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The mammalian target of rapamycin (mTOR) controls T-cell differentiation in response to polarizing cytokines. We previously found that mTOR blockade by rapamycin (RAPA) delays the G1-S cell cycle transition and lymphocyte proliferation. Here, we report that both mTOR complex 1 and mTOR complex 2 are readily activated following TCR/CD28 engagement and are critical for early expression of Ifng, Il4 and Foxp3, and for effector T cell differentiation in the absence of polarizing cytokines. While inhibition of mTOR complex 1 and cell division were evident at low doses of RAPA, inhibition of mTOR complex 2, Ifng, Il4 and Foxp3 expression, and T-cell polarization required higher doses and more prolonged treatments. We found that while T-bet and GATA3 were readily induced following TCR/CD28 engagement, administration of RAPA delayed their expression, and interfered with the loss of DNA methylation within Ifng and Il4 promoter regions. In contrast, RAPA prevented activation-dependent DNA methylation of the Foxp3 promoter favoring Foxp3 expression. As a result, RAPA-cultured cells lacked immediate effector functions and instead were enriched for IL-2+ cells. We propose that mTOR-signaling, by timing the expression of critical transcription factors and DNA methylation of proximal promoter regions, regulates transcriptional competence at immunologically relevant sites and hence lymphocyte differentiation., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Induction of isotype switching to a specific C(H) gene correlates with the transcriptional activation of the same gene in germline (GL) configuration. Expression of correctly spliced GL transcripts is necessary to target a switch region for recombination. In human B cells, the IgE and IgG4 isotypes are both induced by IL-4 through sequential switching, but are functionally antagonistic because IgG4 appears to have IgE-blocking activity. In order to understand the molecular mechanisms that regulate IgG4 production, we undertook a systematic analysis of the gamma4 GL promoter. A HindIII/NaeI region (-421/+474) highly conserved in the human gamma locus mediated the synergistic activation of a reporter gene in response to IL-4 and CD40 cross-linking. STAT6 binding to the proximal gamma4 GL promoter was essential for both IL-4-induced activation and CD40-dependent enhancement of transcription. Of note, a 45bp region (-76/-32) centered around the STAT6 binding motif drove robust synergistic activation of a heterologous fos promoter upon stimulation with IL-4 and CD40 cross-linking. This finding suggested that the (-76/-32) region may contain a novel IL-4/CD40 responsive element (RE). Electrophoretic mobility shift assays (EMSA) analysis using BL-2 nuclear extracts and in vitro translated NF-kappaB/Rel family proteins revealed the presence of a motif that overlaps the 5' end of the STAT6 element and binds selectively c-Rel. A mutation that abrogated c-Rel, but not STAT6, binding strongly impaired the CD40-induced enhancement of IL-4-dependent gamma4 GL transcription in reporter assays. These results indicate that c-Rel is selectively involved in the CD40-dependent activation of the IL-4/CD40 RE in the proximal gamma4 GL promoter.

Parasite immunologists had known for some time that IgE-mediated hypersensitivity reactions are rare in patients with chronic helminth infections, even though basophils and mast cells in these patients are sensitized with antiparasite IgE and exposed, often continuously, to parasite antigens. The inhibition of allergic reactivity in chronic helminth infections is mainly due to IgG4 ‘blocking antibodies’ in the serum of the infected individual. IgG4 do not fix complement and bind weakly to Fcγ receptors. Thus, antigen binding by IgG4, unlike IgE, is likely to have no or minimally harmful consequences. The discovery that, similar to IgE, expression of IgG4 is IL-4-dependent and is an intermediate step in sequential switching from IgM to IgE makes it imperative to understand how the two isotypes are coregulated and whether the two responses can be uncoupled, selectively boosting IgG4 over IgE. The ultimate goal is to apply to allergy the lesson we learnt from helminth infections. [ABSTRACT FROM AUTHOR]

Background: Allergic reactions occur rarely in patients with chronic helminth infections, although FcεRI–bearing cells are sensitized with anti–parasite IgE and are exposed to antigen. The inhibition of allergic reactivity is due to ‘blocking antibodies’, predominantly IgG4. IgG4 antibodies represent 50–95% of anti–filarial IgG, and have blocking activity because they compete with cell–bound IgE for allergen binding. Blocking IgG4 antibodies have also been detected in patients treated with immunotherapy. However, the molecular mechanisms of IgG4 regulation have remained unexplored. We address this issue starting with the IL–4–dependent induction of γ4 germline transcription, an essential step in the commitment to isotype switching. Results: γ4 germline transcripts were cloned and shown to contain Iγ4 spliced to the 5′ portion of Cγ4 using the splice donor site shared by γ1 and γ3 germline transcripts. Sequence analysis located the human Iγ4 exon ≈0.6 kb upstream of the Sγ4 region. Four major transcription start sites located 547–583 bp upstream of the Iγ4 splice donor site were identified by primer extension analysis. A HindIII/NaeI construct (–413/+466) had the highest activity in reporter assays. Transcription was induced 4.6–fold by IL–4, 2.1–fold by CD40 engagement, and 14.5–fold by a combination of the two signals. Thus CD40 crosslinking enhanced IL–4–dependent transcription by 3.1–fold, showing that the two stimuli act synergistically. Conclusion: As we understand more about IgG4 regulation, our hope is to apply to allergy the lesson we learnt from helminth infections. [ABSTRACT FROM AUTHOR]