Ute Schulz

The Role of IL-10 and TGF-β in the Differentiation and Effector Function of T Regulatory Cells

Suppression by T regulatory (Tr) cells is essential for the induction of peripheral tolerance. Many types of CD4+ Tr cells have been described in a number of systems, and although the precise mechanisms which mediate their effects remain to be defined, it is well established that they can suppress immune responses via cell-cell interactions and/or the production of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). Type 1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL-10 and TGF-β, and these cytokines mediate their ability to suppress pathological immune responses in the settings of transplantation, allergy and autoimmune disease. Tr1 cell activity is not necessarily beneficial, and they can also suppress immune responses to antigens from tumours and pathogens. In vivo, the differentiation of Tr1 cells is likely controlled by certain dendritic cells which promote IL-10 production and may express tolerogenic costimulatory molecules. Another subset of CD4+ Tr cells is defined by constitutive expression of CD25, and although these CD4+CD25+ Tr cells appear to suppress via mechanisms which are largely independent of cytokines, they may actively promote the differentiation of Tr1 cells. Many questions about the basic biology of Tr1 cells remain to be answered, but the development of therapeutic strategies designed to harness their immunoregulatory effects can already be contemplated.

Prognostic significance of increased IL-10 production in patients prior to allogeneic bone marrow transplantation.

IL-10 is a potent immunosuppressant which inhibits allo-antigen-specific T cell responses. In addition, IL-10 is a strong endogenous anti-inflammatory cytokine. To investigate the role of IL-10 in the induction of acute GVHD following allogeneic bone marrow transplantation (BMT) we performed a prospective study on spontaneous IL-10 production by peripheral blood mononuclear cells (PBMNC) in 84 patients admitted for allogeneic BMT. High spontaneous IL-10 production by PBMNC at the time of admission and prior to any preparative treatment correlated with a subsequent low incidence of GVHD and transplant-related mortality (8%), as compared to patients with low or intermediate IL-10 production (50%, P < 0.01). Our data demonstrate the prognostic significance of increased IL-10 production in BMT patients and suggest a major role of IL-10 in maintaining immunobalance in the setting of allogeneic BMT. Bone Marrow Transplantation (2000) 25, 237–241.

Interferon (IFN)-gamma is a main mediator of keratinocyte (HaCaT) apoptosis and contributes to autocrine IFN-gamma and tumour necrosis factor-alpha production.

Background  Apoptosis of keratinocytes or intestinal epithelial cells is an important pathophysiological mechanism of organ damage during acute graft‐versus‐host disease.

Molecular and functional characterization of allogantigen specific anergic T cells suitable for cell therapy

Background CD4+ regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies. Design and Methods The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported. Results Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors. Conclusions Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.

Tryptophan catabolism is associated with acute GVHD after human allogeneic stem cell transplantation and indicates activation of indoleamine 2,3-dioxygenase

Induction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway, acts as a potent immunoregulatory loop. To address its role in human allogeneic stem cell transplantation, we measured major tryptophan metabolites, such as quinolinic acid and kynurenine, in serial urine specimens from 51 patients by liquid chromatography-tandem mass spectrometry. Samples were collected between admission and day 90 after transplantation, and metabolite levels were correlated with early clinical events and outcome. In selected patients, IDO gene expression was assessed by quantitative RT-PCR in intestinal biopsies. Surviving patients had significantly lower metabolite levels on days 28, 42, and 90, respectively, compared with patients dying of GVHD and associated complications (n = 10). Kynurenine levels were directly correlated with severity and clinical course of GVHD: Mean urinary quinolinic acid levels were 4.5 ± 0.3 μmol/mmol creatinine in the absence of acute GVHD, 8.0 ± 1.1 μmol/mmol creatinine for GVHD grade 1 or 2, and 13.5 ± 2.7 μmol/mmol creatinine for GVHD grade 3 or 4 (P < .001), respectively. GVHD-dependent induction of IDO was further suggested by increased expression of IDO mRNA in intestinal biopsies from patients with severe GVHD. Our data indicate reactive release of kynurenines in GVHD-associated inflammation.

Reverse signalling of membrane‐integrated tumour necrosis factor differentially regulates alloresponses of CD4+ and CD8+ T cells against human microvascular endothelial cells

Reverse signalling of membrane‐integrated ligands is a common phenomenon in the tumour necrosis factor (TNF) family and contributes to the pleiotropy of this pro‐inflammatory cytokine and to the plasticity of the immune system in general. Transmembrane TNF (mTNF) itself can induce resistance to bacterial endotoxin in monocytes and can stimulate the immune activity of mitogen‐activated, as well as of virus‐infected, T cells. The aim of the present study was to investigate the influence of reverse signalling of mTNF on the allogeneic activity of CD4+ and CD8+ T cells against human microvascular endothelial cells (HMEC), as targets of various inflammatory responses. The proliferative potential of CD4+ T cells towards HMEC was attenuated by mTNF signalling, whereas stimulation of mTNF on CD8+ T cells increased their cytotoxic potential aginst HMEC. These effects were specific for reverse signalling of mTNF, as a blockade of the classical TNF–TNF receptor interaction by a neutralizing TNF receptor antibody had no effect. Cytokine profiling of the effector cells revealed that the anti‐endothelial CD4+ T cells were of a T helper 2 (Th2) phenotype, whereas CD8+ T cells mainly produced cytotox. T cell 1 (Tc1) cytokines. From the results obtained in this study, we conclude that reverse signalling of mTNF differentially modulates CD4+ and CD8+ T‐cell activity against allogeneic endothelial cells, which should be taken into account in settings of therapeutic cytokine antagonisms.

