphoid tissues. To investigate in detail the variability of lymphoid tissues and strain differences of control rats as well as those of immune reactivity and sensitivity to immunosuppression, we performed a histopathological analysis of various lymphoid tissues in conjunction with the evaluation of immune function in a T cell-dependent antibody response (TDAR) assay with cyclophosphamide (CP) in Sprague Dawley (SD) and F344 rats. Six-week-old male SD and F344 rats were orally treated with CP at 0 (control) or 4 mg/kg/day for 28 days; keyhole limpet hemocyanin (KLH) was introduced intravenously on Days 14 and 23, and the serum concentrations of anti-KLH antibodies were measured. HE staining and immunohistochemistry for T-cell (CD3) and B-cell (CD45RA) markers were performed using tissues from the spleen, thymus, and various lymph nodes. In CP-treated rats of both strains, decreased concentrations of anti-KLH antibodies were observed. Histopathological analysis revealed decreased lymphocytes mainly in the B-cell area, and these changes induced by CP treatment were more prominent in the F344 rats than in the SD rats. The present study also demonstrated that some of the lymphoid tissues of the control F344 rats were less developed than those of the control SD rats, suggesting that F344 rats might be easily affected by CP-induced immunosuppression. This information concerning rat strain differences in lymphoid tissues will be useful in histopathological evaluation for drug-induced immunotoxicity.

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.