16 research outputs found

    Fluoxetine reduces murine graft-versus-host disease by induction of T cell immunosuppression

    Get PDF
    Serotonin reuptake inhibitors (SRIs) are widely used drugs in the treatment of depression and anxiety disorders. Although SRIs are generally regarded as safe drugs with relatively few side effects, literature suggests that high concentrations of SRIs may alter immune function. We investigated whether high-dose treatment with fluoxetine was able to suppress acute graft-versus-host disease (GvHD) in a MHC-matched, minor histocompatibility antigen mismatched murine bone marrow transplantation model. We found that high doses fluoxetine induce a significant reduction of clinical symptoms and increase survival of these animals. The amelioration of clinical GvHD was accompanied by a reduced expansion of alloreactive T cells. We further analyzed the direct in vitro effect of six SRIs on the viability and proliferation of human T cells and found an anti-proliferative and pro-apoptotic effect that was significantly larger in activated than in resting T cells. We discuss these results in the light of potential future exploration of SRIs as a novel class of T cell immunosuppressive drugs

    The Dark Side of EGFP: Defective Polyubiquitination

    Get PDF
    Enhanced Green Fluorescent Protein (EGFP) is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-κB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered

    Mechanisms and risks of immunotherapy using blocking anti-CTLA-4 mAb for the induction of antileukemic effects after allogeneic bone marrow transplantation in mice

    No full text
    Voor deze thesis bestudeerden wij het effect van blokkering van de CTLA- 4 receptor op graft-versus-leukemie (GvL) effecten en graft-versus-host ziekte (GVHZ) in een beenmerg transplantatie (BMT) model in de muis met een mineure histocompatibiliteitsantigen-mismatch. We vonden dat blokker ing van de CTLA-4 receptor antileukemisch effect kan induceren zonder Gv HZ, maar dat dit gepaard gaat met mogelijks lethale auto-immuniteit. Wij onderzochten de immuun mechanismen die de pathognese van dit fenomeen k unnen verklaren. Blokkering van de CTLA-4 receptor, op een vroeg tijdstip na de BMT leidd e tot acute GVHZ. Blokkering van de CTLA-4 receptor op een later tijdsti p na de BMT leidde tot een mogelijks lethaal syndroom met lymfoprolifera tie in milt en lymfeknopen, maar anderzijds met een stabiel gemengd T ce ll chimerisme, met onveranderde frekwenties van allo-reactieve T cellen en zonder anti-gastheer of anti-donor reactiviteit in vitro. Wij zage n een lymfoproliferatieve aandoening die verschillende organen aantastte met auto-immune hepatitis en met circulerende auto-antistoffen. De lymf ocyten van behandelde dieren vertoonden ex vivo een spontane prolifer atie en een belangrijke proliferatie als ze gestimuleerd werden met dend ritische cellen (DC) afkomstig van het beenmerg van een gastheer-type mu is en gepulst met weefsel-specifieke antigenen, ook afkomstig van speeks elklieren van een gastheer-type muis. Beide fenomenen werden exclusief g emedieerd door T cellen van gastheer en niet van donor oorsprong, hetgee n een auto-immune pathogenese ondersteunt. Deze specifieke in vitro p roliferatie van T cellen afkomstig van de gastheer vonden we ook terug a ls we dezelfde gastheer DC pulsten met peptides afkomstig van de BW5147 leukemie-cellijn. Deze cellijn werd in vivo gebruikt om na te gaan of we een antileukemisch effect konden induceren door het blokkeren van de CTLA-4 receptor. Wij vonden dat behandelde dieren die BW5147 toegediend kregen niet kwamen te overlijden ten gevolge van leukemie-infiltratie m aar wel ten gevolge van auto-immuniteit. Wij besloten dus dat we inderda ad een antileukemisch effect kunnen induceren door het blokkeren van de CTLA-4 receptor en dat dit niet gepaard ging met het ontstaan of inducer en van GvHZ maar wel tot stand kwam in de context van een auto-immuun sy ndroom. Zowel het antileukemisch als het auto-immune effect waren afhank elijk van de allogene BMT aangezien dieren die een syngene BMT kregen no ch antileukemische activiteit noch overte klinsche auto-immuniteit verto onden. Deze bevindingen tonen aan dat CTLA-4-blokkering potentieel bezit om antileukemische effecten te induceren na allogene BMT op voorwaarde dat de auto-immune effecten onder controle gehouden kunnen worden of con troleerbaar zijn. In patiënten die behandeld worden met anti-CTLA-4 antistoffen voor solid e of hematologische tumoren, worden ook vaak auto-immuun-gemedieerde nev eneffecten gezien maar het mechanisme is nog niet goed gekend. Gegevens van verschillende studies in patiënten en experimenten in de muis wijzen op een mogelijke rol van de regulatoire T cellen (Treg) mn. dat CTLA-4- blokkade een defect zou induceren, maar deze gegevens zijn niet conclusi ef. Wij tonen aan dat de auto-immune chimeren die behandeld werden met a nti-CTLA-4 antistof, een expansie van de CD4+Foxp3+ Treg vertonen. Daarn aast onderdrukten CD4+CD25+ Treg van auto-immune en gezonde chimeren de anti-CD3-geinduceerde proliferatie van CD4+CD25- effector cellen in vitr o even sterk. Bovendien is de capaciteit van anti-CTLA-4-behandelde C D4+CD25+FR4high Treg om de zelf-Ag-specifieke antwoord van auto-immune T cells te onderdrukken, intact en even sterk als die van Treg van contro le dieren. Wij concluderen dus dat beenmerg chimeren, die behandeld word en met anti-CTLA-4 antistoffen en een auto-immuun beeld vertonen, een ve rhoogd aantal Treg hebben in vivo en een normale suppressieve capacit eit in vitro, in het bijzonder tegenover de zelf-specifieke respons v an pathogene auto-immune T cellen. Wij vermoeden dat deze stijging in aa ntal van Treg na CTLA-4-blokkering een compensatoir fenomeen is maar dat het onvoldoende is om de proliferatie van pathologische T cellen in viv o te onderdrukken. De verhoogde expressie van IL-10 en IFN-γ kun nen deze hypothese bevestigen. Momenteel zijn we aan het onderzoeken of blokkering van de CTLA-4 recept or een tijdelijke daling in frekwentie of functie van Treg induceert op een vroeg tijdstip na de start van de behandeling. Daarnaast onderzoeken we ook of de effecten van CTLA-4-blokkkade gemedieerd worden door een i ntrinsiek effect op de effector T cellen.nrpages: 121status: publishe