Interleukin-10 promotes NK cell killing of autologous macrophages by stimulating expression of NKG2D ligands.

Under inflammatory conditions, the pleiotropic cytokine interleukin‐10 (IL‐10) is released in many tissues. It mediates anti‐inflammatory effects in particular by inhibiting the release of T helper type 1 (Th1) cytokines. In contrast, we show here that NK cell cytotoxicity against autologous macrophages is elevated if both cell types are cultured with IL‐10. The expression of most activatory NK receptors is increased after culture in the presence of IL‐10. On the other hand, macrophages cultured in the presence of IL‐10 show elevated expression of the NKG2D ligands major histocompatibility complex (MHC) class 1‐like molecules (MIC) – A and – B, as well as UL‐16 binding proteins (ULBP) – ULBP‐1, ULBP‐2 and ULBP‐3. By masking the interaction of NK cells with macrophages through interruption of the NKG2D receptor with its ligands, we could reverse the IL‐10‐induced lysis of macrophages. Our data therefore reveal that IL‐10 may exert a novel immunomodulatory role by stimulating NKG2D ligand expression on macrophages, thereby rendering them susceptible to NK cell elimination. This suggests that NK cells would delete macrophages and potentially other immature antigen‐presenting cells (APC) or their precursors under inflammatory conditions as a feedback mechanism to shut off uncontrolled immune responses.

Interleukin-10 modulation of alloreactivity and graft-versus-host reactions.

Background. The biological properties of interleukin (IL)-10 in tolerance induction and inhibition of alloreactivity have suggested a therapeutic use of this cytokine as an additional or alternative prophylaxis for graft-versus-host disease (GvHD). However, the effects of exogenous IL-10 on GvHD are mainly studied in animal models, and the results remain conflicting. This study aims to demonstrate, for the first time, whether the addition of exogenous IL-10 can reduce the severity of graft-versus-host reactions (GvHR) in humans. Methods. The regulatory role of exogenous IL-10 in GvHR was investigated using an in vitro human skin explant model. The effects of IL-10 on skin GvHR were tested in parallel with allo-antigen induced T-cell proliferation, cytolytic reactivity, and cytokine production. Results. In the presence of IL-10, the mixed lymphocyte reaction (MLR) primed responder cells showed significantly lower proliferative and cytolytic responses compared with the responder cells from the control MLR carried out in the absence of IL-10. The responder cells from IL-10 containing MLR induced significantly less severe skin GvHR and displayed a significantly reduced T-cell activation and cytokine production. A significant correlation was observed between the levels of TNF-&agr; production and the sensitivity to IL-10 modulation of GvHR. Conclusions. The addition of exogenous IL-10 strongly inhibited the broad alloreactivity initiated by primary MLR and significantly reduced the overall severity of skin GvHR induced by MLR primed responder cells. Responder cells producing high TNF-&agr; following allogeneic stimulation appeared to be less sensitive to IL-10 modulation of GvHR.

Histological correlation between different centers using the skin explant model to predict graft-versus-host disease following bone marrow transplantation.

Acute graft-versus-host disease (GVHD) remains the major complication of allogeneic stem cell transplantation (SCT) with an incidence of 40-60% and a mortality of up to 50%. Several assays have been developed to try to predict the development of GVHD including the mixed lymphocyte culture reaction, cytotoxic and helper T lymphocyte precursor frequency assays. In the Northern region of England we have used an in vitro skin explant model for predicting GVHD in MHC compatible bone marrow transplant recipients since 1988. The aims of the present study was to test the reproducibility of the model in two other bone marrow transplant centers in Europe. The assay consists of incubating patient skin explants with effector cells from mixed donor versus recipient lymphocyte cultures and the subsequent detection of graft-versus-host reactions by histopathological grading (0-IV) of the skin explants. 503 slides from 134 patients were evaluated. All were graded for negative GVHR grade 0-I or positive grade II-IV. Results from control and test slides significantly correlated between centers to the p value of 0.0001 by Fishers exact probability test. These results show that the skin explant assay is reproducible between centers and supports the continued use of the assay to predict GVHD in allogeneic stem cell transplant recipients.

Skin-explant model to evaluate effectiveness of depletion of hla-alloreactive t-cells

Abstract Depletion of host specific alloreactive T-cells from donor lymphocytes is an attractive tool for donor lymphocyte infusions after stem cell grafting. We intended to establish a preclinical method to evaluate effectiveness and safety of this approach. The skin explant model is a powerful method to predict the risk of GvHD in the HLA-identical setting, and we adapted this model to the HLA-haploidentical situation. Unmanipulated donor T-cells are compared with donor T-cells following depletion of host specific T-cells. Prior to depletion donor MNC are incubated in the presence of irradiated recipient MNC and after a period of 5 days activated T-cells, positive for CD25, CD69, CD71 and HLA-DR, are magnetically removed (VarioMacs, Miltenyi). A repeat MLC of unmanipulated donor cells. Fresh skin biopsies are divided into pieces of 1 - 2 mm diameter and incubated with 10 6 cultured T-cells in 20% autologous serum for 72 hours, fixed in formaldehyde and stained with haematoxylin-eosin. Skin alterations are graded according to Learner et al. In 3 separate experiments undepleted cells caused subepidermal cleft formation corresponding to a grade III to IV GvHD reaction. In contrast, depleted cells showed only grade I alterations, as similarly observed in medium control. Our preliminary data suggest that this approach could represent a useful tool to determine effectiveness of magnetic depletion of alloreactive T-cells and be valuable to assess the safety of these cells prior to infusion.