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 TDAR   SLE

T-CELL-DEPENDENT ANTIBODY RESPONSE (TDAR)
OVERVIEW

The immune system has been identified as a target organ for adverse effects caused by test material administration. Because of this, the T-Cell-Dependent Antibody Response (TDAR) assay is used to assess potential immunotoxicity and to determine the impact of a test material on the immune response at the preclinical stage of drug discovery. This assay is the gold-standard for studying these effects due to the requirement of multiple immune functions and cell types to generate an antibody response including antigen uptake and presentation, CD4 T cell help, B cell activation and antibody production.

Biomere has validated multiple TDAR models including non-human primates and rodent models.

TDAR ASSAY ANIMAL MODELS AVAILABLE

Non-Human Primates

• KLH-peptide immunization in cynomolgus macaques

Rodents

• KLH-peptide immunization in C57BL/6 female mice
• OVA-peptide in alhydrogel immunizations in C57BL/6 female mice

TDAR ASSAY READOUTS

• IgG and IgM serum levels by ELISA
• Immune response analysis by flow cytometry
• Hematology analysis by complete blood count (CBC)

Female C57BL/6 mice from JAX were treated with either vehicle or anti-CD154 mAb intraperitoneally (IP) on Day -1. On Day 0, mice were given NP-KLH IP. Serum was collected from mice on Day -1, 7, and 14 and (A) anti-NP IgM and (B) anti-NP IgG ELISAs were performed.

Naïve male and female cynomolgus macaques of Chinese origin were treated with vehicle or anti-CD154 mAb IV on Day 0, 14, and 28. On Day 3 and 31, NHPs received KLH intramuscularly (IM). Serum was collected from animals twice weekly through Day 56 and (A) anti-KLH IgM and (B) anti-KLH IgG ELISAs were performed on serum samples. (C-I) Flow cytometry was performed on blood samples on Day 49. Frequency of (C) CD4+ TH cells, (D) IFNγ+ TH cells, and (E) TNFα+ TH cells. Frequency of (F) CD20+ B cells, (G) Naïve B Cells, (H) Germinal Center B Cells, and (I) Memory B Cells.

Summary

Significant decreases were observed in IgM and IgG antibodies generated in both the mouse and NHPTDAR assays in immune suppressive treatment (α-CD40L) compared to vehicle control. A proportional decrease in germinal center B cells compared to other B cell groups, as well as a decrease in B cells as a total population in α-CD40L versus control were observed. Decreases in the proportion of CD4+ TH cells were also observed in α-CD40L treated versus control, with a decrease in the proportion of IFNγ+ TH cells. From these results, we can see α-CD40L was able to suppress the generation of antibodies against the target antigen and impacted the TH cell and germinal center B cell response.

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MOUSE MODELS OF SYSTEMIC LUPUS
ERYTHEMATOSUS (SLE)

NZB/W F1 MOUSE MODEL OF SLE

Characteristics

• Most commonly used preclinical model for SLE
• Model developed by Helyer and Howie in 1963 and subsequently transferred to Jackson Laboratories
• Develops anti-dsDNA (auto) antibodies at ≥ 16 weeks of age
• Develops 3+ proteinuria after 20 weeks of age
• NZM lines: Genetic susceptibility loci (sle1,2,3)

Clinical Measures

• Proteinuria
• Weekly body weights
• Anti-dsDNA antibody ELISA
• Glomerular filtration rate
• IDEXX clinical analyzer

FLOW CYTOMETRY

• B cells, T cells, dendritic cells, NK cells, NKT cells, apoptosis
• Experienced with multicolor (up to 10 color) panel design
• Validation with human (PMBCs), rat and mouse cells (whole blood, splenocytes and lymph node cells)

HISTOPATHOLOGY

• Glomerulonephropathy
• Dilated tubules
• Degenerate tubules
• Lymphocyte aggregates

NZB/W F1 SLE MOUSE PREVENTION STUDY

• N=10/group with proteinuria = 0+ at 20 weeks of age
• Dose once every two weeks from 20 – 46 weeks of age
• Measure body weights once weekly and proteinuria once every two weeks
• Humane survival end points: ≥ 3+ proteinuria on two consecutive weeks

NZB/W F1 SLE MOUSE REMISSION STUDY

• NZB/W F1 female mice with moderate proteinuria (1 – 2+) were entered into groups at 28 weeks of age (n = 10/group) and initiated treatment (bar)
• Proteinuria was measured once every two weeks (the following week to confirm a ≥ 3+ reading)
• Humane survival end points: ≥ 3+ proteinuria on two consecutive weeks

Both mice reached the same humane endpoint, but lymphocyte aggregates were noticeably more severe in MRL/lpr mice (mean score = 3) compared to NZB/W F1 mice (mean score = 2)

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