In vivo maintenance of human regulatory T cells during CD25 blockade.

Regulatory T cells (Tregs) mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction, and instability are implicated in the pathogenesis of numerous autoimmune diseases. There is considerable interest in therapeutic modulation of the IL-2 pathway to treat autoimmunity, facilitate transplantation tolerance, or potentiate tumor immunotherapy. Daclizumab is a humanized mAb that binds the IL-2 receptor a subunit (IL-2R a or CD25) and prevents IL-2 binding. In this study, we investigated the effect of daclizumab-mediated CD25 blockade on Treg homeostasis in patients with relapsing-remitting multiple sclerosis. We report that daclizumab therapy caused an ~50% decrease in Tregs over a 52-wk period. Remaining FOXP3+ cells retained a demethylated Treg-specific demethylated region in the FOXP3 promoter, maintained active cell cycling, and had minimal production of IL-2, IFN- g, and IL-17. In the presence of daclizumab, IL-2 serum concentrations increased and IL-2R bg signaling induced STAT5 phosphorylation and sustained FOXP3 expression. Treg declines were not associated with daclizumab-related clinical benefit or cutaneous adverse events. These results demonstrate that Treg phenotype and lineage stability can be maintained in the face of CD25 blockade.

Partial and transient modulation of the CD3-T-cell receptor complex, elicited by low-dose regimens of monoclonal anti-CD3, is sufficient to induce disease remission in non-obese diabetic mice.

It has been established that a total of 250 microg of monoclonal anti-mouse CD3 F(ab')(2) fragments, administered daily (50 microg per dose), induces remission of diabetes in the non-obese diabetic (NOD) mouse model of autoimmune diabetes by preventing beta cells from undergoing further autoimmune attack. We evaluated lower-dose regimens of monoclonal anti-CD3 F(ab')(2) in diabetic NOD mice for their efficacy and associated pharmacodynamic (PD) effects, including CD3-T-cell receptor (TCR) complex modulation, complete blood counts and proportions of circulating CD4(+), CD8(+) and CD4(+) FoxP3(+) T cells. Four doses of 2 microg (total dose 8 microg) induced 53% remission of diabetes, similarly to the 250 microg dose regimen, whereas four doses of 1 microg induced only 16% remission. While the 250 microg dose regimen produced nearly complete and sustained modulation of the CD3 -TCR complex, lower doses, spaced 3 days apart, which induced similar remission rates, elicited patterns of transient and partial modulation. In treated mice, the proportions of circulating CD4(+) and CD8(+) T cells decreased, whereas the proportions of CD4(+) FoxP3(+) T cells increased; these effects were transient. Mice with greater residual beta-cell function, estimated using blood glucose and C-peptide levels at the initiation of treatment, were more likely to enter remission than mice with more advanced disease. Thus, lower doses of monoclonal anti-CD3 that produced only partial and transient modulation of the CD3-TCR complex induced remission rates comparable to higher doses of monoclonal anti-CD3. Accordingly, in a clinical setting, lower-dose regimens may be efficacious and may also improve the safety profile of therapy with monoclonal anti-CD3, potentially including reductions in cytokine release-related syndromes and maintenance of pathogen-specific immunosurveillance during treatment.

Well-developed ligand-binding assays demonstrate robust performance using singlet analysis.

Aim: Replicate sample testing has long been regarded as a necessity for bioanalytical laboratory testing, especially in the realm of ligand-binding assays (LBAs). In an era in which results were derived from crude test tube-based assays, the replication of results was warranted. Those assays were often imprecise and required multiple replicates to arrive at results that approached accuracy. However, given technological advancements and excellent accuracy and precision of many modern LBAs, the practice of replicate testing should be re-evaluated. Although most regulatory guidelines allow for singlet testing when sufficient robustness and precision are demonstrated during validation, duplicate testing is still common practice. Recently however, several articles have been published that support singlet analysis for LBAs performed on a platform with automated liquid handling. Results: Data from five pharmacokinetic assay validations and five clinical and preclinical studies originally run in duplicate were re-evaluated in singlet and found to be nearly identical to the original duplicate results. Conclusion: We confirm that well-developed LBAs produce comparable data whether evaluated in singlet or duplicate. Additionally, automation is not requisite for singlet testing.

NFATc2 and T-bet contribute to T-helper-cell-subset-specific regulation of IL-21 expression.

T helper (Th) 2 cells selectively express IL-21 in addition to the classic Th2 cytokines IL-4, IL-5, and IL-13. In contrast to these clustered Th2 cell cytokine genes, the IL-21 gene resides on a different chromosome and is not coordinately regulated by the same locus control region that directs the expression of other Th2 cytokines. We demonstrate that the proximal promoter of IL-21 controls its Th-cell-subset-specific expression through the action of NFATc2 and T-bet. Whereas NFATc2 directly binds to and activates transcription of the IL-21 promoter in Th2 cells, T-bet represses IL-21 transcription by inhibiting the binding of NFATc2 to the promoter in Th1 cells. These data suggest that there are multiple mechanisms by which Th-cell-subset-specific cytokine genes are regulated.

Biology of IL-21 and the IL-21 receptor.

Interleukin-21 (IL-21) is the newest member of the common gamma-chain family of cytokines, which includes IL-2, IL-4, IL-7, IL-9, IL-13, and IL-15. Its private receptor, IL-21R, has been shown to activate the Janus kinase/signal transducers and activators of transcription signaling pathway upon ligand binding. Initial studies have demonstrated that IL-21 has pleiotropic effects on the proliferation, differentiation, and effector functions of B, T, natural killer, and dendritic cells. More recently, the potential therapeutic capacity of IL-21 in the treatment of cancers has been widely investigated. The biological role of IL-21 in the immune system is complex, as IL-21 has been shown to have the ability to both promote and inhibit immune responses. Overall, the current data point to IL-21 being a novel immunomodulatory cytokine, whose regulation of any given immune response is highly dependent on the surrounding environmental context.

IL-21 induces the apoptosis of resting and activated primary B cells.

Cytokines play an important role in regulating the development and homeostasis of B cells by controlling their viability. In this study, we show that the recently described T cell-derived cytokine IL-21 induces the apoptosis of resting primary murine B cells. In addition, the activation of primary B cells with IL-4, LPS, or anti-CD40 Ab does not prevent IL-21-mediated apoptosis. The induction of apoptosis by IL-21 correlates with a down-regulation in the expression of Bcl-2 and Bcl-x(L), two antiapoptotic members of the Bcl-2 family. Furthermore, the reconstitution of Bcl-x(L) or Bcl-2 expression protects primary B cells from IL-21-induced apoptosis. In addition, a short-term preactivation of B cells with anti-CD40 Ab confers protection from IL-21-mediated apoptosis through the up-regulation of Bcl-x(L). These studies reveal a novel pathway that mediates B cell apoptosis via the IL-21R and suggest that IL-21 may play a role in regulating B cell homeostasis.