On human macrophages IL-10 acts as a more potent anti-inflammatory cytokine than IL-6, although both cytokines signal mainly via activation of the transcription factor STAT3. In this study we compare IL-10 and IL-6 signaling in primary human macrophages derived from blood monocytes. Pretreatment of macrophages with PMA or the proinflammatory mediators LPS and TNF-␣ blocks IL-6-induced STAT3 activation, whereas IL-10-induced activation of STAT3 remains largely unaffected. Although LPS induces the feedback inhibitor suppressor of cytokine signaling 3 (SOCS3) in macrophages, inhibition of IL-6 signal transduction by LPS occurs rapidly and does not depend on gene transcription. We also found that pretreatment of macrophages with IL-10 inhibits subsequent STAT3 activation by IL-6, whereas IL-10-induced STAT3 activation is not affected by preincubation with IL-6. This cross-inhibition is dependent on active transcription and might therefore be explained by different sensitivities of IL-10 and IL-6 signaling toward the feedback inhibitor SOCS3, which is induced by both cytokines. In contrast to the IL-6 signal transducer gp130, which has been previously shown to recruit SOCS3 to one of its phosphotyrosine residues (Y759), peptide precipitation experiments suggest that SOCS3 does not interact with phosphorylated tyrosine motifs of the IL-10R. Taken together, different sensitivities of IL-10 and IL-6 signaling toward mechanisms that inhibit the Janus kinase/STAT pathway define an important mechanism that contributes to the different anti-inflammatory potencies of these two cytokines.

The adoptive transfer of donor CD4+CD25+ regulatory T cells has been shown to protect from lethal graft-versus-host disease after allogeneic bone marrow transplantation in murine disease models. Efficient isolation strategies that comply with good manufacturing practice (GMP) guidelines are prerequisites for the clinical application of human CD4+CD25+ regulatory T cells. Here we describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol performed under GMP conditions. The generated cell products contained on average 49.5% CD4+CD25high T cells that phenotypically and functionally represented natural CD4+CD25+ regulatory T cells and showed a suppressive activity comparable to that of CD4+CD25+ regulatory T-cell preparations purified by non-GMP-approved fluorescence-activated cell sorting.

The immediate early response of cells treated with IL-6 (interleukin-6) is the activation of the signal transducer and activator of transcription (STAT)3. The Src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP2 and the feedback inhibitor SOCS3 (suppressor of cytokine signalling) are potent inhibitors of IL-6 signal transduction. Impaired function of SOCS3 or SHP2 leads to enhanced and prolonged IL-6 signalling. The inhibitory function of both proteins depends on their recruitment to the tyrosine motif 759 within glycoprotein gp130. In contrast to inactivation, desensitization of signal transduction is regarded as impaired responsiveness due to prestimulation. Usually, after activation the sensing receptor becomes inactivated by modifications such as phosphorylation, internalization or degradation. We designed an experimental approach which allows discrimination between desensitization and inactivation of IL-6 signal transduction. We observed that pre-stimulation with IL-6 renders cells less sensitive to further stimulation with IL-6. After several hours, the cells become sensitive again. We show that not only signal transduction through previously activated receptors is affected by desensitization but signalling through receptors which were not targeted by the first stimulation was also attenuated ( trans -desensitization). Interestingly, in contrast to inhibition, desensitization does not depend on the presence of functional SHP2. Furthermore, cells lacking SOCS3 show constitutive STAT3 activation which is not affected by pre-stimulation with IL-6. All these observations suggest that desensitization and inhibition of signalling are mechanistically distinct.

3718 CD25+Foxp3+ regulatory T cell (Treg) bear great potential to prevent or treat a variety of immune mediated diseases, including autoimmunity, organ rejection or GvHD. Currently Treg for clinical application can be separated by magnetic cell separation via the CliniMACS® Plus Instrument using CD25 enrichment plus/minus prior depletion of CD8 or CD19 positive cells. With this technology Treg can be enriched to a mean purity of about 50% and first clinical trials for prevention of GvHD show no adverse effects at all. Despite these promising results, concerns have been raised whether in the setting of organ transplantation or autoimmunity higher Treg purities and/or the in vitro expansion of Treg without loss of Foxp3+ expression are required. Therefore, we have optimized the parameters for CD25 enrichment via CliniMACS to achieve higher purity of the isolated Treg. The purity of Treg could be increased by about 20–30% resulting in an average purity of 70–80% of Foxp3+ Treg. We have also developed a protocol for the in vitro expansion of CliniMACS isolated Treg using CD3/CD28 coated MACSiBead™Particles. In the presence of Rapamycin CliniMACS isolated Treg could be expanded about 20 fold with a single round of stimulation. Importantly Foxp3+ expression was not affected by the expansion but remained constant at about 70–80%. Similarly the expression of effector cytokines by expanded Treg was greatly suppressed by Rapamycin. These data show that Treg for clinical application can efficiently be isolated with high purity via CliniMACS and subsequently be expanded in vitro without loss of Foxp3 expression. Disclosures: Conrads: Miltenyi Biotec: Employment. Schmitz:Miltenyi Biotec: Employment. Assenmacher:m: Employment. Niemand:Miltenyi Biotec: Employment. Scheffold:Miltenyi Biotec: Employment.

Several publications during the last few years have reported CD25hiCD4+ regulatory T cells (Tregs) to prevent or to reverse disease in different mouse models of experimental autoimmune encephalomyelitis (EAE), colitis, graft rejection and graft-versus-host-disease (GvHD). As mouse and human Tregs share many phenotypical and functional characteristics, Tregs could provide a promising therapeutic approach for various human autoimmune diseases and pathological alloresponses. Here we have shown that Tregs can be isolated from leukapheresis harvests by CD25 enrichment using the CliniMACS technology (n=13). By this procedure we obtained 2.32x108 (± 1.12x108, range 0.71–4.42x108) cells out of 1010 mononuclear cells with a mean purity of 52.12% (± 12.11%, range 25.48–66.61%) for CD25hiCD4+ cells. Around 90% of enriched cells were CD25+CD4+. Among contaminating CD4− cells most cells were CD25+ which were further characterized by counterstaining to be mainly CD19+ B cells and a few CD8+, CD56+ or CD123+ cells. It is possible to deplete the CD19+ or CD8+ cells by using CD19 Microbeads or CD8 Microbeads respectively with the CliniMACS Instrument before CD25 enrichment. Combined depletion of different cells, e.g. CD19+ and CD8+ cells is conceivable. Isolated cells were phenotypically and functionally characterized. The majority of the CD25hiCD4+ T cells expressed glucocorticoid-induced tumor necrosis factor receptor (GITR), CD62L and CD45RO. In addition, isolated cells were able to suppress the proliferation and activation of cocultured conventional CD4+ cells after polyclonal stimulation with anti-CD3 antibody. We conclude that the large-scale isolation of CD25hiCD4+ regulatory T cells for clinical applications (e.g. therapy of autoimmune diseases, graft rejection or GvHD) is possible by using the CliniMACS CD25 Reagent and the CliniMACS Instrument.