Placenta Derived Adherent Cells Modulate the Allogenic Mixed Lymphocyte Reaction Partially via an Indoleamine 2,3-Dioxygenase Mediated Mechanism.

Abstract Placenta Derived Adherent Cells (PDAC) are multipotent progenitor cells derived from human placental tissues. Previously we have reported that PDACs could suppress T-cell proliferation when added to in-vitro mixed lymphocyte reactions (PDAC-MLR) (Paludan C. et al, Blood. (ASH Annual Meeting Abstracts) 2006 108: Abstract 1737). Here we present aspects of the mechanism of this PDAC suppression property. We have found that PDACs modify cytokine production in the PDAC-MLR reaction in comparison to the MLR; TNF-α and IFN-γ are reduced 75% and 30% while TGF-β production is significantly increased. We have used a transwell assay system to investigate the cell-contact-dependency of the effects of PDACs on T-cell proliferation. The assay system comprised combinations of the MLR in the top chamber together with the PDAC-MLR, PDACs plus naive T cells or PDACs alone in the bottom chamber. Maximum inhibition of T cell proliferation of the MLR in the top insert could be achieved by placing the PDAC-MLR co-culture in the bottom chamber. Minimum suppression was obtained when placing PDACs plus naive T cells or PDACs alone in the bottom chamber. PDAC-MLR conditioned media could partially suppress the MLR reaction. Addition of L-tryptophan into the MLR with PDAC conditioned media completely abolished PDAC-induced suppression of T cell proliferation. Likewise, the addition of the 1-methyl tryptophan to the PDAC-MLR reaction could abolish the PDAC-induced suppression. These results suggested that the suppression of the MLR by PDACs was possibly due to the depletion of the essential amino acid L-tryptophan which could be due to up-regulation of indoleamine 2,3-dioxygenase (IDO). Quantitative RT-PCR analysis of IDO gene expression revealed that IDO was up-regulated by about 4000-fold when PDACs were co-cultured with activated T cells, but not when co-cultured with naive T cells. Experiments are ongoing to confirm the causative role of IDO, and other factors, in PDAC-suppression of T-cell proliferation. In summary, we believe that soluble factors including the production of pro-inflammatory cytokines may contribute to PDAC suppression of the MLR, that induction of soluble factors from PDACs is significantly augmented by T-cell activation and that IDO expression by PDACs during the PDAC-MLR reaction plays a significant and direct role in suppression of T cell proliferation by PDACs.

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Frequencies of FoxP3+ naïve T cells are related to both viral load and naïve T cell proliferation responses in HIV disease

Journal of Leukocyte Biology ◽

10.1189/jlb.1210661 ◽

2011 ◽

Vol 90 (3) ◽

pp. 621-628 ◽

Cited By ~ 3

Author(s):

Benigno Rodriguez ◽

Douglas A. Bazdar ◽

Nicholas Funderburg ◽

Robert Asaad ◽

Angel A. Luciano ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

Viral Load ◽

T Cell ◽

T Cell Proliferation ◽

Hiv Disease ◽

Naive T Cells ◽

Naïve T Cells ◽

Naïve T Cell ◽

Naive T Cell

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The Outcome of Neutrophil-T Cell Contact Differs Depending on Activation Status of Both Cell Types

Frontiers in Immunology ◽

10.3389/fimmu.2021.633486 ◽

2021 ◽

Vol 12 ◽

Author(s):

Danielle Minns ◽

Katie J. Smith ◽

Gareth Hardisty ◽

Adriano G. Rossi ◽

Emily Gwyer Findlay

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Full Range ◽

T Cell Proliferation ◽

Cell Contact ◽

Activation Process ◽

Naive T Cells ◽

Naïve T Cells

Neutrophils and T cells exist in close proximity in lymph nodes and inflamed tissues during health and disease. They are able to form stable interactions, with profound effects on the phenotype and function of the T cells. However, the outcome of these effects are frequently contradictory; in some systems neutrophils suppress T cell proliferation, in others they are activatory or present antigen directly. Published protocols modelling these interactions in vitro do not reflect the full range of interactions found in vivo; they do not examine how activated and naïve T cells differentially respond to neutrophils, or whether de-granulating or resting neutrophils induce different outcomes. Here, we established a culture protocol to ask these questions with human T cells and autologous neutrophils. We find that resting neutrophils suppress T cell proliferation, activation and cytokine production but that de-granulating neutrophils do not, and neutrophil-released intracellular contents enhance proliferation. Strikingly, we also demonstrate that T cells early in the activation process are susceptible to suppression by neutrophils, while later-stage T cells are not, and naïve T cells do not respond at all. Our protocol therefore allows nuanced analysis of the outcome of interaction of these cells and may explain the contradictory results observed previously.

