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The failure of the immune system to provide efficient protection against tumour cells has been considered as a major issue in immunology. It is now well established that inadequate function of the host immune system is one of the main mechanisms by which tumours escape from immune control contributing to the limited success of cancer immunotherapy. Several cell populations have been described which display immunosuppressive properties and may impede tumor-specific immunity. Among them, GR1+CD11b+ immature myeloid suppressor cells and CD4+CD25+ regulatory T cells seem to play an important role. These cells accumulate in the spleens of tumour bearing mice and patients with cancer and contribute to immunosuppression by inhibiting the function of CD8+ T cells and/or by promoting tumour angiogenesis. The aim of our work was to define the mechanisms by which a single dose of cyclophosphamide (CTX), a chemical agent commonly used in chemotherapy treatment, induces the rejection of established P815 mastocytoma. Our data show that CTX treatment leads to the selective loss of GR1medCD11b+ splenic myeloid cell producing TGF-â, a cytokine which is known to suppress antitumoral response. Furthermore, injection of CTX causes a decrease in the number of naturally occurring regulatory T cells (CD4+CD25+Foxp3+) in the spleen and the tumor. Finally, CTX treatment induces the differentiation of GR1highCD11b+ splenic myeloid cells into mature GR1highCD11b+CD11c+ (possibly dendritic cells?) which express high levels of CD11c, MHC class II and CD86 molecules. Of note, these cells are mainly detected in tumour necrosis areas. Collectively, these results suggest that CTX prevents suppressive mechanisms and induces a population of CD11c+ myeloid cells which may present tumor antigens and activate T lymphocytes, an hypothesis in line with the requirement for CD4+ cells in CTX-induced long term resistance.

Background: HIV/AIDS remains a global concern that, although manageable using anti-retroviral therapy (ART), is still eluded by a cure with paucity of knowledge regarding its acquisition and spread especially through the male genital tract (MGT)1–4. Several authors have shown the human foreskin to be an effective mucosal effector site with heterogenous populations of innate and adaptive immune cells, that are permissive to HIV infection5–8. In support of this, medical male circumcision (MMC), has been reported to confer up to 60 % risk reduction in HIV acquisition9–17. Most studies have focused on investigating blood lymphoid immune cells and their interaction with HIV-1, this study sought to elucidate the myeloid cell composition of the inner and outer foreskin, and to investigate the susceptibility of these cells to ex vivo HIV infection by (i) Isolating migratory and non-migratory Langerhans cells (LCs) and “macrophage-like” cells from the foreskin epidermis (ii) Immunophenotyping and characterising foreskin LCs and “macrophage-like” cells using CD4+CCR5+ as proxy for HIV susceptibility, HLA-DR+CD80/86+ for maturation, and the mannose receptor, DC-SIGN and Siglec-1 as HIV attachment factors and (iii) Investigating the HIV susceptibility of foreskin epidermal cells using an optimised ex vivo pluricellular foreskin infection model of suspension cells. Methodology: Foreskin specimen were obtained from 60 seronegative adult South African men (aged 18-35 years) undergoing voluntary medical male circumcision (vMMC). Migratory and non-migratory foreskin cells were isolated from the inner and outer foreskin using spontaneous migration and enzymatic digestion of remnant epidermal tissue respectively, and subsequently immunophenotyped using multiparameter flow cytometry (n=31). The optimal HIV infection model was determined through assessment of different infection models inclusive of i) epidermal sheets, ii) foreskin explants and iii) pluricellular suspension cells (n=5). Using the ex vivo pluricellular foreskin infection model of suspension cells (n=17), Subtype C transmitted founder (T/F) and chronic infection derived (CC) infectious molecular clones (IMCs) were used alongside Subtype B NL4-3 IMCs with CCR5, CXCR4 and BaL envelopes. The extent of HIV infection was quantified by measurement of p24 in different immune cell subsets over a time-course. The different HIV infected cell subsets were characterized using CD45, CD207, CD1a, CD11c, CD14, CD3, HLA-DR, CD80/86, CD209, CD206, CD169, CD4 and CCR5. Results: Foreskin myeloid cells contained a rare population of LCs (1.11 % ± 1.02 %;) that was predominantly migratory (p = 0.0084) and “macrophage-like” cells (9.87 % ± 9.64 %) that, in addition to being 8-fold more abundant (p