    Transient expansion of Mac1+Ly6-G+Ly6-C+ early myeloid cells with suppressor activity in spleens of murine radiation marrow chimeras: possible implications for the graft-versus-host and graft-versus-leukemia reactivity of donor lymphocyte infusions

    No full text
    A murine model of minor histocompatibility antigen (miHCag)-mismatched bone marrow transplantation (BMT) was used to study the development of immunoregulatory cells in the posttransplantation period and their possible involvement in the dissociated graft-versus-host (GVH) and graft-versus-leukemia (GVL) reactivity of posttransplantation donor lymphocyte infusions (DLIs). DLI, applied immediately after BMT, induced GVH disease (GVHD), but when DLI was delayed for 3 weeks, GVHD was avoided while a distinct GVL response was allowed to develop. A population of Mac1+Ly6-G+Ly6-C+ immature myeloid cells, found in small numbers in normal mice, strongly expanded in spleens of chimeras, reaching a maximum level at week 3 and returning to base level by week 12. Upon isolation, these cells exhibited interferon-gamma (IFN-gamma)-dependent, nitric oxide (NO)-mediated suppressor activity toward in vitro alloresponses, suggesting that, after in vivo DLI, they are activated by IFN-gamma to produce NO and suppress GVH reactivity. Because not only alloactivated T-cell proliferation but also leukemia cell growth was found susceptible to inhibition by exogenous NO, in vivo activation of these cells after DLI may explain the occurrence of a GVL effect despite suppression of GVHD. This suggested sequence of events was supported by the finding that the ex vivo antihost proliferative response of spleen cells, recovered shortly after in vivo DLI, was characterized by strong mRNA production of the monokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha) and of inducible nitric oxide synthase (iNOS). Our data suggest that transiently expanding Mac1+Ly6-G+Ly6-C+ immature myeloid cells (probably as a result of extramedullary myelopoiesis) may play a role in controlling GVH while promoting GVL reactivity of DLI after allogeneic BMT.status: publishe

