Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) have been extensively studied in the past 30-40 years. Their potent suppressive capacity shown in several pathological and clinical settings, such as cancer and transplantation, has made it evident that better understanding their development and function is critical. Specifically, Tregs play a pivotal role in preventing autoimmunity, graft-versus-host disease (GvHD), and organ graft rejection. We previously demonstrated that germline or induced SH2 domain-containing inositol 5-phosphatase (SHIP) deficiency in the host abrogates GvHD. Here we show that SHIP-deficiency promotes an increase of FoxP3+ cells in both the CD4+CD25+ and the CD4+CD25- T cell compartments with increased expression of Treg-associated markers. Importantly, SHIP-deficiency does not compromise Treg function. Interestingly, like conventional Tregs, SHIP-/- CD4+CD25- T cells are unresponsive to allogeneic stimulators and suppress allogeneic responses by T cells in vitro, and can mediate reduced lethal GvHD in vivo. Thus, SHIP limits the immunoregulatory capacity of CD4+ T cell, particularly in allogeneic settings. SHIP-deficiency expands the number of immunoregulatory cells in both the T lymphoid and myeloid lineages. Here, we examined if these increases are interrelated. Specifically, we found that myeloid specific SHIP-deficiency leads to expansion of both MDSC and Treg numbers. Conversely, T lineage specific ablation of SHIP leads to expansion of Treg numbers, but not expansion of MDSC, indicating an intrinsic role for SHIP in limiting Treg numbers. Interestingly, MDSC lack SHIP expression suggesting that another SHIP-deficient myeloid cell promotes MDSC and Treg expansion. Also, increased levels of G-CSF, a myelopoietic growth factor, in SHIP-/- mice may extrinsically promote MDSC expansion since we found that G-CSF is required for the expansion of splenic MDSC in mice with induced SHIP-deficiency. MDSC consist of two distinct subsets, granulocytic-MDSC (G-MDSC), and monocytic-MDSC (M-MDSC) that differ in morphology, phenotype, suppressive capacity and differentiation potential. Importantly, M-MDSC can further differentiate into dendritic cells, macrophages and preferentially into G-MDSC, in the presence of tumor-derived factors (TDF). The retinoblastoma gene (Rb1), a tumor suppressor gene and central regulator of the cell cycle and differentiation, has been shown to influence monocytic and neutrophilic lineage commitment and to limit myeloproliferative disease. Here, we examined the role of Rb1 in the biology of MDSC subsets in tumor-bearing mice. Firstly, M-MDSC expressed high levels of Rb1 which remained relatively stable in culture with GM-CSF. Conversely, freshly isolated G-MDSC initially expressed undetectable levels of Rb1 that increased over time in culture, which correlated with increased histone acetylation at the Rb1 promoter. This increased Rb1 expression and histone acetylation was accelerated by histone deacetylase inhibitors (HDACi) treatment, suggesting Rb1 expression may be controlled by histone modification. Furthermore, when treated with HDACi, M-MDSC did not differentiate into G-MDSC in culture, even with TDF present. Finally, induced Rb1 deficiency in vivo promoted an expansion of splenic CD11b+Ly6G+Ly6Clo cells, similar to G-MDSC in tumor-bearing mice. Although further studies are required, these results strongly suggest that Rb1, like SHIP, plays a role in MDSC accumulation, particularly G-MDSC in cancer.