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The outcome of neutrophil-T cell contact differs depending on activation status of both cell types

10.1101/2020.11.05.370171 ◽

2020 ◽

Author(s):

Danielle Minns ◽

Katie J Smith ◽

Gareth Hardisty ◽

Adriano Rossi ◽

Emily Gwyer Findlay

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Full Range ◽

T Cell Proliferation ◽

Cell Contact ◽

Activation Process ◽

Naive T Cells ◽

Naïve T Cells

AbstractNeutrophils and T cells exist in close proximity in lymph nodes and inflamed tissues during health and disease. They are able to form stable interactions, with profound effects on the phenotype and function of the T cells. However, the outcome of these effects are frequently contradictory; in some systems neutrophils suppress T cell proliferation, in others they are activatory or present antigen directly. Published protocols modelling these interactions in vitro do not reflect the full range of interactions found in vivo; they do not examine how activated and naïve T cells differentially respond to neutrophils, or whether de-granulating or resting neutrophils induce different outcomes. Here, we established a culture protocol to ask these questions with human T cells and autologous neutrophils. We find that resting neutrophils suppress T cell proliferation, activation and cytokine production but that de-granulating neutrophils do not, and neutrophil released intracellular contents are pro-activatory. Strikingly, we also demonstrate that T cells early in the activation process are susceptible to suppression by neutrophils, while later-stage T cells are not, and naïve T cells do not respond at all. Our protocol therefore allows nuanced analysis of the outcome of interaction of these cells and may explain contradictory results observed previously.

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Human Mesenchymal Stem Cells Exert Immunosuppressive Capacities by Induction of Regulatory T Cells

Blood ◽

10.1182/blood.v112.11.2414.2414 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2414-2414

Author(s):

Kirsten Canté-Barrett ◽

Jessica M.E. van den Oever ◽

Willem E. Fibbe

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Fold Increase ◽

T Cell Proliferation ◽

Soluble Factors ◽

Cell Divisions ◽

And Function ◽

Cells Cultured

Abstract It is widely accepted that Mesenchymal Stem Cells (MSCs) exhibit immunosuppressive capacities in vitro and in vivo. In an effort to understand the mechanism of suppression, co-cultures of MSCs with several types of immune cells have been studied. MSCs inhibit B- and T cell proliferation and inhibit the generation, maturation and function of dendritic cells. In the present study, we evaluated the effect of MSCs on the expansion and function of regulatory T cells (T regs). Human bone-marrow derived MSCs were cultured and expanded in low-glucose DMEM containing 10% FCS and frozen until used in experiments. Freshly isolated, MACS selected human CD4+CD25+ T regs were cultured in IMDM, supplemented with 10% pooled human serum and 300 units/ml IL-2, in the presence or absence of irradiated MSCs (60 Gy) (ratio MSCs: T regs = 1:5). Results are derived from 3 to 15 independent experiments. In the presence of IL-2 and MSCs, the percentage of FOXP3+ CD4+CD25+ T cells increased from 26.8% ±2.2 (no MSCs) to 42.3% ±2.4 (with MSCs) over a period of 5 days, representing a 1.6 (±0.1)-fold induction. Moreover, a distinct CD4+CD25+ population with high FOXP3 expression appeared after 5 days of culture in the presence of MSCs (23.3% ±2.5 in the presence of MSCs versus 7.2% ±1.0 in the absence of MSCs). This CD4+CD25+FOXP3hi population was not observed after co-culturing MSCs and CD4+CD25− T cells (1.7% ±0.6). To show that the MSC-induced T regs were functionally suppressive, freshly isolated CFSE-labeled CD4+ T cells were stimulated with PHA (0.8 μg/ml). After 3 days, about 40% of the T cells had undergone one or more cell divisions as measured by CFSE dilution. Addition of MSC-induced T regs (in a 1:1 ratio) resulted in a 50% reduction of the proliferation of CFSE-labeled T cells (down to 15–20% of cells undergoing one or more cell divisions). Control CD4+CD25+ T cells cultured in the absence of MSCs did not suppress T cell proliferation. These results indicate that MSC-induced CD4+CD25+ FOXP3hi cells exert regulatory function. To study whether the induction of T regs by MSCs was dependent on cell-cell contact, co-culture experiments were performed in transwells where MSCs were physically separated from T cells. CD4+CD25+ T cells co-cultured with MSCs in transwells showed a 1.4 (±0.1)-fold increase in the percentage of FOXP3hi cells, in comparison with T cells cultured in the absence of MSCs. Similarly, direct co-cultures of MSCs and T cells resulted in a 1.8 (±0.2)-fold increase in the percentage of FOXP3hi T regs. In addition, medium derived from co-cultures of MSCs and CD4+CD25+ T cells and added to freshly isolated CD4+CD25+ T cells resulted in a 2.6 (±0.6) fold increase in the percentage of CD4+CD25+FOXP3hi T regs, implicating that FOXP3hi induction by MSCs was mediated by soluble factors. Since T Cell Receptor-stimulated naïve T cells (as well as natural T regs, generated in the thymus) require both IL-2 and TGF-β to become induced T regs, we analyzed the involvement of TGF-β. Addition of the pharmacological inhibitor of the TGF-β receptor (SB431542) only marginally reduced FOXP3 induction in the presence of MSCs, suggesting that MSC-mediated expression of high FOXP3 levels requires alternate or additional cytokines. In conclusion, we show that MSCs promote the induction of CD4+CD25+ T cells that express high levels of FOXP3 and these MSC-induced T regs suppress proliferation of PHA-stimulated CD4+ T cells. These effects are mediated by soluble factors produced during the co-culture of MSCs and T cells. The cytokines involved are presently unknown, but likely do not involve TGF-β.

