Birgit Sawitzki

Alloantigen-Induced CD25+CD4+ Regulatory T Cells Can Develop In Vivo from CD25−CD4+ Precursors in a Thymus-Independent Process

The capacity of naturally occurring autoreactive CD25+CD4+ regulatory T cells (Treg) to control immune responses both in vivo and in vitro is now well established. It has been demonstrated that these cells undergo positive selection within the thymus and appear to enter the periphery as committed CD25+CD4+ Treg. We have shown previously that CD25+CD4+ Treg with the capacity to prevent skin allograft rejection can be generated by pretreatment with donor alloantigen under the cover of anti-CD4 therapy. Here we demonstrate that this process does not require an intact thymus. Furthermore, generation of these Treg is not dependent on the expansion of CD25+CD4+ thymic emigrants, because depletion of CD25+ cells before pretreatment does not prevent Treg development, and Treg can be generated from CD25−CD4+ precursors. Taken together, these results clearly demonstrate that CD25+CD4+ Treg can be generated in the periphery from CD25−CD4+ precursors in a pathway distinct to that by which naturally occurring autoreactive CD25+CD4+ Treg develop. These observations may have important implications for the design of protocols, both experimental and clinical, for the induction of tolerance to autoantigens or alloantigens in adults with limited thymic function.

Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells.

Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-β was highly dependent on NFAT expression because the ability of CD4+ T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-β–induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.

Fas ligand breaks tolerance to self-antigens and induces tumor immunity mediated by antibodies

The role of Fas ligand (FasL) in programmed cell death via interaction with its receptor Fas is well characterized. It has been proposed that expression of FasL can confer immune privilege to some organs, allowing them to kill infiltrating lymphocytes and inflammatory cells. However, a number of studies have shown that when tumors or transplants express FasL, rejection often occurs as a consequence of proinflammatory functions of FasL. Here we demonstrate that FasL elicits tumor immunity in a murine melanoma model with weak immunogenicity and low expression of major histocompatibility complex (MHC) class I. We show that protected mice recognize melanocyte differentiation self-antigens. Importantly, tumor immunity is mediated by antibodies, as it can be transferred by serum from protected mice.

Clinical Use of Tolerogenic Dendritic Cells-Harmonization Approach in European Collaborative Effort.

The number of patients with autoimmune diseases and severe allergies and recipients of transplants increases worldwide. Currently, these patients require lifelong administration of immunomodulatory drugs. Often, these drugs are expensive and show immediate or late-occurring severe side effects. Treatment would be greatly improved by targeting the cause of autoimmunity, that is, loss of tolerance to self-antigens. Accumulating knowledge on immune mechanisms has led to the development of tolerogenic dendritic cells (tolDC), with the specific objective to restrain unwanted immune reactions in the long term. The first clinical trials with tolDC have recently been conducted and more tolDC trials are underway. Although the safety trials have been encouraging, many questions relating to tolDC, for example, cell-manufacturing protocols, administration route, amount and frequency, or mechanism of action, remain to be answered. Aiming to join efforts in translating tolDC and other tolerogenic cellular products (e.g., Tregs and macrophages) to the clinic, a European COST (European Cooperation in Science and Technology) network has been initiated—A FACTT (action to focus and accelerate cell-based tolerance-inducing therapies). A FACTT aims to minimize overlap and maximize comparison of tolDC approaches through establishment of minimum information models and consensus monitoring parameters, ensuring that progress will be in an efficient, safe, and cost-effective way.