Chronic myeloid leukaemia (CML) is characterised by the constitutively active tyrosine kinase BCR-ABL. Standard treatment with tyrosine kinase inhibitors (TKI) in the chronic phase (CP) of CML conveys excellent long-term prognosis but is associated with side effects and costs. Treatment free remission (TFR) is possible in a proportion of patients discontinuing treatment after obtaining deep treatment responses but it is not fully known how to select the right patients for stopping attempts. Treatment of accelerated phase (AP) and blast crisis (BC) is more complicated and the prognosis more dismal. In this thesis, we have studied factors of importance for outcome in CML patients with focus on immunological factors and clinical management. In a cohort of 32 newly diagnosed CP-CML patients, evidence of active immune escape mechanisms were found. These declined with the course of TKI treatment and at the same time, effector lymphocyte responses were elicited. These anti-leukaemia immune responses might help in the long-term control of CML. Multiple plasma protein markers were also measured with three multiplex platforms in a smaller cohort of patients (n=14). Inflammatory cytokines and other plasma proteins were affected by TKI treatment and multiplexing seems useful for finding potential biomarkers with biologic or prognostic significance in CML. Patients progressing to AP/BC were studied in a population-based material from the Swedish CML register. Approximately 4% of TKI-treated CP-CML patients transformed to AP/BC within 2 years of diagnosis. Monitoring of treatment responses was suboptimal in 1/3 of these patients and the median survival was 1.4 years after diagnosis of AP/BC. Thus, minimising the risk of disease progression through strict adherence to guidelines for monitoring and treatment is essential. In a cohort of patients (n=50) discontinuing TKI treatment within a large European trial, musculoskeletal pain was reported by 30% of patients, starting within 1- 6 weeks of TKI discontinuation and spontaneously resolving over time in most cases. Patients (n=56) were also evaluated with a multiplex platform with a total of 162 inflammation- and cancer-related plasma proteins. No predictive protein biomarkers for successful TKI discontinuation could be found. However, profound effects of TKI-treatment were seen and plasma proteomics could be useful for understanding effects of long-term TKI-treatment.

T-cells are present in the immune system to fight against invaders. Once their job is done, suppressing their activity is an important step in maintaining a proper immune response. Myeloid derived suppressor cells (MDSCs) are immune cells that suppress T-cell activity. Currently, MDSCs are defined as a heterogeneous population of immature cells that are derived in the bone marrow and travel to the site of inflammation or cancer. Two major subtypes of MDSCs have been identified in mice and humans, monocyte-like MDSCs (M-MDSC) and granulocyte MDSCs (G-MDSC). G-MDSCs typically make up the majority of the total population of MDSCs but are less T-cell suppressive than M-MDSCs. One of the major problems in the study of MDSCs is that the current marker system for subtypes does not differentiate between precursor MDSCs (lacking suppressive ability) and functional MDSCs (those with suppressive ability). Therefore, using cancer models in mice, we investigated the development and potential to classify precursor MDSCs from functional MDSCs. While MDSCs have been highlighted as a target cell to inhibit in cancer, in other conditions, such as pregnancy, MDSCs have been shown to be beneficial in maintaining a normal pregnancy. Therefore, targeting the increase of MDSCs in abnormal pregnancy conditions like pre-eclampsia may act as a prevention or therapeutic strategy. Finally, it is known that many dietary components can act as modulators of immune cells. Specifically, the polyphenol like phytochemical, curcumin has been shown to act as an anti-inflammatory agent with the potential to modulate multiple immune cells. Therefore, we propose two different studies to investigate the potential of curcumin as either an inhibitor and/or promotor of MDSCs in a disease-specific context. Together the role of phytochemicals as immunomodulators of MDSCs is still very young, in part due to the complexity of phytochemicals themselves, but the studies cited here provide evidence that the field is ripe for additional questions to be asked.

No description available.

T cells play a pivotal role in controlling viral infection; however, the precise mechanisms responsible for regulating T‐cell differentiation and function during infections are incompletely understood. In this study, we demonstrated an expansion of myeloid‐derived suppressor cells (MDSC s), in particular the monocytic MDSC s (M‐MDSC s; CD 14+ CD 33+ CD 11b+ HLA ‐DR −/low), in patients with chronic hepatitis C virus (HCV ) infection. Notably, HCV ‐induced M‐MDSC s express high levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT 3) and interleukin‐10 (IL ‐10) compared with healthy subjects. Blocking STAT 3 signalling reduced HCV ‐mediated M‐MDSC expansion and decreased IL ‐10 expression. Importantly, we observed a significant increase in the numbers of CD 4+ CD 25+ Foxp3+ regulatory T (Treg) cells following incubation of healthy peripheral blood mononuclear cells (PBMC s) with MDSC s derived from HCV ‐infected patients or treated with HCV core protein. In addition, depletion of MDSC s from PBMC s led to a significant reduction of Foxp3+ Treg cells developed during chronic HCV infection. Moreover, depletion of MDSC s from PBMC s significantly increased interferon‐γ production by CD 4+ T effector (Teff) cells derived from HCV patients. These results suggest that HCV ‐induced MDSC s promote Treg cell development and inhibit Teff cell function, suggesting a novel mechanism for T‐cell regulation and a new strategy for immunotherapy against human viral diseases.

No description available.

