Immunology

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4⁺ and CD8⁺ populations and assessed for the ability to process and present particulate antigen to CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ DCs both processed exogenous particulate antigen, but CD8⁺ DCs were much more efficient than CD4⁺ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross‐presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8⁺ DCs, our studies also revealed an important contribution of CD24. CD8⁺ DCs were also more efficient than CD4⁺ DCs in inducing naïve T cells to acquire certain effector T‐cell functions, for example generation of cytotoxic CD8⁺ T cells and interferon (IFN)‐γ‐producing CD4⁺ T cells. In summary, CD8⁺ DCs are particularly potent antigen‐presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.

Dendritic cells (DCs) in mesenteric lymph nodes (MLNs) induce Foxp3⁺ regulatory T cells to regulate immune responses to beneficial or non‐harmful agents in the intestine, such as commensal bacteria and foods. Several studies in MLN DCs have revealed that the CD103⁺ DC subset highly induces regulatory T cells, and another study has reported that MLN DCs from programmed death ligand 1 (PD‐L1) ‐deficient mice could not induce regulatory T cells. Hence, the present study investigated the expression of these molecules on MLN CD11c⁺ cells. Four distinct subsets expressing CD103 and/or PD‐L1 were identified, namely CD11b⁺ CD103⁺ PD‐L1^High, CD11b⁻ CD103⁺ PD‐L1^High, CD11b⁻ CD103⁺ PD‐L1^Low and CD11b⁺ CD103⁻ PD‐L1^Int. Among them, the CD11b⁻ CD103⁺ PD‐L1^High DC subset highly induced Foxp3⁺ T cells. This subset expressed Aldh1a2 and Itgb8 genes, which are involved in retinoic acid metabolism and transforming growth factor‐β (TGF‐β) activation, respectively. Exogenous TGF‐β supplementation equalized the level of Foxp3⁺ T‐cell induction by the four subsets whereas retinoic acid did not, which suggests that high ability to activate TGF‐β is determinant for the high Foxp3⁺ T‐cell induction by CD11b⁻ CD103⁺ PD‐L1^High DC subset. Finally, this subset exhibited a migratory DC phenotype and could take up and present orally administered antigens. Collectively, the MLN CD11b⁻ CD103⁺ PD‐L1^High DC subset probably takes up luminal antigens in the intestine, migrates to MLNs, and highly induces regulatory T cells through TGF‐β activation.

Highly differentiated CD8⁺ CD28⁻ CD27⁻ T cells have short telomeres, defective telomerase activity and reduced capacity for proliferation. In addition, these cells express increased levels of inhibitory receptors and display defective Akt(ser⁴⁷³) phosphorylation following activation. It is not known whether signalling via programmed death 1 (PD‐1) contributes to any of the attenuated differentiation‐related functional changes in CD8⁺ T cells. To address this we blocked PD‐1 signalling during T‐cell receptor (TCR) activation using antibodies against PD‐1 ligand 1 (PDL1) and PDL2. This resulted in a significant enhancement of Akt(ser⁴⁷³) phosphorylation and TCR‐induced proliferative activity of highly differentiated CD8⁺ CD28⁻ CD27⁻ T cells. In contrast, the reduced telomerase activity in these cells was not altered by blockade of PDL1/2. We also demonstrate that PD‐1 signalling can inhibit the proliferative response in primary human CD8⁺ T cells from both young and older humans. These data collectively highlight that some, but not all, functional changes that arise during progressive T‐cell differentiation and during ageing are maintained actively by inhibitory receptor signalling.