Alpha-1,2-Mannosidase and Hence N-Glycosylation Are Required for Regulatory T Cell Migration and Allograft Tolerance in Mice

Background Specific immunological unresponsiveness to alloantigens can be induced in vivo by treating mice with a donor alloantigen in combination with a non-depleting anti-CD4 antibody. This tolerance induction protocol enriches for alloantigen reactive regulatory T cells (Treg). We previously demonstrated that alpha-1,2-mannosidase, an enzyme involved in the synthesis and processing of N-linked glycoproteins, is highly expressed in tolerant mice, in both graft infiltrating leukocytes and peripheral blood lymphocytes. Principal Findings In this study we have identified that alpha-1,2-mannosidase expression increases in CD25+CD4+ Treg when they encounter alloantigen in vivo. When alpha-1,2-mannosidase enzyme activity was blocked, Treg retained their capacity to suppress T cell proliferation in vitro but were unable to bind to physiologically relevant ligands in vitro. Further in vivo analysis demonstrated that blocking alpha-1,2-mannosidase in Treg resulted in the migration of significantly lower numbers to the peripheral lymph nodes in skin grafted mice following adoptive transfer, where they were less able to inhibit the proliferation of naïve T cells responding to donor alloantigen and hence unable prevent allograft rejection in vivo. Significance Taken together, our results suggest that activation of alloantigen reactive Treg results in increased alpha-1,2-mannosidase expression and altered N-glycosylation of cell surface proteins. In our experimental system, altered N-glycosylation is not essential for intrinsic Treg suppressive capacity, but is essential in vivo as it facilitates Treg migration to sites where they can regulate immune priming. Migration of Treg is central to their role in regulating in vivo immune responses and may require specific changes in N-glycosylation upon antigen encounter.

Bag-1 up-regulation in anti-CD4 mAb treated allo-activated T cells confers resistance to apoptosis

The nondepleting anti‐CD4 mAb RIB5/2 is a powerful inducer of tolerance to MHC‐incompatible renal and heart allografts in rat recipients. In vitro the mAb blocks the proliferation andcytokine production of alloreactive T cells. To learn more about the mechanism of anti‐CD4‐mediated suppression, we applied differential display reverse transcription‐PCR to identify differences atmRNA level between T cells stimulated by alloantigen in the presence or absence of anti‐CD4 mAb. A sequence alignment of a 550‐bp DNA fragment appearing only in anti‐CD4 mAb‐treated cells resulted in at least 95% homology to a mouse cDNA encoding for the anti‐apoptotic protein Bag‐1. Further investigation of Bag‐1 expression during mixed lymphocyte reactions revealed a three‐ to fourfold up‐regulation of Bag‐1 mRNA expression in anti‐CD4 mAb‐treated allogeneic cultures which was confirmed at protein level. Bag‐1 up‐regulation was associated with an increase resistance to apoptosis of T cells from anti‐CD4 mAb‐treated cultures. Application of antisense oligonucleotides specific for Bag‐1 reduced Bag‐1 protein expression and restored susceptibility to apoptosis. In addition, up‐regulationof Bag‐1 mRNA could also be detected in graft‐infiltrating T cells from anti‐CD4 mAb‐treated rats in vivo. Thus, the expression of Bag‐1 in a subset of anti‐CD4 mAb‐treated alloreactive T cells conferred resistance against apoptosis, potentially contributing to the long‐term survival of these cells.

CD45RA Distinguishes CD4+CD25+CD127-/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression.

BackgroundAdoptive transfer of forkhead box protein (FOX)3+ regulatory T (Treg) cells offers a promising strategy to reduce damage to an allograft by the recipients immune system. Identification of cell surface markers sufficient to purify Treg cells expanded ex vivo to remove cellular contaminants requires optimization. Furthermore, the expanded Treg must be able to survive, expand, and suppress in allograft recipients exposed to immunosuppressants, such as tacrolimus (TAC). Reduced CD127 expression enhances identification of Treg in the human CD4+CD25+ population. CD45RA expression identifies naive CD4+CD25+ Treg with an enhanced stability of Treg phenotype. MethodsWe combine an analysis of CD45RA, CD25, and CD127 expression to identify subpopulations of CD4+CD127−/loCD25+ cells. Regulatory T cells were sorted according to expression of CD25 and CD45RA and expanded in the presence of a physiological relevant concentration of TAC. Regulatory T cell–specific demethylation region (TSDR) demethylation, FOXP3 expression, and suppression were analyzed. ResultsCD4+CD127−/loCD25+CD45RA+ Treg cells had a stable TSDR demethylated FOXP3+ phenotype after expansion whereas CD4+CD127lo/−CD25+CD45RA− Treg cell lost the TSDR demethylated phenotype. CD45RA− Treg had a greater capacity to suppress after expansion with TAC. ConclusionsAlthough CD45RA− Treg retained a greater suppressive capacity when expanded with TAC, the marked loss of the TSDR demethylated status highlights the potential for loss of stability of these cells in transplant recipients treated with TAC based immunosuppression. We show that a population of CD4+CD127−/loCD45RA+ Regulatory T cell may offer the best compromise between susceptibility to loss of suppression when exposed to TAC and maintenance of a TSDR demethylated phenotype following in vitro expansion.