Influenza A virus (IAV) infection is a highly contagious respiratory disease, which can cause substantial morbidity and occasionally death. Invariant natural killer T (iNKT) cells, a subset of CD1d-restricted T lymphocytes, have been identified as important modulators of immunity, mediating both pro- and anti-inflammatory responses. We show that iNKTs play an important role for the generation of protective innate and adaptive immune responses to IAV, and enhance heterotypic immunity to influenza virus following vaccination with a novel pseudotyped virus, S-FLU, which lacks HA expression. iNKT-deficient mice (Jα18^-/-) showed increased susceptibility and lung pathology following IAV infection, which correlated with an exaggerated accumulation of inflammatory monocytes and neutrophils in the lung. Consistent with these findings, we demonstrated in IAV-infected CD1d^-/-:CD1d^{+/+} mixed bone marrow chimeric mice, that the lack of CD1d expression on myeloid cells purified from the lungs of IAV-infected mice significantly increased expression of genes linked to cell activation, survival and polarisation between pro- and antiinflammatory responses. We extended these results by examining the role of chemokine signalling during IAV infection, and identified a novel role for fractalkine (CX3CL1) and its receptor (CX3CR1) in innate immune cell recruitment. The use of CX3CR1-iNKT cell double knockout mice revealed that, although upregulated in Jα18^-/- mice, CX3CR1-CX3CL1 signalling is not required for cell migration during exacerbated IAV-responses. Finally, we showed that iNKT-deficient mice displayed reduced longevity of peripheral IAVspecific CD8^+ T cells following S-FLU vaccination, compared with wild-type mice. S-FLU vaccination protected mice following 5 day heterotypic challenge, however vaccinated mice exhibited reduced viral clearance, and importantly a significant reduction in IAV specific memory T cell response, suggesting a possible role of iNKT cells during T cell priming in modulating the lifespan of antigen-specific T cell responses. Although additional experiments are warranted, these results suggest that harnessing iNKT cells should be considered to modulate the innate and adaptive immune response to optimise heterotypic vaccine design and for therapeutic intervention against acute influenza infection.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20229号 / 医博第4188号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 原田 浩, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

INTRODUCTION: Previous studies have shown that osteocytes, the matrix-embedded cells in bone, control bone modeling and remodeling through direct contact with adjacent cells and via secreted factors that can reach cells in the bone marrow microenvironment (BMM). Osteocytes express several receptors including G protein-coupled receptors (GPCRs) and mice lacking the stimulatory subunit of G-proteins (Gsα) in osteocytes have abnormal myelopoiesis, skeletal abnormalities and reduced adipose tissue. This study aimed at evaluating the effects of osteocyte-secreted factors on myeloid cell proliferation and differentiation in vitro. To investigate cross-talk between osteocytes and the BMM, we established osteocytic cell lines lacking Gsα expression to study the molecular mechanisms by which osteocytes control myeloid cell proliferation and differentiation. METHODS: CRISPR/Cas9 was used to knockout Gsα in the osteocytic cell line Ocy454. Conditioned media (CM) from differentiated Ocy-GsαCtrl and Ocy-GsαKO cells were used to treat myeloid cells and bone marrow mononuclear cells (BMNCs) isolated from long bones of 6-8-week-old C57/BL6 mice. BMNCs were cultured with Macrophage Colony Stimulating Factor (M-CSF), Receptor Activator of Nuclear Factor Kappa β Ligand (RANKL) to induce osteoclast differentiation. Proliferation, TRAP staining, TRAP activity, resorption pit assay, F-actin ring formation and mRNA expression were used to evaluate cell proliferation, differentiation and function of the induced osteoclasts. Proteomics analysis of CM was performed to identify osteocyte-secreted factors capable of controlling myelopoiesis and osteoclastogenesis. RESULTS: Myeloid cells treated with CM from Ocy-GsαKO showed a significant increase in cell proliferation compared to Ocy-GsαCtrl CM and non-treated control. BMNCs treated with CM from Ocy-GsαCtrl and Ocy-GsαKO showed a significant increase in cell proliferation as compared to non-treated control. Osteoclast differentiation was significantly suppressed by CM from Ocy-GsαCtrl and further suppressed by CM from Ocy-GsαKO compared to non-treated control. Osteoclasts exposed to CM from Ocy-GsαKO showed a significant defect in activity and function as compared to cells exposed to CM from Ocy-GsαCtrl and non-treated cells. Osteoclast apoptosis was significantly enhanced by Ocy-GsαCtrl and Ocy-GsαKO CM compared to non-treated control. Among osteocyte secreted factors, we identified neuropilin-1 (NRP-1) as a Gsα-dependent osteocytic factor capable of suppressing osteoclastogenesis. CM from Ocy-GsαKO in which M-CSF was reduced by shRNA demonstrated decrease in BMNCs proliferation, demonstrating that osteocytes are also important sources of this cytokine. CONCLUSIONS: Osteocytes produce several Gsα-dependent and -independent secreted factors capable of supporting myelopoiesis, promoting macrophage proliferation and suppressing osteoclast formation. We identified osteocyte-derived NRP-1 as a novel factor capable of decreasing osteoclastogenesis. In addition, we found that M-CSF secreted by osteocytes is responsible in part for BMNC proliferation. Future studies should focus on determining the role of osteocyte-mediated NRP-1 and other secreted factor(s) in control of myelopoiesis and osteoclastogenesis. / 2020-06-26T00:00:00Z

