Popliteal lymph node assay: facts and perspectives

Abstract

The popliteal lymph node assay (PLNA) derives from the hypothesis that some supposedly immune-mediated adverse effects induced by certain pharmaceuticals involve a mechanism resembling a graft-versus-host reaction. The injection of many but not all of these compounds into the footpad of mice or rats produces an increase in the weight and/or cellularity of the popliteal lymph node in the treated limb (direct PLNA). Some of the compounds known to cause these adverse effects in humans, however, failed to induce a positive PLNA response, leading to refinements of the technique to include pretreatment with enzyme inducers, depletion of CD4(+) T cells or additional endpoints such as histological examination, lymphocyte subset analysis and cytokine fingerprinting. Alternative approaches have been used to improve further the predictability of the assay. In the secondary PLNA, the test compound is injected twice in order to illicit a greater secondary response, thus suggesting a memory-specific T cell response. In the adoptive PLNA, popliteal lymph node cells from treated mice are injected into the footpad of naive mice; a marked response to a subsequent footpad challenge demonstrates the involvement of T cells. Finally, the reporter antigens TNP-Ficoll and TNP-ovalbumin are used to differentiate compounds that induce responses involving neo-antigen help or co-stimulatory signals (modified PLNA). The PLNA is increasingly considered as a tool for detection of the potential to induce both sensitization and autoimmune reactions. A major current limitation is validation. A small inter-laboratory validation study of the direct PLNA found consistent results. No such study has been performed using an alternative protocol. Other issues include selection of the optimal protocol for an improved prediction of sensitization vs autoimmunity, and the elimination of false-positive responses due to primary irritation. Finally, a better understanding of underlying mechanisms is essential to determine the most relevant endpoints. The confusion resulting from use of the PLNA to predict autoimmune-like reactions as well as sensitization should be clarified. Interestingly, most drugs that were positive in the direct PLNA are also known to cause drug hypersensitivity syndrome in treated patients. This observation is expected to open new avenues of research.

Development and partial validation of a mouse model for predicting drug hypersensitivity reactions

“…The popliteal lymph node assay (PLNA), first published in 1981, is the most thoroughly investigated model for predicting drugs that are associated with autoimmune and hypersensitivity responses in the clinical setting (Aida et al, 1998;Gleichmann, 1981;Pieters, 2001;Ravel & Descotes, 2005;Shinkai et al, 1999). The model itself has been modified to include several approaches (indirect and adoptive transfer PLNA) (Pieters, 2001;Ravel & Descotes, 2005).…”

Autoimmune Models*

“…There is evidence that chemical-self-antigen conjugates can influence T-cell responses (Goebel et al 1996;Griem et al 1998a, b;Pollard et al 2000). It has been proposed that the popliteal lymph node assay (PLNA) is an indicator of responses, particularly T-cell responses, against drug-self complexes (Pieters et al 2002;Ravel and Descotes 2005;Rubin 2005). However, the PLNA, in its basic form as a first-tier screen, is a simple measure of immunosensitization.…”

Profiling Adverse Immune Effects

“…The translation from in vitro to disease models can be made via simple straightforward in vivo methods such as the popliteal lymph node assay (PLNA) [83]. By using this method the T cell sensitizing capacity of compounds can be easily assessed by detecting T cell activation (proliferation, cytokine production) in response to footpad injection of the compound.…”