Interaction of Fas/APO‐1 (CD95) and its ligand (FasL) plays an important role in the activation‐induced cell death (AICD) of T lymphocytes. In the present work, the contribution of soluble FasL to AICD of the human T‐cell line Jurkat has been studied. Jurkat cells prestimulated with phytohaemagglutinin (PHA) induced the death of non‐activated Jurkat cells, and also of L1210Fas, but not that of Fas‐negative L1210 cells. Culture supernatants from prestimulated Jurkat cells were highly toxic to their non‐activated counterparts. Time–course analysis revealed that PHA‐stimulated Jurkat cells quickly release (less than 15 min) to the medium a toxic molecule following a biphasic pattern, with maximal cytotoxic activities at 1 hr and 7 hr after stimulation. The cytotoxic effect of those supernatants was prevented by the addition of a blocking anti‐Fas monoclonal antibody, suggesting that PHA‐stimulated Jurkat cells exert Fas‐based cytotoxicity mainly through the release of soluble FasL. The constitutive intracellular expression of FasL in non‐activated Jurkat cells and its release as a consequence of PHA activation were detected by immunostaining and immunoblotting using an anti‐FasL antibody. These data indicate that, at least in Jurkat cells, AICD is mainly mediated by the rapid release of preformed FasL in soluble form upon stimulation.

To assess the role of the macrophage scavenger receptor type A (SRA) in immune activation by CpG DNA, cytokine induction and DNA uptake were tested in vitro and in vivo using SRA knockout (SRA^−/−) and wild type (WT) mice. As a source of CpG DNA, Escherichia coli DNA (EC DNA) and a 20‐mer phosphorothioate oligodeoxynucleotide with two CpG motifs (CpG ODN) were used. In vitro, both EC DNA and the CpG ODN induced dose‐dependent increases of interleukin (IL)‐12 production by spleen cells and bone‐marrow‐derived macrophages (BMMΦ) from both SRA^−/− and WT mice. The levels of cytokines produced by SRA^−/− spleen cells and BMMΦ were similar to those of WT spleen cells and BMMΦ. When injected intravenously with CpG ODN and EC DNA, both SRA^−/− and WT mice showed elevated serum levels of IL‐12. To investigate further the role of the SRA, flow cytometry and confocal microscopy were performed to examine the uptake of fluorescently labelled oligonucleotides. SRA^−/− and WT BMMΦ showed similarity in the extent of uptake and distribution of oligonucleotides as assessed by these two techniques. Together, these findings indicate that, while the SRA may bind DNA, this receptor is not essential for the uptake of CpG DNA or its immunostimulatory activity.

Alemtuzumab is a humanized monoclonal antibody against CD52, an antigen found on the surface of normal and malignant lymphocytes. It is approved for the treatment of B‐cell chronic lymphocytic leukaemia and is undergoing Phase III clinical trials for the treatment of multiple sclerosis. The exact mechanism by which alemtuzumab mediates its biological effects in vivo is not clearly defined and mechanism of action studies have been hampered by the lack of cross‐reactivity between human and mouse CD52. To address this issue, a transgenic mouse expressing human CD52 (hCD52) was created. Transgenic mice did not display any phenotypic abnormalities and were able to mount normal immune responses. The tissue distribution of hCD52 and the level of expression by various immune cell populations were comparable to those seen in humans. Treatment with alemtuzumab replicated the transient increase in serum cytokines and depletion of peripheral blood lymphocytes observed in humans. Lymphocyte depletion was not as profound in lymphoid organs, providing a possible explanation for the relatively low incidence of infection in alemtuzumab‐treated patients. Interestingly, both lymphocyte depletion and cytokine induction by alemtuzumab were largely independent of complement and appeared to be mediated by neutrophils and natural killer cells because removal of these populations with antibodies to Gr‐1 or asialo‐GM‐1, respectively, strongly inhibited the activity of alemtuzumab whereas removal of complement by treatment with cobra venom factor had no impact. The hCD52 transgenic mouse appears to be a useful model and has provided evidence for the previously uncharacterized involvement of neutrophils in the activity of alemtuzumab.

