Search

Figure S1 Generation of a murine EMT6-hHER2 breast cancer cell line. A, Human-HER2 expression in EMT6-hHER2 or EMT6-WT tumor cell line determined by FACS. B, H&E (top panel) and 〈-hHER2 (bottom panel) immunohistochemical staining of orthotopic EMT6-WT and EMT6-hHER2 tumors. C, Cell proliferation assay showing EMT6-WT and EMT6-hHER2 growth inhibition after T-PNU or T-DM1 treatment in vitro. Figure S2 Trastuzumab, free PNU or mitoxantrone treatment is ineffective in promoting EMT6-hHER2 tumor growth inhibition. A, Therapeutic response of EMT6-hHER2 tumors exposed to T-PNU (1 mg/kg, 2x), free PNU (6 and 8 μg/kg, 2x), trastuzumab (20 mg/kg, 4x, once per week) and mitoxantrone (4 mg/kg 1x). B, Survival curves of A. Figure S3 T-PNU samples cluster apart from untreated, trastuzumab and T-DM1 treated samples based on gene expression. A, Sample similarity in a 2D projection by multi-dimensional scaling. Only the top 1000 differentially expressed genes (DEGs) are taken into account. B, Heatmap of the gene expression vs sample matrix. Displayed are custom selected genes plus the top 1000 DEGs (rows) of all samples (columns). Figure S4 Heatmap PID CD8 TCR downstream pathway. Figure S5 Gene signatures of innate immune and cytokine pathways. A, Network cluster of gene sets with overlapping genes with shared function of inflammatory pathway. The network includes 36 gene sets and 7551 genes (see Table S2). B-D, Heatmaps of BIOCARTA inflammatory, dendritic cell (DC) and cytokine pathway, respectively. Asterisks denote low-responding T-PNU samples. Figure S6 Characterization of intratumoral T cells upon treatment. *p ≤ 0.05, **p ≤ 0.01. A, MvA plot depicting expression and effect size of selected key immune genes. B, Validation by FACS of selected CD8 T cell markers of functional activation and proliferation identified in A in between the comparisons. Table S1 Gene sets of network TCR pathway. Table S2 Gene sets of network activated TLR pathway. (PDF 10859 kb)

Background Gram-negative bacteremia (GNB) is a major cause of illness and death after hematopoietic stem cell transplantation (HSCT), and updated epidemiological investigation is advisable. Methods We prospectively evaluated the epidemiology of pre-engraftment GNB in 1118 allogeneic HSCTs (allo-HSCTs) and 1625 autologous HSCTs (auto-HSCTs) among 54 transplant centers during 2014 (SIGNB-GITMO-AMCLI study). Using logistic regression methods. we identified risk factors for GNB and evaluated the impact of GNB on the 4-month overall-survival after transplant. Results The cumulative incidence of pre-engraftment GNB was 17.3% in allo-HSCT and 9% in auto-HSCT. Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were the most common isolates. By multivariate analysis, variables associated with GNB were a diagnosis of acute leukemia, a transplant from a HLA-mismatched donor and from cord blood, older age, and duration of severe neutropenia in allo-HSCT, and a diagnosis of lymphoma, older age, and no antibacterial prophylaxis in auto-HSCT. A pretransplant infection by a resistant pathogen was significantly associated with an increased risk of posttransplant infection by the same microorganism in allo-HSCT. Colonization by resistant gram-negative bacteria was significantly associated with an increased rate of infection by the same pathogen in both transplant procedures. GNB was independently associated with increased mortality at 4 months both in allo-HSCT (hazard ratio, 2.13; 95% confidence interval, 1.45–3.13; P Conclusions Pre-engraftment GNB is an independent factor associated with increased mortality rate at 4 months after auto-HSCT and allo-HSCT. Previous infectious history and colonization monitoring represent major indicators of GNB. Clinical Trials registration NCT02088840.

