[Show abstract][Hide abstract] ABSTRACT: Accumulation of CD3+TCRαβ+CD4-CD8- double negative T (DNT) cells is a hallmark of autoimmune lymphoproliferative syndrome (ALPS). DNT origin and differentiation pathways remain controversial. Here we show that human ALPS DNT have features of terminally differentiated effector memory CD45RA+ (TEMRA) cells, but are CD27+CD28+KLRG1- and do not express the transcription factor T-bet. This unique phenotype was also detected among CD4+ or CD8+ ALPS TEMRA cells. TCRβ deep sequencing revealed a significant fraction of shared CDR3 sequences between ALPS DNT and both CD4+ and CD8+TEMRA cells. Moreover, in ALPS patients with a germline FAS mutation and somatic loss of heterozygosity, in whom biallelic mutant cells can be tracked by absent Fas expression, Fas negative T-cells accumulated not only among DNT, but also among CD4+ and CD8+TEMRA cells. These data indicate that in human Fas deficiency DNT can not only derive from CD8+, but also from CD4+ T-cells. Furthermore, defective Fas signalling leads to aberrant transcriptional programs and differentiation of subsets of CD4+ and CD8+ T-cells. Accumulation of these cells before their double negative state appears to be an important early event in the pathogenesis of lymphoproliferation in ALPS patients.
[Show abstract][Hide abstract] ABSTRACT: Adoptive transfer of third-party mesenchymal stromal cells (MSCs) has emerged as a promising tool for the treatment of steroid-refractory GVHD. Despite numerous in vitro studies and preclinical models, little is known about their effects on the patients' immune system. We assessed immune alterations in the T-cell, B-cell, NK-cell, dendritic cell, and monocytic compartments of steroid-refractory GVHD patients 30, 90, and 180 days after MSC (n=6) or placebo (n=5) infusion respectively. Infused MSCs were bioactive as suggested by the significant reduction of epithelial cell death, which represents a biomarker for acute GVHD. There were several indications that MSCs shift the patients' immune system towards a more tolerogenic profile. Most importantly infusion of MSCs was associated with increased levels of regulatory (FOXP3(+) and IL-10(+) ) T-cells, reduced pro-inflammatory IL-17(+) T(Th17)-cells, and skewing towards type-2 T-helper cell responses. Furthermore IL-2, which has been recently shown to exert a positive immune modulating effect in GVHD patients, was higher in the MSC patients at all evaluated time points during six months after MSC-infusion. Overall, our findings will contribute to the refinement of monitoring tools, for assessing MSC treatment-efficacy and increase our understanding regarding the MSCs' in vivo effects.
[Show abstract][Hide abstract] ABSTRACT: Upon specific interaction with APCs, T cells capture membrane fragments and surface molecules in a process termed trogocytosis. In this study, we demonstrate that human Ag-specific CD8(+) T cells acquire the coinhibitory molecule programmed death ligand 1 (PD-L1) from mature dendritic cells (mDC) and tumor cells in an Ag-specific manner. Immature dendritic cells were less effective in transferring surface molecules onto CD8(+) T cells than mDCs. Interestingly, trogocytosis of PD-L1 requires cell-cell contact and cannot be induced by uptake of soluble proteins obtained from mDC lysates. The transfer process is impaired by inhibition of vacuolar ATPases in T cells as well as by fixation of dendritic cells. Of importance, CD8(+) T cells that acquired PD-L1 complexes were able to induce apoptosis of neighboring programmed death 1-expressing CD8(+) T cells. In summary, our data demonstrate that human CD8(+) T cells take up functionally active PD-L1 from APCs in an Ag-specific fashion, leading to fratricide of programmed death 1-expressing, neighboring T cells. The transfer of functionally active coinhibitory molecules from APCs onto human CD8(+) T cells could have a regulatory role in immune responses.