We previously identified a cis‐acting element within the 3′ untranslated region of CD40 ligand messenger RNA (mRNA) that is composed of three complex binding sites and acts to increase mRNA stability in both in vitro and in vivo systems. We now demonstrate the functional consequences of the three binding sites with respect to increasing both luciferase activity and mRNA stability in a heterologous transcript expressed in a T‐cell line. The internal region B was shown to be a bona fide stability element because its presence increased luciferase activity fourfold over the unmodified transcript and its removal from the XbaI–HaeIII region resulted in rapid degradation of the transcript. Region A contained both a binding site for a polypyrimidine‐tract‐binding protein (PTB)‐mediated complex (Complex I) and an upstream, adjacent sequence that was a negative regulator of mRNA stability. Region C bound Complex II, which contained both PTB and heterogeneous nuclear ribonucleoproteinL (hnRNPL), and was less effective as a stability element on its own compared to region B. Our findings demonstrate differential levels of activity for the three binding sites relative to the turnover of CD40 ligand mRNA, suggesting that the lack of binding of Complex I/II during the early stages of T‐cell activation contributes to the rapid degradation of the CD40 ligand mRNA transcript.

Effector memory T helper 2 (Th2) cells that accumulate in target organs (i.e. skin or bronchial mucosa) have a central role in the pathogenesis of allergic disorders. To date, the factors that selectively trigger local production of Th2‐attracting chemokines remain poorly understood. In mucosa, at the sites of allergen entry, immature dendritic cells (DC) are in close contact with mast cells. Histamine and prostaglandin E₂ (PGE₂) are two mediators released by allergen‐activated mast cells that favour the polarization of maturing DC into Th2‐polarizing cells. We analysed here the effects of histamine and PGE₂ on the prototypic, Th2‐(CCL17, CCL22) versus Th1‐(CXCL10) chemokine production by human DC. We report that histamine and PGE₂ dose‐dependently up‐regulate CCL17 and CCL22 by monocyte‐derived immature DC. These effects were potentiated by tumour necrosis factor‐α, still observed in the presence of the Th1‐cytokine interferon‐γ (IFN‐γ) and abolished by the immunomodulatory cytokine interleukin‐10. In addition, histamine and PGE₂ down‐regulated IFN‐γ‐induced CXCL10 production by monocyte‐derived DC. These properties of histamine and PGE₂ were observed at the transcriptional level and were mediated mainly through H2 receptors for histamine and through EP2 and EP4 receptors for PGE₂. Finally, histamine and PGE₂ also up‐regulated CCL17 and CCL22 and decreased IFN‐γ‐induced CXCL10 production by purified human myeloid DC. In conclusion, these data show that, in addition to polarizing DC into mature cells that promote naïve T‐cell differentiation into Th2 cells, histamine and PGE₂ may act on immature DC to trigger local Th2 cell recruitment through a selective control of Th1/Th2‐attracting chemokine production, thereby contributing to maintain a microenvironment favourable to persistent immunoglobulin E synthesis.

Toll‐like receptors (TLRs) signal through two main pathways: a myeloid differentiation factor (MyD)88‐dependent pathway that acts via nuclear factor κB (NF‐κB) to induce proinflammatory cytokines such as tumour necrosis factor‐α (TNF‐α) and a MyD88‐independent pathway that acts via type I interferons to increase the expression of interferon‐inducible genes. Repeated signalling through TLR4 and a number of other TLRs has been reported to result in a reduction in the subsequent proinflammatory cytokine response, a phenomenon known as TLR tolerance. In this study we have shown that, whilst NF‐κB activation and production of TNF‐α and interleukin‐12 by murine RAW264.7 and J774.2 cells in response to stimulation by TLR4, ‐5, ‐7 or ‐9, was reduced by prior stimulation with TLR4, ‐5, ‐7 or ‐9 ligands, the primary stimulation of TLR3, which does not use the MyD88 pathway, did not reduce the TNF‐α or interleukin‐12 responses to subsequent TLR stimulation. The response to TLR3 stimulation was not diminished by prior TLR ligand exposure. Furthermore, the production of interferon‐β (IFN‐β) following stimulation of TLR3 or ‐4, which is MyD88‐independent, was increased by prior activation of TLR4, ‐5, ‐7 or ‐9. In contrast, TLR9 ligand‐induced IFN‐β production, which is MyD88‐dependent, was tolerized by prior TLR stimulation. These results are consistent with differential regulation of MyD88‐dependent and MyD88‐independent cytokine production following serial activation of TLRs.