Jan Kubach

Human CD25+ regulatory T cells: two subsets defined by the integrins α4β7 or α4β1 confer distinct suppressive properties upon CD4+ T helper cells

Down‐regulation of autoreactive T cell responses in vivo includes cell‐contact‐dependent as well as contact‐independent mechanisms. Infectious tolerance is a contact‐dependent mechanism used by naturally occurring CD25+ T regulatory cells (Tregs) to confer suppressive activity upon conventional CD4+ T cells thereby generating secondary T helper suppressor cells(Thsup), which inhibit T cell activation via soluble mediators. Here, we describe two distinct subsets of human Tregs, characterized by expression of either the α4β7 integrin or the α4β1 integrin. Upon activation, both subsets show an enhanced expression of FoxP3, recently described as a key transcription factor of murine Tregs. In addition, both are able to convey suppressive capacity to conventional CD4+ T cells. However, the properties of Treg subsets are rather distinct: α4β7+Tregs induce IL‐10‐producing Thsup (Tr1‐like), whereas α4β1+ Tregs induce TGF‐β‐producing Thsup (Th3‐like). Our findings reconcile conflicting results by clearly demonstrating that suppression through naturally occurring CD25+ Tregs is primary cell‐contact‐dependent but is subsequently followed by cell‐contact‐independent T cell inhibition mediated by second‐generation Tr1‐ and Th3‐like Thsup via the soluble factors IL‐10 and TGF‐β.

miR-155 Inhibition Sensitizes CD4+ Th Cells for TREG Mediated Suppression

Background In humans and mice naturally occurring CD4+CD25+ regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance. Principal Findings DNA-Microarray analyses of human as well as murine conventional CD4+ Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4+ Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4+ Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression. Conclusion Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4+ Th cells to nTreg cell-mediated suppression.

Protection from graft-versus-host disease by HIV-1 envelope protein gp120-mediated activation of human CD4+CD25+ regulatory T cells

Naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) represent a unique T-cell lineage that is endowed with the ability to actively suppress immune responses. Therefore, approaches to modulate Treg function in vivo could provide ways to enhance or reduce immune responses and lead to novel therapies. Here we show that the CD4 binding human immunodeficiency virus-1 envelope glycoprotein gp120 is a useful and potent tool for functional activation of human Tregs in vitro and in vivo. Gp120 activates human Tregs by binding and signaling through CD4. Upon stimulation with gp120, human Tregs accumulate cyclic adenosine monophosphate (cAMP) in their cytosol. Inhibition of endogeneous cAMP synthesis prevents gp120-mediated Treg activation. Employing a xenogeneic graft versus host disease model that has been shown to be applicable for the functional analysis of human Tregs in vivo, we further show that a single dose of gp120 is sufficient to prevent lethal graft versus host disease and that the tolerizing effect of gp120 is strictly dependent on the presence of human Tregs and their up-regulation of cAMP upon gp120-mediated activation. Our findings demonstrate that stimulation via the CD4 receptor represents a T-cell receptor-independent Treg activating pathway with potential to induce immunologic tolerance in vivo.

Dendritic Cells: Sentinels of Immunity and Tolerance

The induction of effective antigen-specific T-cell immunity to pathogens without the initiation of autoimmunity has evolved as a sophisticated and highly balanced immunoregulatory mechanism. This mechanism assures the generation of antigen-specific effector cells as well as the induction and maintenance of antigen-specific tolerance to self-structures of the body. As professional antigen-presenting cells of the immune system, dendritic cells (DC) are ideally positioned throughout the entire body and equipped with a unique capability to transport antigens from the periphery to lymphoid tissues. There is growing evidence that DC, besides their well-known immunostimulatory properties, also induce and regulate T-cell tolerance in the periphery. This regulatory function of DC is strictly dependent on their different stages of maturation and activation. Additionally, immunosuppressive agents and cytokines further influence the functions of maturing DC. The regulatory properties of DC include induction of T-cell anergy, apoptosis, and the generation of T-cells with regulatory capacities. This brief review summarizes the current knowledge about the immunoregulatory role of DC as guardians for the induction of T-cell immunity and tolerance.

CD4-mediated functional activation of human CD4 + CD25 + regulatory T cells

Naturally occurring CD4+CD25+FoxP3+ regulatory T cells (CD25+ Tregs) constitute a specialized population of T cells that is essential for the maintenance of peripheral self‐tolerance. The immune regulatory function of CD25+ Tregs depends upon their activation. We found that anti‐CD4 antibodies activate the suppressive function of human CD25+ Tregs in a dose‐dependent manner. We demonstrate that CD4‐activated CD25+ Tregs suppress the proliferation of CD4+ and CD8+ T cells, their IL‐2 and IFN‐γ production as well as the capacity of CD8+ T cells to re‐express CD25. By contrast, anti‐CD4 stimulation did not induce suppressive activity in conventional CD4+ T cells. These results identify CD4 as a trigger for the suppressive function of CD25+ Tregs and suggest a possible CD4‐mediated exploitation of these cells.

A specific CD4 epitope bound by tregalizumab mediates activation of regulatory T cells by a unique signaling pathway

CD4+CD25+ regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti‐CD4 monoclonal antibodies (mAbs), the humanized CD4‐specific monoclonal antibody tregalizumab (BT‐061) is able to selectively activate the suppressive properties of Tregs in vitro. BT‐061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT‐061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti‐CD4 mAbs. We found that, due to this special epitope binding, BT‐061 induces a unique phosphorylation of T‐cell receptor complex‐associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT‐061 does not induce the release of pro‐inflammatory cytokines. These results demonstrate that BT‐061 stimulation via the CD4 receptor is able to induce T‐cell receptor‐independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.

IgG1 anti-epidermal growth factor receptor antibodies induce CD8-dependent antitumor activity

Anti‐EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR+ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody‐dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab‐mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA‐A2 and adoptively transferred human HLA‐A2+ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti‐EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti‐EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8+ T effector cells. This novel mechanism of ADCC conducted by CD8+ T effector cells was restricted to IgG1 anti‐EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8+ T effector cell‐mediated ADCC was enhanced in the presence of IL‐15 and strongly improved after glycosylation of anti‐EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.

Identification of transcriptional pathways in naturally occurring human regulatory T cells

Naturally occurring CD4+CD25 Tregs (nTregs) are essential for T cell homeostasis and the maintenance of peripheral tolerance. They prevent the activation of autoaggressive T cells in the context of autoimmune diseases and suppress inadequate allergen specific T cells. On the opposite, nTregs inhibit also effective immune responses against tumors such as melanoma. A detailed understanding of molecular mechanisms that control the functional properties of human nTregs is mandatory for the development of novel immunotherapies against allergy, autoimmunity and cancer. Therefore, we initiated a genomic, proteomic and kinome profiling of human nTregs to identify key molecules in human nTregs associated with their functional activation which are responsible for their state of anergy and/or their suppressive activity. We started with large-scale isolation of nTregs using whole leukapheresis products followed by polyclonal stimulation and analysis at different time points. As a result, we identified a distinct molecular activation pattern specific for the activation state of human nTregs. The impact of identified key molecules was tested in functional assays using specific inhibitors and siRNA mediated knockdown of these targets. A general transcriptional network analysis is currently under investigation and will be presented on the meeting. The main objective of our analysis is the identification of novel targets for the immunotherapeutic intervention of dysregulated immune responses in the near future. from 12th Joint Meeting of the Signal Transduction Society (STS). Signal Transduction: Receptors, Mediators and Genes Weimar, Germany. 29–31 October 2008