The Journal of Immunology 12/2011; 188(2):744-52. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (Tregs) play an important role in the maintenance of immune tolerance to self-antigens and are involved in modulating immune responses in autoimmunity, transplant rejection, and tumor immunity. Recently, a novel subset of TCR-αβ(+) CD4(-) CD8(-) (double negative, DN) T cells has been described to specifically suppress T-cell responses in mice. Here, we demonstrate that human DN T cells are highly potent suppressors of both CD4(+) and CD8(+) T-cell responses. In contrast to naturally occurring CD4(+) CD25(+) Tregs, DN T cells have to be activated by antigen-presenting cells (APCs) to induce their regulatory potential. The suppressive activity of DN T cells is neither mediated indirectly by modulation of APCs nor by competition for T-cell growth factors. Furthermore, DN T-cell-mediated suppression toward responder T cells is TCR dependent and requires novel protein synthesis. In contrast to murine DN T cells, which eliminate effector T cells via Fas/FasL or perforin/granzyme, human DN T cells suppress proliferation of responder T cells by cell contact-dependent mechanisms. Taken together, our data indicate that human DN T cells exert strong immunosuppressive effects on both CD4(+) and CD8(+) T cells and may serve as a new therapeutic approach to treat autoimmunity and transplant rejection.
European Journal of Immunology 03/2011; 41(3):739-48. · 4.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Effective immunotherapy using T cell receptor (TCR) gene-modified T cells requires an understanding of the relationship between
TCR affinity and functional avidity of T cells. In this study, we evaluate the relative affinity of two TCRs isolated from
HLA-A2-restricted, gp100-reactive T cell clones with extremely high functional avidity. Furthermore, one of these T cell clones,
was CD4−CD8− indicating that antigen recognition by this clone was CD8 independent. However, when these TCRs were expressed in CD8− Jurkat cells, the resulting Jurkat cells recognized gp100:209–217 peptide loaded T2 cells and had high functional avidity,
but could not recognize HLA-A2+ melanoma cells expressing gp100. Tumor cell recognition by Jurkat cells expressing these TCRs could not be induced by exogenously
loading the tumor cells with the native gp100:209–217 peptide. These results indicate that functional avidity of a T cell
does not necessarily correlate with TCR affinity and CD8-independent antigen recognition by a T cell does not always mean
its TCR will transfer CD8-independence to other effector cells. The implications of these findings are that T cells can modulate
their functional avidity independent of the affinity of their TCRs.
Cancer Immunology and Immunotherapy 05/2009; 58(5):719-728. · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Novel immunosuppressive strategies are targeting for an antigen-specific deletion of T cells responsible for organ damage in autoimmunity and allograft rejection. Here, we present a new flow cytometry-based assay that allows the reliable and efficient detection of T cells that were eliminated in an antigen-specific fashion. A stable cell-labelling technique utilizing the two membrane dyes PKH26 and PKH67 has been combined with annexin V and 7-aminoactinomycin (7-AAD) staining to detect apoptotic cells. A differential gating strategy enabled us to determine the viability/apoptosis for each PKH-stained T cell subpopulation independently. The capability to simultaneously analyze apoptosis within T cell mixtures of different antigen specificities establishes this assay as a superior tool for the further development of novel antigen-specific immunosuppressive approaches.
Journal of immunological methods 04/2009; 344(2):98-108. · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The immune attack against malignant tumors require the concerted action of CD8+ cytotoxic T lymphocytes (CTL) as well as CD4+ T helper cells. The contribution of T cell receptor (TCR) alphabeta+ CD4- CD8- double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here, we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-gamma and TNF. Although lacking the CD8 molecule the gp100-specific DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2+ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo.
Cancer Immunology and Immunotherapy 11/2008; 58(5):709-18. · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A characteristic feature of tumors is high production of lactic acid due to enhanced glycolysis. Here, we show a positive correlation between lactate serum levels and tumor burden in cancer patients and examine the influence of lactic acid on immune functions in vitro. Lactic acid suppressed the proliferation and cytokine production of human cytotoxic T lymphocytes (CTLs) up to 95% and led to a 50% decrease in cytotoxic activity. A 24-hour recovery period in lactic acid-free medium restored CTL function. CTLs infiltrating lactic acid-producing multicellular tumor spheroids showed a reduced cytokine production. Pretreatment of tumor spheroids with an inhibitor of lactic acid production prevented this effect. Activated T cells themselves use glycolysis and rely on the efficient secretion of lactic acid, as its intracellular accumulation disturbs their metabolism. Export by monocarboxylate transporter-1 (MCT-1) depends on a gradient between cytoplasmic and extracellular lactic acid concentrations and consequently, blockade of MCT-1 resulted in impaired CTL function. We conclude that high lactic acid concentrations in the tumor environment block lactic acid export in T cells, thereby disturbing their metabolism and function. These findings suggest that targeting this metabolic pathway in tumors is a promising strategy to enhance tumor immunogenicity.
