Fully human anti-BAFF inhibitory monoclonal antibody tabalumab does not adversely affect T-dependent antibody responses in cynomolgus monkey (<i>Macaca fasicularis</i>): A summary of three pre-clinical immunotoxicology evaluations

<div><p></p><p>The potential immunotoxicity of tabalumab was assessed as a component of standard pre-clinical toxicology studies in cynomolgus monkeys. To evaluate potential tabalumab-associated immunosuppression after antigen challenge, cynomolgus monkeys were administered placebo control or tabalumab in three immunotoxicological safety studies. Study 1, a 4-week pilot study, evaluated biweekly intravenous (IV) control, and 0.3, 1.0, 5.0, and 15.0 mg/kg tabalumab doses. Study 2 evaluated IV control, and 0.1, 1.0, and 30.0 mg/kg tabalumab doses biweekly for 6 weeks. Study 3 evaluated IV control and 0.1, 1.0, 30.0 mg/kg, and subcutaneous (SC) 30.0 mg/kg tabalumab biweekly for 6 months, with recovery (16 weeks) to monitor standard immunotoxicity endpoints. T-cell dependent primary and secondary antibody responses to tetanus toxoid antigen challenge (4-week and 6-week studies) or keyhole limpet hemocyanin (KLH; 6-week and 6-month studies) were evaluated as a measure of immunocompetence, together with quantitation of T- and B-cell subsets. In addition, anti-tabalumab antibody formation (6-week and 6-month studies) was assessed. The results indicated that, despite expected decreases in circulating B-cell populations, no changes in follicle histopathology or organ weights, except decreases in spleen weight (after 6-months of 30 mg/kg IV/SC treatment only), were attributed to tabalumab. Non-adverse microscopic decreases in size or number of germinal centers in spleen, mesenteric, and mandibular lymph nodes occurred, but without an effect on antibody responses to KLH or tetanus. At 16-weeks recovery, microscopic compound-related changes observed after 6 months of treatment were completely reversed (0.1 mg/kg group) and partially reversed (1.0 and 30.0 mg/kg groups), while peripheral blood B cells remained 66–72% reduced from baseline. Despite reduced germinal centres in lymphoid organs, and reductions in circulating B cells, T-cell-dependent humoral immunity was maintained following tabalumab administration in three safety studies in cynomolgus monkeys.</p></div

Deletion of a Conserved <i>cis</i>-Element in the <i>Ifng</i> Locus Highlights the Role of Acute Histone Acetylation in Modulating Inducible Gene Transcription

<div><p>Differentiation-dependent regulation of the <i>Ifng</i> cytokine gene locus in T helper (Th) cells has emerged as an excellent model for functional study of distal elements that control lineage-specific gene expression. We previously identified a <i>cis</i>-regulatory element located 22 kb upstream of the <i>Ifng</i> gene (<u>C</u>onserved <u>N</u>on-coding <u>S</u>equence -22, or CNS-22) that is a site for recruitment of the transcription factors T-bet, Runx3, NF-κB and STAT4, which act to regulate transcription of the <i>Ifng</i> gene in Th1 cells. Here, we report the generation of mice with a conditional deletion of CNS-22 that has enabled us to define the epigenetic and functional consequences of its absence. Deletion of CNS-22 led to a defect in induction of <i>Ifng</i> by the cytokines IL-12 and IL-18, with a more modest effect on induction via T-cell receptor activation. To better understand how CNS-22 and other <i>Ifng</i> CNSs regulated <i>Ifng</i> transcription in response to these distinct stimuli, we examined activation-dependent changes in epigenetic modifications across the extended <i>Ifng</i> locus in CNS-22-deficient T cells. We demonstrate that in response to both cytokine and TCR driven activation signals, CNS-22 and other <i>Ifng</i> CNSs recruit increased activity of histone acetyl transferases (HATs) that transiently enhance levels of histones H3 and H4 acetylation across the extended <i>Ifng</i> locus. We also demonstrate that activation-responsive increases in histone acetylation levels are directly linked to the ability of <i>Ifng</i> CNSs to acutely enhance Pol II recruitment to the <i>Ifng</i> promoter. Finally, we show that impairment in IL-12+IL-18 dependent induction of <i>Ifng</i> stems from the importance of CNS-22 in coordinating locus-wide levels of histone acetylation in response to these cytokines. These findings identify a role for acute histone acetylation in the enhancer function of distal conserved <i>cis</i>-elements that regulate of <i>Ifng</i> gene expression.</p></div

Deletion of CNS-22 leads to prominent defects in IL-12+IL-18 dependent modulation of histone hyperacetylation.

