Hepatic NK cells in liver transplantation

The study described in this thesis represents an attempt to identify possible factors determining the induction of operational tolerance after LTX. Current beliefs propose that the establishment of tolerance is a multifactorial phenomenon in which the combined action of several factors conveys tolerance towards the grafted organ. Among these factors, the transfer of passenger donor leukocytes into the recipient after transplantation and the secretion of specific donor-derived soluble MHC molecules seem to play a relevant role. In this thesis we considered two components as possible inducers of LTX tolerance: donor NK cells (as part of the pool of passenger donor leukocytes) and HLA-G (as soluble HLA molecule). We analyzed different aspects of donor hepatic NK cells as to investigate their contribution to tolerance induction in LTX. Additionally, we laid the foundations for a critical assessment of the role of HLA-G as a tolerogenic molecule in LTX. Our results suggest that hepatic NK cells in adults derive from hematopoietic CD34+ stem cells that are continuously recruited from peripheral blood (Chapter 5). Hematopoietic CD34+ stem cells are known to be periodically released from the bone marrow into peripheral blood. This continuous release of cells into peripheral blood seems to constitute a mechanism to maintain full occupancy of hematopoietic stem cells niches not only in bone marrow cavities but also in peripheral tissues, including the adult liver. Once relocated in the liver, these early progenitors may develop, under the influence of the local microenvironment, into liver-specific NK cells (Chapter 5). Liver-specific NK cells contain a unique subset of CD56bright NK cells that are physiologically activated and highly cytotoxic (Chapter 2). These characteristics of hepatic CD56bright NK cells differ from the functional properties attributed to the same subset of NK cells found in blood or in other peripheral tissues, such as lymph nodes or uterus. After LTX, part of donor hepatic NK cells detach from the grafted organ and are detectable in the recipient circulation for approximately two weeks (Chapter 2). Besides this portion of migrating cells, donor hepatic NK cells remain in the grafted liver for at least two years after LTX (Chapter 5). This long-term chimerism can be explained by two scenarios. First, donor hepatic NK cells and their precursors may relocate, after LTX, to specific sites in the recipient (such as bone marrow, gut-associated lymphoid tissues or spleen, see Chapter 2) and from there contribute to long-term donor NK-cell chimerism. Alternatively, the donor NK cells found long after LTX within the liver graft may constitute a pool of long-living NK cells similar to the ones recently described in mice liver by the groups of von Andrian, Lanier and Cooper. Overall these observations in mice indicate that NK cells activated specifically by a chemical hapten or a virus, or non-specifically by a state of lymphopenia or by inflammatory cytokines acquire classical characteristics of immune memory that include an extended lifespan, the ability to self-renew, and the capacity to mount robust and protective recall responses. However, as specific markers for human memory NK cells are not known yet, we could not test whether donor hepatic NK cells long after LTX actually represent a pool of NK cells that acquired immune memory. With regard to the role of these donor-derived NK cells in the induction of allograft tolerance after LTX, we could not find firm evidence that NK cells play a prominent role in tolerance induction (Chapter 3; Chapter 4). In fact neither donor nor recipient NK-cell alloreactivity, as predicted by considering donor and recipient HLA/KIR genotypes, was found to correlate with LTX survival or the induction of allograft tolerance (Chapter 3). Additionally, specific depletion of hepatic donor NK cells prior to LTX in a spontaneously tolerant rat model, did not affect the establishment of tolerance (Chapter 4). Altogether, based on these results we reject the hypothesis that cytotoxic NK cells from the donor liver play a main role in inhibiting the immune response towards the allogeneic graft by killing alloreactive T cells and APCs from the recipient. Therefore, these results exclude the possibility of exploiting the cytotoxic properties of donor hepatic NK cells for tolerance induction in clinical LTX. In addition to the study of NK cells, we here presented our initial attempts to assess the role of soluble HLA-G in LTX tolerance. In contrast to what was suggested by few original studies on this topic, our first results seem to associate HLA-G with states of inflammation rather than tolerance induction (Chapter 6). These results will be further expanded and additional experiments are needed to draw more definite conclusions on this subject