[Show abstract][Hide abstract] ABSTRACT: In eukaryotic cells the phospholipid phosphatidylserine (PS) is restricted to the inner plasma-membrane leaflet. This lipid asymmetry, which is maintained by the concerted action of phospholipid transport proteins, is mainly lost during apoptosis. Here, we demonstrate that primary human CD8+ cytotoxic T lymphocytes (CTLs) expose PS on T-cell receptor (TCR)-mediated antigen (Ag) recognition. In contrast to PS externalization on apoptotic cells, activation-induced PS exposure is less pronounced and reversible. Fluorescence microscopic analysis revealed that PS is distributed nonhomogenously over the plasma membrane and concentrated in membrane lipid raft domains at the immunologic synapse. By studying the activity of PS transport proteins using a fluorescence-labeled PS analogue, we found that activation of CTLs inhibited the flippase-mediated inward-directed PS transport without affecting the outward transport. Shielding of exposed PS by annexin V protein during Ag recognition diminished cytokine secretion, activation, and cell-to-cell clustering of Ag-specific CTLs. In summary, our data demonstrate for the first time that externalized PS on Ag-stimulated CTLs is linked to T-cell activation and probably involved in cell-to-cell contact formation at the immunologic synapse.
[Show abstract][Hide abstract] ABSTRACT: Human tumors frequently escape immune destruction, despite the presence of cytotoxic T cells (CTL) recognizing tumor-associated antigens (TAA). We have previously shown that programmed death ligand-1 (PD-L1), a recently identified ligand of the B7 superfamily, is expressed on murine tumors and can inhibit antitumor immune responses. To evaluate the clinical relevance of our animal model findings, we examined human tumors and tumor-specific T cells. We found PD-L1 to be constitutively expressed on human renal cell carcinoma (RCC) cell lines and upregulated on human melanoma cell lines upon exposure to interferon-gamma. Similarly, we found binding of anti-PD-L1 monoclonal antibody (mAb) on frozen sections from RCC and melanomas, but not on normal tissues. The corresponding inhibitory receptor of PD-L1, PD-1, revealed a higher expression on tumor-infiltrating lymphocytes than on peripheral blood lymphocytes (PBL) from melanoma patients upon specific antigen stimulation. Stimulation of PBL from healthy donors with peptide-loaded dendritic cells in the presence of anti-PD-L1 mAb altered neither the total T cell numbers after expansion, nor the percentage of peptide-specific CTL, when providing a T cell help by addition of cytokines. However, when stimulating TAA-specific CTL and T helper cells with Ag-pulsed dendritic cells in the absence of exogenous cytokines, PD-L1 blockade increased the cytokine production. Similar to the data achieved in the murine system, the blockade of PD-L1 on human tumors resulted in enhanced cytolytic activity of TAA-specific CTLs and cytokine production of TAA-specific T helper cells when interacting directly with the tumor. In summary, our data suggest that PD-L1/PD-1 interactions negatively regulate T cell effector functions predominantly in the absence of exogenous cytokine support, indicating an important role for this pathway in tumor evasion.
International Journal of Cancer 08/2006; 119(2):317-27. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor-alpha (TNFalpha) is a potent inhibitor of renin gene expression in renal juxtaglomerular cells. We have found that TNFalpha suppresses renin transcription via transcription factor NFkappaB, which targets a cAMP responsive element (CRE) in the renin promoter. Here we aimed to further clarify the role of NFkappaB and the canonical CRE-binding proteins of the CRE-binding protein/activating transcription factor (CREB/ATF) family in the inhibition of renin gene expression by TNFalpha in the juxtaglomerular cell line As4.1. TNFalpha caused a moderate decrease in the binding of CREB1 to its cognate CRE DNA binding site. On the other hand, NFkappaB-p65 transcriptional activity was substantially reduced by TNFalpha, which targeted a trans-activation domain at the very C terminus of the p65 molecule. Our results suggest that TNFalpha inhibits renin gene expression by decreasing the transactivating capacity of NFkappaB-p65 and partially by attenuating CREB1 binding to CRE.
