Characterization of the draining lymph node response in the mouse drug allergy model: A model for drug hypersensitivity reactions.

The mouse drug allergy model (MDAM) was developed as a tool to predict the potential of systemically administered drugs to produce hypersensitivity reactions (HR). Drugs associated with HR in the clinic produce a marked increase in the cellularity of the draining lymph nodes (DLN) in the MDAM. The objective of this study was to characterize the changes in the DLN following exposure to drugs associated with HR and to investigate whether lymphocyte migration and/or proliferation play a role in the response. These endpoints were also investigated in the local lymph node assay (LLNA) to determine whether responses between the two assays occur via similar mechanisms. Results demonstrated that total numbers of T- and B-cells were proportionally increased in the DLN of mice treated with positive control drugs (i.e. abacavir, amoxicillin, ofloxacin, and sulfamethoxazole) compared to animals administered the vehicle or negative control drugs (metformin and cimetidine). In contrast, a significant increase in the B-cell population of the DLN was observed for 2,4-dinitrofluorobenzene (DNFB) following the LLNA protocol. Down-regulation of CD62L and up-regulation of CCR7 were observed for T-cells from the DLN of the positive control treated mice in the MDAM, but not with DNFB in the LLNA. A mild increase in T-cell proliferation was observed in the MDAM with positive control drugs, while DNFB in the LLNA induced proliferation within the B-cell population only. Anti-CD40L antibody administration inhibited MDAM responses to positive control drugs, but did not affect DNFB-induced increases in total cell number in the LLNA. These results suggest that the increased cellularity of the DLN in the MDAM may be the result of drug-induced alterations in lymphocyte migration and/or effects on lymphocyte proliferation. Moreover, it appears that different mechanisms may be involved in driving the MDAM and LLNA responses.

The CD40 agonist antibody CP-870,893 enhances dendritic cell and B-cell activity and promotes anti-tumor efficacy in SCID-hu mice.

CD40 is a member of the TNF family of receptors that has been shown to play a crucial role in enhancing dendritic cell activity and fostering anti-tumor immune responses. In this study, we demonstrate the in vitro properties and in vivo efficacious activity of the CD40 agonist antibody, CP-870,893. CP-870,893 is a fully human, IgG2 antibody that selectively interacts with CD40 at a site distinct from its ligand-binding region with a KD of 0.4 nM. It enhances the expression of MHC class II, CD54, CD86, and CD23 on human B cells in vitro. CP-870,893 also enhances dendritic cell activity as evidenced by cytokine secretion (IL-12, IL-23, IL-8), the upregulation of CD86 and CD83, and the ability to prime T cells to secrete IFNγ. In SCID-beige mice, a single parenteral injection of CP-870,893 was therapeutically effective against several CD40(pos) human tumors (B-cell lymphoma, breast, colon, and prostate) indicating direct effects on tumor cell survival and/or growth. When mice were co-implanted with human T cells and dendritic cells, the activity of CP-870,893 against CD40(pos) tumors increased, and efficacy was also observed against CD40(neg) and CD40(low) tumors demonstrating the ability of CP-870,893 to enhance anti-tumor immune function in vivo. These studies suggest that CP-870,893 has the potential to be efficacious against a wide range of tumor types through both direct and immune-mediated effects.

Human lymphocyte activation assay: an in vitro method for predictive immunotoxicity testing.