Post-transplant relapse is still a major cause of treatment failure in high-risk acute leukemia (AL) patients. Attempts to manipulate donor T cell alloreactivity to spare normal tissues while killing leukemic cells have been largely unsuccessful. In the search for strategies to separate the GvL effect from GVHD, we investigated the role of a thymic-derived CD4+CD25+ FoxP3+ regulatory T-cell subpopulation (Tregs) that physiologically helps maintain immunological self-tolerance and immune homeostasis. Evidence from murine bone marrow (BM) transplantation across major histocompatibility barriers showed co-infusion of conventional T lymphocytes (Tcons) with Tregs suppressed lethal GVHD without impairing Tcon activity against malignant diseases. In 69 high-risk AL patients who had received an HLA haploidentical T cell-depleted hematopoietic transplant and no post-transplant immunosuppressive GvHD prophylaxis, adoptive immunotherapy with donor Tregs (2 × 106/kg) and Tcons (1 × 106/kg) protected patients from GvHD (Di Ianni et al., Blood 2011) and largely prevented post-transplant leukemia relapse. In fact, only 5% of patients relapsed (Martelli et al., Blood 2014). However, also because such patients had advanced stage disease, TRM was still in the range of that of T cell-depleted haplo transplants, (i.e. 40%, Aversa et al., JCO 2005). A current cohort of AL patients was conditioned with total body irradiation (TBI) (8Gy as single dose TBI or 13.5 Gy as fractionated TBI), cyclophosphamide (30mg/kg), thiotepa (8mg/kg) and fludarabine (200mg/m2). To date, 24 high-risk AL patients (7 ALL, 17 AML) have been enrolled. Twenty-two of the 24 patients engrafted, one AML patient relapsed, 4/22 developed aGvHD (4/4 are alive) and one developed cGvHD, TRM was exceptionally low (Fig. 1). At a median follow up of 2 years, 19 patients are alive. Consequently, probabilities of leukemia-free and leukemia/cGVHD-free survivals are good (Fig. 1). Hypotheses have been put forward to explain the ability of Tregs to prevent of GvHD while preserving the GvL effect mediated by Tcons. However, the mechanism is still unknown. In humans, naïve CD45RA+ Tregs express CXCR4 BM homing receptor and preferentially localize to the BM while memory CD45RO+ Tregs display lower CXCR4 expression and home to the periphery (Booth et al., J Immunol. 2010). Since Tregs that are recovered from peripheral blood and are used for adoptive immunotherapy are CD45RO+ and largely CxCR4 negative, we hypothesized that GvL without GvHD might be due to unopposed Tcon alloreactivity in the BM combined with regulated T cell alloreactivity at the periphery. In order to verify such hypothesis, we infused NSG mice with human leukemia and human Tregs and Tcons. Mice that received leukemia and haploidentical Tcons (without Tregs) cleared leukemia but died of GvHD. T cells harvested from their BM, spleen and liver were predominantly CD8+ and displayed alloreactivity against leukemia. Mice that received leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. T cells harvested from spleen and liver were composed of CD8+ T cells (40%) and CD4+T cells (60%). Purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction. Purified CD8+ T cells displayed no alloreactivity against leukemia. In contrast, T cells harvested from BM were still predominantly CD8+ and still displayed alloreactivity against leukemia suggesting Tcons had retained their alloantigen recognition. Finally, in mice treated with Tregs alone, T cells were recovered only in the spleen and liver, they displayed a CD4+ phenotype and inhibited mixed lymphocyte reaction. No T cells were found in the BM. In contrast, when we infused human purified CD45RA+ naïve Tregs (that display the CXCR4 BM homing receptor), mice died of leukemia progression despite the co-infusion of large doses of Tcons. In the BM we isolated CD8+ T cells that displayed no ability to kill leukemia and CD4+ T cells that displayed regulatory function. Thus, naïve Tregs homed to the BM and blocked the GvL effect of T cons. When CXCR4 was blocked with an anti-CXCR4 antibody, naïve Tregs could not home to the BM and T cons killed leukemia. In conclusion, Treg-Tcon adoptive immunotherapy confines the graft-versus-host alloreaction to the hematopoietic system and, consequently, allows a GvL effect without GvHD. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

This study shows that teriparatide promotes the circulating osteoblast (OB) precursor degree of maturation in patients affected by postmenopausal osteoporosis. Anabolic treatment with teriparatide has proven effective for the therapy of postmenopausal osteoporosis and significantly reduces the risk of non-vertebral fragility fractures. The aim of this study was to investigate the effect of teriparatide on circulating OB precursors. We evaluated by flow cytometry and real-time PCR the expression of OBs typical markers in peripheral blood mononuclear cells during treatment with teriparatide plus calcium and vitamin D, raloxifene plus calcium and vitamin D or calcium and vitamin D alone at various time points. Serum bone alkaline phosphatase and osteocalcin (OC) were measured as markers of bone turnover. Our results show that circulating OB precursors are more numerous and more immature in patients affected by fragility fractures than in osteoporotic patients without fractures. We also show that teriparatide treatment increases the expression of alkaline phosphatase and of OC in OB precursors; thus, it increases their degree of maturation. We suggest that teriparatide acts as anabolic agents also by promoting the maturation of OB precursors.

