Specificity of SN50 for NF-κB?

In the January issue of Nature Immunology, Ray and coworkers report data that shows a diminution in Gata3 expression and IL-4 production by T cells after activation of p50-deficient T cells in vitro¹. They also found that challenge of p50-deficient mice, in a manner that causes allergic inflammation in wild-type animals, led to an even greater decrease in T_H2 cytokines in the bronchoalveolar spaces¹. These data provide important evidence that supports previous indications of a role for p50 (also called NF-κB1) in helper T cell effector functions^2,3.

However, the use of an inhibitory peptide (SN50) derived from the p50 protein was presented as evidence that NF-κB is the intermediate link between the TCR and selective regulation of T_H2 development (see Fig. 3)¹. Although it was originally reported that this cell-permeable peptide inhibits nuclear induction of the NF-κB proteins, follow-up work showed that the SN50 peptide is not necessarily specific for p50 or for the NF-κB transcription factors in general^4,5. Rather, because it competes for proteins generally involved in nuclear import, SN50 also blocks the nuclear induction of the transcription factors STAT, AP-1 and NFAT⁴. Although the peptide may be more specific at lower concentrations, 75 μg/ml of SN50 inhibited binding of nuclear extracts to AP-1 and NFAT probes, as well as the κB probe in a study with primary T cells⁵. At 37.5 μg/ml, increased AP-1 and decreased NFAT were observed⁵.” Induction of AP-1 and NFAT is also linked to the TCR. STAT6, on the other hand, regulates GATA3, which, along with NFATc, plays a key role in T_H2 development^6,7. In this context, it should also be noted that the diminution in nuclear GATA-3 that Ray and coworkers observed (Fig. 3c)¹ might be due to a generalized effect of 100 μg/ml of SN50 on nuclear-import processes^4,5.

The partial inhibition obtained at the effective concentration of 100 μg/ml of SN50 (Fig. 3)¹ suggests that comparison of the results to those obtained with other approaches to the inhibition of NF-κB (Rel) family dimers would be valid. When nuclear induction of multiple NF-κB subunits in T cells was inhibited with the use of a transgenic approach, the result was a T cell–intrinsic inhibition of IFN-γ production after antigen restimulation⁸. With immunization techniques similar to those used by Ray and coworkers¹, a modest increase in antigen-specific IgE (whose production in vivo depends on T_H2 responses) was observed, and no decrease in antigen-specific production of the T_H2 cytokine IL-4 was detected⁸. Of course, the use of a trans-dominant inhibitor cannot be equated with the targeted inactivation of a single NF-κB subunit. However, other articles on the role of NF-κB proteins in effector T cell function document a defect in IFN-γ production by T cells, and even an increase in IL-4 production^9,10. These reports include findings on RelB and the p50-related factor p52 (NF-κB2). If SN50 inhibited only NF-κB dimers, the basis for the difference in these results is not clear. In regard to the role of RelA, although IgE production is highly dependent on T_H2 cytokines, IgE expression in RAG-deficient mice reconstituted with RelA-deficient lymphoid cells was the same as in wild-type controls¹¹. In considering the activity of p50 revealed in the T_H2 component of immune responses^1,2,3, it is worth noting that signaling by regulatory coreceptors such as IL-1R and T1/ST2^12,13 may play a crucial role, which perhaps parallels a role played by IL-18– induced NF-κB in T_H1 responses. Overall, three points are suggested in addition to the issue of SN50 specificity. First, although particular subunits of NF-κB may play specific roles when knocked-out, collectively NF-κB in T cells may regulate both T_H1 and T_H2 responses. Therefore, the phrase “NF-κB deficiency does not affect IFN-γ production”¹ may not fully reflect the roles of NF-κB. Second, these studies indicate that individual deficiencies may reveal subunit-specific roles in effector T cells, but it is unclear whether the TCR selectively regulates just one subunit or a limited set of dimers (for example, p50-p50 and p50-RelA, but not p52-containing dimers). Third, the complex interrelationships among these subunits make it difficult to extrapolate from the results obtained with p50-deficient mice¹ to a general role for TCR-induced NF-κB. As noted by the authors, p50 can influence cells as an inhibitory p50 dimer¹. Accordingly, it is possible that the basis for the findings of Ray and coworkers¹ reflects either a removal of repression by TCR-independent p50 homodimers already present in the nucleus of resting T cells or a stoichiometric change to p52-dominated heterodimers, rather than a role for TCR-induced heterodimers containing p50. Further studies and highly specific inhibition of NF-κB dimers will help to clarify such issues in vitro and in vivo.

See Response to 'Specificity of SN50 for NF-κB?' by Anuradha Ray and the A critical role for NF-κB in Gata3 expression and TH2 differentiation in allergic airway inflammation by Jyoti Das.