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Antigen-specific T-T interactions regulate CD4 T-cell expansion

Blood ◽

10.1182/blood-2007-09-114389 ◽

2008 ◽

Vol 112 (4) ◽

pp. 1249-1258 ◽

Cited By ~ 40

Author(s):

Julie Helft ◽

Alexandra Jacquet ◽

Nathalie T. Joncker ◽

Isabelle Grandjean ◽

Guillaume Dorothée ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Feedback Loop ◽

Cd4 T Cell ◽

T Cell Proliferation ◽

Naive T Cells ◽

Naïve T Cells ◽

T Cell Regulation ◽

Peptide Complexes

Abstract The regulation of CD4 T-cell numbers during an immune response should take account of the amount of antigen (Ag), the initial frequency of Ag-specific T cells, the mix of naive versus experienced cells, and (ideally) the diversity of the repertoire. Here we describe a novel mechanism of T-cell regulation that potentially deals with all of these parameters. We found that CD4 T cells establish a negative feedback loop by capturing their cognate major histocompatibility class (MHC)/peptide complexes from Ag-presenting cells and presenting them to Ag-experienced CD4 T cells, thereby inhibiting their recruitment into the response while allowing recruitment of naive T cells. The inhibition is Ag specific, begins at day 2 (long before Ag disappearance), and cannot be overcome by providing new Ag-loaded dendritic cells. In this way, CD4 T-cell proliferation is regulated in a functional relationship to the amount of Ag, while allowing naive T cells to generate repertoire variety.

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Potentiating Vγ9Vδ2 T cell proliferation and assessing their cytotoxicity towards adherent cancer cells at the single cell level

Biology Open ◽

10.1242/bio.059049 ◽

2022 ◽

Author(s):

Chenxiao Liu ◽

Karolina Skorupinska-Tudek ◽

Sven-Göran Eriksson ◽

Ingela Parmryd

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cancer Cells ◽

T Cell Proliferation ◽

Adherent Cells ◽

Cell Level ◽

Cell Immunotherapy ◽

Vγ9vδ2 T Cells ◽

T Cell Immunotherapy

Vγ9Vδ2 T cells is the dominant γδ T cell subset in human blood. They are cytotoxic and activated by phosphoantigens whose concentrations are increased in cancer cells, making the cancer cells targets for Vγ9Vδ2 T cell immunotherapy. For successful immunotherapy, it is important both to characterise Vγ9Vδ2 T cell proliferation and optimise the assessment of their cytotoxic potential, which is the aim of this study. We found that supplementation with freshly-thawed human serum potentiated Vγ9Vδ2 T cell proliferation from peripheral mononuclear cells (PBMCs) stimulated with (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and consistently enabled Vγ9Vδ2 T cell proliferation from cryopreserved PBMCs. In cryopreserved PBMCs the proliferation was higher than in freshly prepared PBMCs. In a panel of short-chain prenyl alcohols, monophosphates and diphosphates, most diphosphates and also dimethylallyl monophosphate stimulated Vγ9Vδ2 T cell proliferation. We developed a method where the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells is assessed at the single cell level using flow cytometry, which gives more clear-cut results than the traditional bulk release assays. Moreover, we found that HMBPP enhances the Vγ9Vδ2 T cell cytotoxicity towards colon cancer cells. In summary we have developed an easily interpretable method to assess the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells, found that Vγ9Vδ2 T cell proliferation can be potentiated media-supplementation and how misclassification of non-responders may be avoided. Our findings will be useful in the further development of Vγ9Vδ2 T cell immunotherapy.