Acute myeloid leukemias (AML) with complex karyotype (≥ 3 abnormalities) and/or monosomy 7/del(7q) are a subset of AML with extremely poor prognosis. Hematopoietic stem cell transplantation (HSCT) is the only potentially curative treatment, but relapse rate is high (most often above 50%) and negatively impacts on patients' survival. (Ciurea et al. Cancer 2018) Such poor results are even questioning the role of HSCT and rise the need of new transplant procedures for these patients. To this end, we are performing a clinical trial of haploidentical HSCT with adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) in the absence of post-transplant pharmacologic immune suppression (Treg-Tcon haplo-HSCT). Such approach allows for an extremely low relapse rate in patients with high-risk acute leukemias transplanted in complete remission. (Martelli et al. Blood 2014) In this study we analyzed the outcome of all the patients that were referred to our center from January 1st 2007 to May 31st 2018 with a diagnosis of AML with complex karyotype and/or monosomy 7/del(7q) and that were fit for receiving an induction treatment. 49 patients were included. Median age at diagnosis was 52; 22 were female, 27 were male. 20/49 had chromosome 7 abnormalities (isolated in 12 patients; in a complex karyotype in 8 patients). 11 patients received a hypomethylating agent as first line treatment, while the other 38 patients received high-dose chemotherapy. We performed 30 HSCT procedures in 25 patients; 5 patients received a second transplant (4 after disease relapse and 1 after rejection). 6 underwent a HLA-matched HSCT, 6 a T-depleted haploidentical HSCT, and 18 a Treg-Tcon haplo-HSCT. Median age at transplant was 42 with a median follow up of 40 months. Regarding disease status at transplant, 26 HSCT were performed in patients that were in 1st or 2nd hematological remission and 4 in patients with refractory disease. Cytogenetic analysis (karyotype and/or FISH analysis) revealed the presence of abnormal clones in 11 patients and multiparametric flow cytometry analysis detected minimal residual disease in 16 patients. Thus, most of the patients (17/30, 57%) were transplanted with detectable disease. 15/49 patients were disease-free and alive at the time of the analysis, all of them after receiving HSCT (HSCT vs Non-HSCT, p In conclusion, our results not only confirm that HSCT is the only potentially curative treatment for AML with complex karyotype and/or chromosome 7 involvement, but also demonstrate that Treg-Tcon haplo-HSCT allows for a strong antileukemic effect, the only needed for a long term control of such unfavorable AMLs with high disease burden at transplant. While it is necessary to confirm these results in a larger cohort of patients, Treg-Tcon haplo-HSCT could represent the best transplant option for this subset of very high-risk AML patients. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Bone metastases are frequent and debilitating consequence for many tumors, such as breast, lung, prostate, and kidney cancer. Many studies report the importance of the immune system in the pathogenesis of bone metastasis. Indeed, bone and immune system are strictly linked to each other because bone regulates the hematopoietic stem cells from which all cells of the immune system derive, and many immunoregulatory cytokines influence the fate of bone cells. Furthermore, both cytokines and factors produced by immune and bone cells promote the growth of tumor cells in bone, contributing to supporting the vicious cycle of bone metastasis. This review summarizes the current knowledge on the interactions among bone, immune, and tumor cells aiming to provide an overview of the osteoimmunology field in bone metastasis from solid tumors.

Laminopathies are a wide and heterogeneous group of rare human diseases caused by mutations of the LMNA gene or related nuclear envelope genes. The variety of clinical phenotypes and the wide spectrum of histopathological changes among patients carrying an identical mutation in the LMNA gene make the prognostic process rather difficult, and classical genetic screens appear to have limited predictive value for disease development. The aim of this study was to evaluate whether a comprehensive profile of circulating cytokines may be a useful tool to differentiate and stratify disease subgroups, support clinical follow-ups and contribute to new therapeutic approaches. Serum levels of 51 pro- and anti-inflammatory molecules, including cytokines, chemokines and growth factors, were quantified by a Luminex multiple immune-assay in 53 patients with muscular laminopathy (Musc-LMNA), 10 with non-muscular laminopathy, 22 with other muscular disorders and in 35 healthy controls. Interleukin-17 (IL-17), granulocyte colony-stimulating factor (G-CSF) and transforming growth factor beta (TGF-&beta, 2) levels significantly discriminated Musc-LMNA from controls, interleukin-1&beta, (IL-1&beta, ), interleukin-4 (IL-4) and interleukin-8 (IL-8) were differentially expressed in Musc-LMNA patients compared to those with non-muscular laminopathies, whereas IL-17 was significantly higher in Musc-LMNA patients with muscular and cardiac involvement. These findings support the hypothesis of a key role of the immune system in Musc-LMNA and emphasize the potential use of cytokines as biomarkers for these disorders.