    Crucial role of timing of donor lymphocyte infusion in generating dissociated graft-versus-host and graft-versus-leukemia responses in mice receiving allogeneic bone marrow transplants

    No full text
    A murine model of minor histocompatibility antigen-mismatched bone marrow transplantation (BMT) was used to study the role of timing of donor lymphocyte infusion (DLI) in eliciting graft-versus-host (GVH) and graft-versus-leukemia (GVL) reactivity. We gave DLI at weeks 3 and 12 after BMT and related its ability to induce a GVL effect with (1) evolution of T cell chimeric status and (2) the extent to which DLI could elicit lymphohematopoietic GVH (LHGVH) reactivity. All mice remained free of GVH disease, but only week 3 DLI chimeras exhibited a significant GVL response when challenged with host-type leukemia cells. In these week 3 DLI chimeras, host-reactive T cells were found to proliferate in vivo (5- [and-6]-carboxyfluorescein diacetate, succinimidyl esther [CFSE]-labeled DLI inocula, TCR-Vbeta6(+) T-cell frequency) and T-cell chimerism rapidly converted from mixed into complete donor type, indicating the occurrence of LHGVH reactivity. In week 12 chimeras, DLI elicited none of the activities noted at week 3. Yet, in both instances, splenocytes, recovered following DLI, generated an equally strong antihost proliferative response in a mixed lymphocyte reaction, thereby arguing against a decisive role of regulatory cells. The lack of in vivo LHGVH reactivity after week 12 DLI was associated with a substantially increased level of pre-existing host-type T-cell chimerism. We conclude that elicitation of a GVL effect may require LHGVH reactivity and that the reason why timing of DLI was critical for obtaining LHGVH reactivity and the desired GVL effect may lie in the evolution of chimeric status. A possible direct involvement of residual host-type antigen-presenting cells in eliciting LHGVH reactivity after DLI should be studied using models that allow chimerism analysis in non-T-cell lineages.status: publishe

    Murine bone marrow chimeras developing autoimmunity after CTLA-4-blockade show an expansion of T regulatory cells with an activated cytokine profile

    No full text
    Autoimmune adverse events are a concern in patients treated with blocking anti-CTLA-4-mAb for solid and hematological tumors. Patient and mouse data on the contribution of a quantitative or qualitative defect of regulatory T cells (T(reg)) in this autoimmune phenomenon are conflicting. We have previously shown that a treatment course with blocking anti-CTLA-4-mAb in murine allogeneic bone marrow chimeras induces an antileukemic response in close association with systemic autoimmunity. Here, we used this model to investigate the effect of CTLA-4-blocking therapy on the kinetics of T(reg) frequency and function. As previously published, CTLA-4-blocking treatment, initiated on day 20 after bone marrow transplantation, led to overt autoimmunity by day 35. CD4(+)Foxp3(+) T(reg) frequency was determined (flowcytometry) on day 21, 23, 25 and 35: treated chimeras showed an expansion of CD4(+)Foxp3(+) T(reg) frequencies on day 25 and 35, without a prior frequency decrease. The T(reg) expansion occurred selectively in the recipient-derived CD4+ T-cell compartment. In vitro, purified CD4(+)CD25(+)FR4(high) T(reg) from 'day 35' autoimmune and control chimeras showed equal suppressive effects towards self-antigen-specific autoimmune T cells. Purified CD4(+)CD25(high)FR4(high) T(reg) from 'day 35' treated chimeras showed increased IL-10 and IFN-gamma mRNA-expression (RT-PCR) relative to control chimeras. In this model of CTLA-4-blockade-induced autoimmunity after allogeneic bone marrow transplantation, anti-CTLA-4-mAb gives rise to a progressive expansion - without a prior transient reduction - of T(reg) cells. T(reg) of autoimmune animals do not show a defect in in vitro suppressive function but show an in vivo activated cytokine profile, suggesting that the expansion occurs as a compensatory phenomenon to control autoimmunity.status: publishe
    corecore