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Monocytic Myeloid Derived Suppressor CELLS (M-MDSC) As Prognostic Factor in Chronic Myeloid Leukemia Patients Treated with Dasatinib

Blood ◽

10.1182/blood.v126.23.2767.2767 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2767-2767 ◽

Cited By ~ 1

Author(s):

Cesarina Giallongo ◽

Nunziatina L Parrinello ◽

Daniele Tibullo ◽

Piera La Cava ◽

Alessandra Romano ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Chronic Myeloid Leukemia ◽

Prognostic Factor ◽

Clinical Response ◽

Myeloid Leukemia ◽

Suppressor Cells ◽

T Cell Proliferation ◽

Soluble Factors

Abstract INTRODUCTION. Myeloid suppressor cells are a heterogeneous group of myeloid cells that are increased in most cancer patients. Recently, we and another group demonstrated that MDSC play an important role of immune escape in chronic myeloid leukemia (CML) patients inducing T cell tolerance. The aim of this study was to investigate the effect of the tyrosine kinase inhibitors (TKI) therapy on MDSC and possible correlation with clinical response. METHODS. MDSCs were analyzed in peripheral blood of 20 healthy donors (HD) and 30 CML patients at diagnosis. MDSCs were also measured during TKI treatment (18 patients treated with imatinib and 10 patients with dasatinib). Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (M-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of MDSCs with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. Exosomes were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. RESULTS. G-MDSCs and M-MDSCs percentages in CML patients were greater than HD (respectively 82.5±9.6% vs 56.2±5.4% and 33.6±19% vs 5.9±4%, p<0.0001). Both isolated subpopulations showed expression of BCR/ABL and were able to inhibit T cells proliferation in comparison to positive control (from 48±7.6% to 25±5% for G-MDSC, p=0.0057 and 16.7±0.6% for M-MDSC, p<0.0001). No suppressive effect was observed in co-cultures with G-MDSC and M-MDSC obtained from HD. In addition, M-MDSC percentage correlated with BCR-ABLABL transcript levels in patients at diagnosis (r=0.5816, p=0.0006). Evaluating the effect of TKI therapy on MDSC levels, we found that both imatinib (IM) and dasatinib (DAS) induced a significant reduction of G-MDSC percentage at 6 months (from 82.5±9.6% to 55±17.3% after IM and 48.7±13% after DAS, p<0.0001) and 12 months (61.2±9.7% after IM and 33.4±14% after DAS, p<0.0001) of treatment. The levels of M-MDSCs significantly decreased only after DAS therapy (from 33.6±19% to 6.8±12.6% at 6 months, p=0.014 and 11.4±12.3% at 12 months, p=0.008); while there was a mild reduction after IM treatment (22.2±24.5% and 22.3±21.7% respectively at 6 and 12 months) although a great variability was observed among patients. Subsequently, correlation of MDSC reduction and clinical response to TKI therapy was investigated. We found that in DAS, but not in IM treated patients, a correlation between percentage of Major Molecular Response (MMR) and number of persistent M-MDSCs was found.. In fact, a significant difference was recorded by comparing M-MDSC levels in the MMR group (n=6) versus no MMR (n=4) at 6 or 12 months (p=0.0034). In addition, to evaluate if leukemic cells are able to expand MDSC by releasing soluble factors or exosomes, we incubated monocytes obtained from HD with sera or exosomes from CML patients at diagnosis or healthy subjects. M-MDSCs percentage significantly increased only in conditions with CML serum ( 29±13%; p=0.0006) or exosomes (8 ±2,8%; p=0.01) while no effect was observed on G-MDSC percentage. CONCLUSION: Therapy with TKI reduces the percentage of MDSCs and levels of the monocytic subset correlates with MMR in patients treated with dasatinib, suggesting their importance in clinical investigation as prognostic factor. Moreover, our data suggest the possible development in CML patients of a circuit primed by tumor cells that, through the release of soluble factors and exosomes, are able to expand M-MDSCs, creating an immunotolerant environment that results in T cell anergy and facilitates tumor growth. Disclosures No relevant conflicts of interest to declare.