<p>(<b>A</b>) Th1 cells generated from either WT or CNS-22^−/− mice were either left unstimulated or activated with IL-12+IL-18 for 1.5 h or anti-CD3 and anti-CD28 for 3 h and then subject to ChIP-chip with antibodies against H4K12ac. Levels of H4K12ac were analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003969#pgen-1003969-g003" target="_blank">Fig. 3</a> and visualized using the IGB browser. (<b>B</b>) H4K12ac levels across the <i>Ifng</i> locus documented in WT Th1 cells were normalized against H4K12ac levels documented in CNS-22^−/− Th1 cells to visualize in a semi-quantitative manner the magnitude to which locus-wide acquisition of H4K12ac marks was impaired in the absence of CNS-22. (<b>C</b>) Th1 cells generated from WT or CNS-22^−/− mice were restimulated with anti-CD3+anti-CD28 for 3 h or IL-12+IL-18 for 1.5 h and recruitment of RNA-Pol II to the <i>Ifng</i> gene was assessed by ChIP-qPCR. RNA-Pol II recruitment is shown as a percentage of input DNA. Data represent means from at least three independent experiments. Statistical analyses were carried out on means and standard errors from three independent experiments * p<0.01, # p<0.05, stimulated WT versus CNS-22^−/−.</p

Identification of putative regulatory elements greater than 100<i>Ifng</i> gene.

<p>(<b>A–B</b>) Naïve CD4⁺ T cells from OT-II transgenic WT mice were differentiated under Th1, Th2 or Th17 polarizing conditions. Cells were left unstimulated or activated with IL-12+IL-18 for 1.5 h and subject to ChIP-chip. To identify STAT4, Smc3 and CTCF binding sites and to define H4K12ac-enriched regions across the <i>Ifng</i> locus, peak calling was carried out using a previously described algorithm for capturing microarray enrichment (ACME) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003969#pgen.1003969-Crawford2" target="_blank">[49]</a>. Peak-calling thresholds were set to a confidence limit of 95% and visualized with the IGB browser <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003969#pgen.1003969-Nicol1" target="_blank">[48]</a>. Data are representative of at least two independent experiments.</p

IL-12+IL-18 driven <i>Ifng</i> transcription is compromised in CNS-22^−/− T cells and NK cells.

<p>(<b>A</b>) CD4⁺ T cells derived from OT-II transgenic WT and CNS-22^−/− mice were differentiated with 2 ng/ml IL-12, 5 µg/ml ova-peptide (ISQAVHAAHAEINEAGR) and cultured with CD4-depleted irradiated feeder cells derived from <i>Il12a</i>^−/− mice. Cells were reactivated for 4 h as described in methods and expression of IFN-γ was assessed by flow cytometric analysis following intracellular staining. Percentages of viable, IFN-γ⁺ T cells are indicated by black numbers and mean fluorescence intensities (MFI) of IFN-γ⁺ cells are indicated in grey. Data are representative of at least three independent experiments. (<b>B</b>) CD8⁺ T cells isolated from WT and CNS-22^−/− mice were differentiated with 2.5 µg/ml of anti-CD3 antibody, 2 ng/ml IL-12 for 3 days and reactivated for 4 h with IL-12+IL-18 and subject to intracellular staining. For evaluating IFN-γ expression in NK cells, following depletion of both CD4⁺ and CD8⁺ T cells bulk splenocytes were activated with IL-12+IL-18 for 4 h and subject to intracellular staining. Percentages of viable, IFN-γ⁺ T/NK cells are indicated in black and MFI of IFN-γ⁺ cells are indicated in grey. Data are representative of at least three independent experiments. (<b>C</b>) Total RNA from anti-CD3+anti-CD28 or IL-12+IL-18 stimulated WT and CNS-22^−/− Th1 cells was isolated at indicated time points and reverse-transcribed to generate cDNA. Transcript levels were measured by RT-PCR and relative levels of spliced transcripts were calculated by normalization against levels of spliced transcripts in resting Th1 cells. Data represent means from at least three independent experiments. Statistical analyses were carried out on means and standard errors from three independent experiments * p<0.01, # p<0.05, stimulated WT versus CNS-22^−/−.</p