CXCR6 marks a novel subset of T-bet(lo)Eomes(hi) natural killer cells residing in human liver

This work was funded by a Wellcome Trust Investigator Award to MKM funding KS and LP, MRC Career Development Award to CD, MRC Clinical Research Training Fellowship to DP, Wellcome Trust Henry Dale Fellowship to VM

Defining Early Human NK Cell Developmental Stages in Primary and Secondary Lymphoid Tissues

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56brightCD16+/− and CD56dimCD16+ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body

Ototoxicity of cisplatin plus standard radiation therapy vs. accelerated radiation therapy in glioblastoma patients

Purpose : To assess the effect of cisplatin (CDDP) plus concurrent radiation therapy on hearing loss. Methods : 451 patients with glioblastoma multiforme (GBM) were randomly assigned after surgery to: Arm A: Carmustine (BCNU) + standard radiation therapy (SRT); Arm B: BCNU + accelerated radiation therapy (ART: 160 cGy twice daily for 15 days); Arm C: CDDP + BCNU + SRT; or Arm D: CDDP + BCNU + ART. Patients on arms C and D received audiograms at baseline, and prior to the start of RT, and prior to cycles 3 and 6. Otologic toxicities were recorded at each visit. Results : 56% of patients had hearing loss at baseline. 13% and 50% of patients experienced worsening ototoxicity after 1 year of treatment in arms A and B vs. C and D, respectively, with 13% of those on arms C and D experiencing significant ototoxicity (≥ grade 3) at 6 months. Increasing age was associated with an increased risk of ototoxicity. Conclusions : Increased exposure to CDDP increases the risk of ototoxicity over time. Older patients are more susceptible to hearing loss with CDDP. The low proportion of patients with clinically significant ototoxicity suggests that baseline screening is unnecessary in GBM patients.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43476/1/11060_2005_Article_9049.pd

Hitomi (ASTRO-H) X-ray Astronomy Satellite

The Hitomi (ASTRO-H) mission is the sixth Japanese x-ray astronomy satellite developed by a large international collaboration, including Japan, USA, Canada, and Europe. The mission aimed to provide the highest energy resolution ever achieved at E  >  2  keV, using a microcalorimeter instrument, and to cover a wide energy range spanning four decades in energy from soft x-rays to gamma rays. After a successful launch on February 17, 2016, the spacecraft lost its function on March 26, 2016, but the commissioning phase for about a month provided valuable information on the onboard instruments and the spacecraft system, including astrophysical results obtained from first light observations. The paper describes the Hitomi (ASTRO-H) mission, its capabilities, the initial operation, and the instruments/spacecraft performances confirmed during the commissioning operations for about a month

First draft genome sequence of a strain from the genus Fusibacter isolated from Salar de Ascotán in Northern Chile

Abstract Fusibacter sp. 3D3 (ATCC BAA-2418) is an arsenate-reducing halotolerant strain within the Firmicutes phylum, isolated from the Salar de Ascotán, a hypersaline salt flat in Northern Chile. This high-Andean closed basin is an athalassohaline environment located at the bottom of a tectonic basin surrounded by mountain range, including some active volcanoes. This landscape can be an advantageous system to explore the effect of salinity on microorganisms that mediate biogeochemical reactions. Since 2000, microbial reduction of arsenic has been evidenced in the system, and the phylogenetic analysis of the original community plus the culture enrichments has revealed the predominance of Firmicutes phylum. Here, we describe the first whole draft genome sequence of an arsenic-reducing strain belonging to the Fusibacter genus showing the highest 16S rRNA gene sequence similarity (98%) with Fusibacter sp. strain Vns02. The draft genome consists of 57 contigs with 5,111,250 bp and an average G + C content of 37.6%. Out of 4780 total genes predicted, 4700 genes code for proteins and 80 genes for RNAs. Insights from the genome sequence and some microbiological features of the strain 3D3 are available under Bioproject accession PRJDB4973 and Biosample SAMD00055724. The release of the genome sequence of this strain could contribute to the understanding of the arsenic biogeochemistry in extreme environments