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Anti-Human T-Lymphocyte Immunoglobulin (ATG)-Induced T Regulatory Cells and Their Soluble Factors Suppress T Cell Proliferation: Potential Role in Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v126.23.1889.1889 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1889-1889

Author(s):

Katia Beider ◽

David Naor ◽

Valeria Voevoda ◽

Olga Ostrovsky ◽

Hanna Bitner ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Lymphocyte ◽

Treg Cells ◽

T Cell Proliferation ◽

Regulatory Cells ◽

Soluble Factors ◽

Human T Lymphocyte

Abstract Polyclonal anti-human T-lymphocyte immunoglobulin(ATG) have been recently shown, in two randomized studies, to significantly reduce the incidence of graft versus host disease (GVHD) post allogeneic stem cell transplantation (HSCT) from both sibling and unrelated donors. Induction of regulatory T cells is suggested as one of the possible mechanisms involved. The aim of our current study was to further characterize the T cell populations induced by ATG treatment and to delineate the mechanisms involved in ATG-induced tolerance in patients receiving intravenous ATG (ATG-Fresenius ® S, Neovii Biotech) as part of their pre HSCT conditioning. Phenotypic characterization of regulatory cells markers revealedthattwo days culture of peripheral blood mononuclear cells (PBMCs) with ATG-F (30-120 µg/ml) resulted in significant increase in CD25 expression on CD4+ T cells. The percentages of cells expressing CTLA4, GITR, CD95 and FoxP3 was also significantly elevated on CD4+ cells compared to rabbit IgG-treated PBMCs. In addition, expression of CD127 and VLA-4 molecules was significantly decreased on CD4+CD25+ cells upon ATG-F treatment (p<0.01). Next, tolerance ability of ATG-F-induced cells was examined. Addition of ATG-F-treated cells to autologous PBMCs stimulated with antiCD3/antiCD28 antibodies resulted in significant (50-75%) inhibition of cell proliferation (p<0.001), measured by CFSE and Ki67 staining. Moreover, CD69 cell expression and interferon-γ (IFNγ) proinflammatory cytokine secretion were reduced by 50-60% and 65-90%, respectively, in the presence of ATG-F-activated Treg cells (p<0.01). Importantly, addition of cyclosporine A to the induction culture with ATG-F interfered with the ATG-induced regulatory phenotype acquisition, suggesting the involvement of interleukin-2 in ATG-mediated activity. In order to purify the tolerizing population, sorting of CD4+CD25+CD127-low cells (considered as viable regulatory T cells) from ATG-F-treated culture was performed. Sorted cells demonstrated greater suppressive potency than bulked pre-sorted cell population when added to autologous stimulated PBMCs. Of note, Treg-depleted fraction was still able to suppress the proliferation, albeit less efficiently then sorted Treg cells, suggesting that ATG-F is capable to induce multiple immune suppressive cell populations that should be further defined. To explore the possible involvement of soluble factor(s)-mediated mechanisms, in addition to the involvement of cell to cell contact mechanism, conditioned medium (CM) produced by ATG-F-primed cells was applied on stimulated autologous PBMCs. Addition of CM produced by ATG-F-treated cells, but not IgG-treated cells, resulted in significant suppression (30-65%, p<0.01) of T cell proliferation and activation, indicating the presence of soluble factors secreted by ATG-F-primed suppressive cells. Indeed, significant dose- and time-dependent induction of TGFβ secretion was observed in ATG-treated cells. To this end, addition of TGFβ receptor kinase inhibitor SB-431542 interfered with suppressive activity of ATG-F-primed cells, enabling partial rescue of proliferation and IFNγ secretion in response to antiCD3/antiCD28 activation. Similar results were obtained with anti-TGFβ neutralizing antibodies. Finally, characterization of phenotype and frequencies of regulatory immune populations in peripheral blood of 26 patients undergoing allogeneic transplantation with conditioning regimen including ATG-F (15mg/kg) was performed. Consistent with our ex vivo results, transient increase in percent of circulating CD4+CD25+CD127-low cells was detected in the ATG-F treated patients on days 14, 21 and 28 after HSCT. Furthermore, elevated levels of TGFβ were detected in the patients' plasma at day 28 and remaining high at day 60 post HSCT. Our results demonstratethat in vitro treatment with ATG-F is capableto induce functional Treg cells. Suppressive ability of ATG-F-induced cells was both contact and soluble-factors dependent and was partially promoted by TGFβ. Patients undergoing allogeneic HSCT with ATG-F-including conditioning demonstrated increased frequencies of circulating Treg cells and elevated plasma levels of TGFβ. Altogether, our data further support the use of ATG-F, a potent inducer of Treg cells, for prevention of GVHD post HSCT and potentially other therapeutic applications. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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