<p>We showed previously that histone deacetylase (HDAC) inhibition with MS-275 in the context of boosting oncolytic immunotherapy can drive heightened antitumor responses, leading to increased survival in mouse intracranial melanoma models. However, it is currently unclear how the co-administration of MS-275 directly impacts tumor growth. Here, we investigated the role of MS-275 in preventing the outgrowth of antigen-deficient tumor variants as a result of suboptimal treatment protocols. By adoptively transferring tumor antigen-specific memory T cells (Tm) that were expanded <em>in vivo</em> with recombinant Vesicular Stomatitis Virus (VSV-gp33), we observed complete regression of 5-day old, intradermal B16-gp33 tumors (B16-F10 overexpressing the LCMV GP33-41 epitope); however, the tumors relapsed within a month of treatment. Relapsing tumor explants were able to grow in mice that were prophylactically immunized with recombinant Adenovirus (Ad-gp33), indicating that the tumor could no longer be recognized. Strikingly however, there was zero tumor recurrence if MS-275 was co-administered with Tm and VSV-gp33, suggesting that MS-275 may prevent the emergence and/or escape of antigen loss variants. Such a benefit is lost if the administration of the drug is delayed as little as five days post VSV treatment, suggesting that its synergistic effects coincide with early immune responses and oncolytic activity. Furthermore, transplantation studies of relapsing tumor explants showed that combination treatment was unable to provide tumor protection, confirming that the mechanisms by which MS-275 prevents tumor recurrence are unlikely through direct up-regulation of antigen presentation in low- or non-antigen-expressing variants <em>in vivo</em>. Indeed, CD4 depletion in the absence of MS-275 resulted in sustained tumor regression, implying that immunoregulatory cells such as CD4+ Treg play a prominent role in sustaining tumor regression. Moreover, MS-275 modulates the phenotypic status of tumor-infiltrating MDSCs toward the differentiation of inflammatory macrophages. Taken together, the data suggests that combination therapy with HDACi with oncolytic immunotherapy mediates a synergized immune attack against the tumor through subversion of immunomodulatory mechanisms.</p> / Master of Science in Medical Sciences (MSMS)

A endometriose (EDT) é caracterizada pela presença de tecido endometrial fora da cavidade uterina, e afeta mulheres em idade reprodutiva. Postulamos que alterações na frequência de células T reguladoras (Treg), natural killer (NK), supressoras mielóides (MDSC) e dendríticas (DC) no peritônio justificariam a redução da capacidade do sistema imune de reagir contra as células endometriais, permitindo sua implantação em locais ectópicos. Aqui, células Treg, NK, MDSC e DC foram quantificadas no fluido peritoneal (FP) e sangue de mulheres com EDT, a fim de associa-las ao desenvolvimento da doença; níveis de citocinas também foram avaliados. Na EDT, observou-se aumento na frequência de Treg, MDSC e DC no sangue e aparente redução destas no FP; ainda, a concentração de IL-12 foi menor no sangue comparadas ao grupo controle. Não foram observadas diferenças quanto às células NK e as outras citocinas analisadas. Os resultados indicam aumento da frequência de populações reguladoras em amostras de sangue de pacientes EDT, entretanto esses resultados não são refletidos no FP. / Endometriosis (EDT) is a gynecological disease characterized by the presence of endometrial cells out of the uterine cavity, which affects women in reproductive age. We postulated that alterations in the frequencies of regulatory T cells (Treg), natural killer cells (NK), myeloid-derived suppressor cells (MDSC) and dendritic cells (DC) in the peritoneum could justify the reduced capacity of the immune system to react to these ectopic endometrial cells, allowing them to invade distant tissues. Here, Treg, NK, MDSC and DC were quantified in the peritoneal fluid (PF) and peripheral blood (PB) of women with EDT, in order to associate them with the development of EDT; cytokine levels were also assessed. In EDT, higher frequencies of Treg, MDSC and DC in the PB and apparent lower frequencies of these cells in the PF were observed; IL-12 concentration was smaller in PB of EDT compared to control. No differences between groups were observed for NK cells and the other cytokines evaluated. The results indicate higher frequencies of regulatory cells in PB samples of EDT patients, although these findings were not reflected in PF samples.