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The Gadd45 family of proteins is known to play a central role as cellular stress sensors that modulate the response of mammalian cells to different stressors, including oncogenic stress. Gadd45a expression is regulated during myeloid cell differentiation, and is also induced in response to acute stimulation with cytokines, myeloablation and inflammation. The proto-oncogene C-myc plays a pivotal role in growth control, differentiation and apoptosis in hematopoietic cells. Deregulated Myc in hematopoietic cells blocks the differentiation program and prevents normal homeostatic cellular apoptosis, which alters the balance of cell populations, often participating in leukemogenesis. The status of Gadd45a expression has been shown to impact on different cancers, including breast cancer and leukemia. How the stress response gene Gadd45a modulates oncogenic stress imparted by deregulated c-Myc in myeloid cells has not been investigated. We hypothesized that Gadd45a and its interacting partner proteins can modulate specific pro-survival or pro-apoptotic signaling pathways, altering the cellular response to oncogenic myc in myeloid cells. Gadd45a may play different roles in proliferating and differentiating cells, and myeloid cells in vivo are at all stages of myeloid development. Therefore, to understand how Gadd45a status impacts on proliferating and differentiating myeloid cells, we decided to study the effect of loss of Gadd45a in myc-expressing cells that are either proliferating or stimulated to undergo differentiation. Therefore, to address this issue we utilized bone marrow from wild-type (wt) and Gadd45a null mice, and retrovirally infected these cells to express constitutive Myc or empty vector control. Using these cells we have shown that bone marrow deficient in Gadd45a and expressing constitutive Myc, display decreased apoptosis under proliferating conditions, yet increased apoptosis in media containing the differentiation inducing cytokine GM-CSF. We show that in expansion media loss of Gadd45a in the presence of Myc elicits its function through the activation of p38, with evidence supporting a role for PU.1 and Mcl-1 expression, which are downstream of p-p38. In contrast, deregulated C-Myc and loss of Gadd45a does not signal through p-38 in GM-CSF, but surprisingly there is a decrease in cytokine receptor expression. This data demonstrates that Gadd45a may be required for optimal cytokine receptor expression in myeloid cells, which can impact on survival of the cells. Although in primary bone marrow Gadd45a status had no effect on differentiation of Myc expressing cells, the loss of Gadd45a in Hoxb8 generated cell lines shifted differentiation towards increased neutrophils. Determining the role of Gadd45a on the biological outcome of myeloid cells in response to deregulated c-Myc will provide vital information in understanding the function of Gadd45a in the development and progression of Myc expressing myeloid leukemia. / Molecular Biology and Genetics

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