Journal of Biological Chemistry 08/2005; 280(26):24356-62. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor-α (TNFα) is a potent inhibitor of renin gene expression in renal juxtaglomerular cells. We have found
that TNFα suppresses renin transcription via transcription factor NFκB, which targets a cAMP responsive element (CRE) in the
renin promoter. Here we aimed to further clarify the role of NFκB and the canonical CRE-binding proteins of the CRE-binding
protein/activating transcription factor (CREB/ATF) family in the inhibition of renin gene expression by TNFα in the juxtaglomerular
cell line As4.1. TNFα caused a moderate decrease in the binding of CREB1 to its cognate CRE DNA binding site. On the other
hand, NFκB-p65 transcriptional activity was substantially reduced by TNFα, which targeted a trans-activation domain at the
very C terminus of the p65 molecule. Our results suggest that TNFα inhibits renin gene expression by decreasing the transactivating
capacity of NFκB-p65 and partially by attenuating CREB1 binding to CRE.
Journal of Biological Chemistry 06/2005; 280(26):24356-24362. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Down-regulation of immune responses by regulatory T (Treg) cells is an important mechanism involved in the induction of tolerance to allo-antigens (Ags). Recently, a novel subset of Ag-specific T-cell receptor (TCR)alpha beta+ CD4(-)CD8- (double-negative [DN]) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of antigraft-specific CD8+ T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3+ T cells, and consist of both naive and Ag-experienced cells. Similar to murine DN Treg cells, human DN Treg cells are able to acquire peptide-HLA-A2 complexes from antigen-presenting cells by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8+ T cells specific for the HLA-A2-restricted self-peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A-HLA-A2 complexes and revealed a reduced proliferative response. These results demonstrate for the first time that a sizable population of peripheral DN Treg cells, which are able to suppress Ag-specific T cells, exists in humans. DN Treg cells may serve to limit clonal expansion of allo-Ag-specific T cells after transplantation.
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor-alpha (TNFalpha) is known to inhibit renin gene expression in juxtaglomerular cells, which are the main source of renin in vivo. In the present study we aimed to characterize the intracellular mechanisms of TNFalpha signaling to renin gene in the mouse juxtaglomerular cell line As4.1. TNFalpha was found to activate NFkappaB, which is one of the principal intracellular mediators of TNFalpha signal transduction. Constitutive activation of NFkappaB suppressed renin gene transcription, but NFkappaB appeared not to target the NFkappaB binding sites in the renin promoter. Thus, NFkappaB, but not the canonical NFkappaB binding sequences in the renin promoter, seemed to be involved in the suppression of renin transcription by TNFalpha. Deletion/mutation analysis revealed that the effect of TNFalpha on renin gene is transmitted by a cAMP-responsive element (CRE) located at -2697 to -2690. Mobility shift/supershift assays evidenced for the presence of NFkappaB proteins in the complex that binds to mouse renin CRE. Our results strongly suggest that NFkappaB mediates the effect of TNFalpha on renin transcription targeting a CRE in the mouse renin promoter.
Journal of Biological Chemistry 02/2004; 279(2):1458-67. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Down-regulation of immune responses by regulatory T (Treg) cells is an impor- tant mechanism involved in the induction of tolerance to allo-antigens (Ags). Re- cently, a novel subset of Ag-specific T- cell receptor (TCR) CD4CD8 (double-negative (DN)) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of antigraft-spe- cific CD8 T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3 T cells, and consist of bothnaiveandAg-experiencedcells.Simi- lar to murine DN Treg cells, human DN Treg cells are able to acquire peptide- HLA-A2 complexes from antigen-present- ing cells by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8 T cells specific for the HLA-A2-restricted self- peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A-HLA-A2 com- plexes and revealed a reduced prolifera- tive response. These results demonstrate for the first time that a sizable population of peripheral DN Treg cells, which are able to suppress Ag-specific T cells, ex- ists in humans. DN Treg cells may serve to limit clonal expansion of allo-Ag- specific T cells after transplantation. (Blood. 2005;105:2828-2835)