Preclinical immunotoxicity assessments may be performed during pharmaceutical drug development in order to identify potential cause for concern prior to use in the clinic. The in vivo T-dependent antibody response (TDAR) is widely used in this regard, given its sensitivity to known immunosuppressive compounds, but may be impractical early in drug development where quantities of test article are limited. The goal of the current work is to develop an in vitro human cell-based assay that is sensitive to immunosuppression, uses relatively small quantities of test article, and is simple to perform with moderate to high throughput. Ideally, this assay would require the cooperation of multiple cellular compartments to produce a response, similar to the TDAR. Although the Mishell-Dutton assay (in vitro mouse splenic sheep red blood cell response) has been used for this purpose, it shows considerable inter-laboratory variability, and rodent cells are used which leads to potential difficulty in translation of findings to humans. We have developed an assay that measures an influenza antigen-specific response using frozen-stored human peripheral blood mononuclear cells, which we have termed the human lymphocyte activation (HuLA) assay. The HuLA assay is sensitive to cyclosporine, dexamethasone, rapamycin, mycophenolic acid, and methotrexate at concentrations within their respective therapeutic ranges. Although proliferation is the primary endpoint, we demonstrate that flow cytometry approaches may be used to characterize the proliferating lymphocyte subsets. Flu antigen-specific proliferation in the HuLA assay primarily involves both CD4+ and CD8+ T-lymphocytes and B-lymphocytes, although other lymphocyte subsets also proliferate. In addition, flu-specific antibody-secreting cells can be measured in this assay by ELISPOT, a response that is also sensitive to known immunosuppressive compounds. The HuLA assay represents a relatively straightforward assay with the capability of detecting immune suppression in human cells and can be applied to compound ranking and immunotoxicity assessment.

Essential role of CCR6 in directing activated T cells to the skin during contact hypersensitivity.

CCR6 is expressed in a number of dermatological inflammatory diseases. Here, we report that mice sensitized with the hapten oxazolone had increased numbers of CCR6+ T cells in the draining lymph nodes. Using CCR6-/- mice, we assessed the role of CCR6 on the development of contact hypersensitivity. After hapten sensitization and re-challenge, ear swelling in CCR6-/- animals was reduced 80% as compared with wild-type (WT) control mice. This decreased level of inflammation was not related to an inhibition in T-cell activation, because CCR6-/- lymph node cells from sensitized mice produced threefold higher levels of IFN-gamma in culture than cells from sensitized WT mice and, when these cells were directly injected into the site of hapten challenge, induced a robust inflammatory response. However, intravenous injection of CCR6-/- lymph node cells from sensitized mice were unable to prime naive mice to re-challenge whereas cells from primed WT mice were able to sensitize animals. These results suggest that CCR6 plays an important role in directing the trafficking of activated T cells into the skin and suggests that a CCR6 antagonist could be useful to treat skin-mediated inflammatory reactions.

The human specific CCR1 antagonist CP-481,715 inhibits cell infiltration and inflammatory responses in human CCR1 transgenic mice.

We previously described the in vitro characteristics of the potent and selective CCR1 antagonist, CP-481,715. In addition to being selective for CCR1 vs other chemokine receptors, CP-481,715 is also specific for human CCR1 (hCCR1), preventing its evaluation in classical animal models. To address this, we generated mice whereby murine CCR1 was replaced by hCCR1 (knockin) and used these animals to assess the anti-inflammatory properties of CP-481,715. Cells isolated from hCCR1 knockin mice were shown to express hCCR1 and migrate in response to both murine CCR1 and hCCR1 ligands. Furthermore, this migration is inhibited by CP-481,715 at dose levels comparable to those obtained with human cells. In animal models of cell infiltration, CP-481,715 inhibited CCL3-induced neutrophil infiltration into skin or into an air pouch with an ED50 of 0.2 mg/kg. CP-481,715 did not inhibit cell infiltration in wild-type animals expressing murine CCR1. In a more generalized model of inflammation, delayed-type hypersensitivity, CP-481,715 significantly inhibited footpad swelling and decreased the amount of IFN-gamma and IL-2 produced by isolated spleen cells from sensitized animals. It did not, however, induce tolerance to a subsequent challenge. These studies illustrate the utility of hCCR1 knockin animals to assess the activity of human specific CCR1 antagonists; demonstrate the ability of the CCR1 antagonist CP-481,715 to inhibit cell infiltration, inflammation, and Th1 cytokine responses in these animals; and suggest that CP-481,715 may be useful to modulate inflammatory responses in human disease.