A endometriose (EDT) é caracterizada pela presença de tecido endometrial fora da cavidade uterina, e afeta mulheres em idade reprodutiva. Postulamos que alterações na frequência de células T reguladoras (Treg), natural killer (NK), supressoras mielóides (MDSC) e dendríticas (DC) no peritônio justificariam a redução da capacidade do sistema imune de reagir contra as células endometriais, permitindo sua implantação em locais ectópicos. Aqui, células Treg, NK, MDSC e DC foram quantificadas no fluido peritoneal (FP) e sangue de mulheres com EDT, a fim de associa-las ao desenvolvimento da doença; níveis de citocinas também foram avaliados. Na EDT, observou-se aumento na frequência de Treg, MDSC e DC no sangue e aparente redução destas no FP; ainda, a concentração de IL-12 foi menor no sangue comparadas ao grupo controle. Não foram observadas diferenças quanto às células NK e as outras citocinas analisadas. Os resultados indicam aumento da frequência de populações reguladoras em amostras de sangue de pacientes EDT, entretanto esses resultados não são refletidos no FP. / Endometriosis (EDT) is a gynecological disease characterized by the presence of endometrial cells out of the uterine cavity, which affects women in reproductive age. We postulated that alterations in the frequencies of regulatory T cells (Treg), natural killer cells (NK), myeloid-derived suppressor cells (MDSC) and dendritic cells (DC) in the peritoneum could justify the reduced capacity of the immune system to react to these ectopic endometrial cells, allowing them to invade distant tissues. Here, Treg, NK, MDSC and DC were quantified in the peritoneal fluid (PF) and peripheral blood (PB) of women with EDT, in order to associate them with the development of EDT; cytokine levels were also assessed. In EDT, higher frequencies of Treg, MDSC and DC in the PB and apparent lower frequencies of these cells in the PF were observed; IL-12 concentration was smaller in PB of EDT compared to control. No differences between groups were observed for NK cells and the other cytokines evaluated. The results indicate higher frequencies of regulatory cells in PB samples of EDT patients, although these findings were not reflected in PF samples.

Le sepsis est à l’origine d’une dysfonction immunitaire prolongée responsable d’infections nosocomiales et d’une mortalité tardive élevée. Sa physiologie complexe demeure mal connue et il n’existe aucun traitement spécifique en dehors de l’antibiothérapie et des thérapeutiques de suppléance d’organes. Nous nous sommes intéressés au rôle des cellules myéloïdes dans cette dysfonction immunitaire. Nous avons pu montrer qu’il existe chez les patients atteints de sepsis une augmentation du nombre de cellules suppressives d’origine myéloïde monocytaires (M-MDSC) CD14+HLA-DRlow/- et granulocytaires (G-MDSC) identifiées comme des granulocytes de faible densité CD14-CD15+. Ces cellules sont responsables d’une activité Indoléamine 2,3-dioxygénase (IDO) et arginase 1, et leur déplétion permet de restaurer la prolifération des lymphocytes T in vitro. L’augmentation précoce des G-MDSC prédit la survenue ultérieure d’infections nosocomiales. De même, l’augmentation de l’activité IDO et de l’arginase 1 plasmatique sont associées à un mauvais pronostic. Au total, nous avons pu démontrer que les cellules myéloïdes acquièrent un phénotype suppresseur en partie responsable de l’immunodépression acquise et du pronostic péjoratif chez les patients septiques. Afin de restaurer les capacités immunitaires des patients, les MDSC pourraient devenir une future cible thérapeutique. / Sepsis results in a sustained immune dysfunction responsible for poor prognosis and nosocomial infections. Sepsis physiology remains poorly understood and no treatment exists currently, excepted from antibiotherapy and life-support techniques. We asked if myeloid cells could play a role in this sustained immune dysfunction. We demonstrated that Peripheral CD14+HLA-DRlow/- monocytic-myeloid-derived suppressor cells (MDSCs) and CD14-CD15+ low-density granulocytes identified as granulocytic- (G-)MDSCs were increased in septic patients. In vitro, arginase and IDO activities relied on MDSCs and depletion of both subsets restored T-cell proliferation. The initial proportion of G-MDSC predicted occurrence of nosocomial infections. Similarly, high plasma Indoleamine 2,3-dioxygenase (IDO) activity and arginase 1 level were associated with poor outcome. Altogether, our results demonstrate that myeloid cells acquire suppressive functions during sepsis, partially responsible for the sustained immune dysfunction and poor outcome. MDSCs may become a future therapeutic target to restore the immune capacities of septic patients.