Bag-1 up-regulation in anti-CD4 mAb-treated allo-activated T cell confers resistance to activation-induced cell death (AICD)

The non-depleting anti-CD4 monoclonal antibody (mAb) RIB5/2 is a powerful inducer of tolerance to major histocompatibility complex (MHC)-incompatible allografts in rat recipients. The unresponsiveness induced is characterized by the persistence (over 300 days) of donor-reactive regulatory T cells within the graft. We applied differential-display reverse-transcription polymerase chain reaction (RT-PCR) to identify differences at the mRNA level between graft-infiltrating cells of anti-CD4 mAb-treated and non-treated control rats at day 5 after kidney transplantation. A 550-bp DNA fragment appearing only in anti-CD4 mAb-treated rats is identical with the anti-apoptotic protein Bag-1. A further investigation of Bag-1 expression during mixed lymphocyte reactions (MLR) revealed a three-four-fold up-regulation of Bag-1 mRNA expression in anti-CD4 mAb-treated allogeneic cultures. Bag-1 up-regulation is associated with higher protection against apoptosis of anti-CD4 mAb-treated cultures. Application of antisense oligonucleotides specific for Bag-1 leads to both a reduction in Bag-1 expression and sensibility against apoptosis. Thus, the expression of Bag-1 in anti-CD4 mAb-treated alloreactive T cells conferred resistance against apoptosis, which may contribute to the long-term survival of tolerance-mediating T cells in vivo.

EVIDENCE THAT TREATMENT WITH DHRS9+ REGULATORY MACROPHAGES INDUCED BY FCγRIII LIGATION AND IFN-γ STIMULATION PROMOTES RENAL ALLOGRAFT TOLERANCE IN PATIENTS: 3261

J.A. Hutchinson1, P. Riquelme2, D.P. Brown1, B. Sawitzki3, M. Rehli4, S. Tomiuk5, J. Schröder4, A. Sotnikova6, P. Miqueu7, M. Zuhayra6, H.H. Oberg6, A. Pascher8, U. Lützen6, U. Janßen5, F. Thaiss9, E. Scheuermann10, E. Henze6, L. Chatenoud11, H. Volk12, R.I. Lechler13, K.J. Wood14, D. Kabelitz6, H.J. Schlitt15, F. Fändrich6, E.K. Geissler2 1Allgemeine Chirurgie, Universitätsklinikum Regensburg, Regensburg/ GERMANY, 2General And Experimental Surgery, University Hospital Regensburg, Regensburg/GERMANY, 3, Institute for Medical Immunology, Berlin/GERMANY, 4, Universitätsklinikum Regensburg, Regensburg/GERMANY, 5, Miltenyi GmbH, Bergisch Gladbach/ GERMANY, 6, Universitätsklinikum Schleswig-Holstein, Kiel/ GERMANY, 7, TcLand Expression, Nantes/FRANCE, 8Department Of General, Visceral And Transplant Surgery, Charité Campus Virchow, Berlin/GERMANY, 9Nephrologie, UKE, Hamburg/GERMANY, 10, Univ. Frankfurt, Frankfurt/GERMANY, 11, Rene Descartes University, Paris/ FRANCE, 12, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin/GERMANY, 13, King’s College London, London/ UNITED KINGDOM, 14Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, Oxford/UNITED KINGDOM, 15, Univ. Regensburg, Regensburg/GERMANY

Minimum information about T regulatory cells: A step toward reproducibility and standardization

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.