Diffusion-Weighted imaging and diffusion tensor imaging detect delayed graft function and correlate with allograft fibrosis in patients early after kidney transplantation.

PURPOSE: To combine diffusion-weighted imaging (DWI) and diffusion tensor imaging (DTI) for detection of allograft dysfunction in patients early after kidney transplantation and to correlate diffusion parameters with renal function and renal histology of allograft biopsies. MATERIALS AND METHODS: Between day 4 and 11 after kidney transplantation 33 patients with initial graft function and 31 patients with delayed graft function (DGF) were examined with a 1.5T magnetic resonance imaging (MRI) scanner. DTI and DWI sequences were acquired and fractional anisotropy (FA), apparent diffusion coefficient (ADCmono), pure diffusion (ADCdiff ), and the perfusion fraction (Fp) were calculated. Kidney biopsies in 26 patients were analyzed for allograft pathology, ie, acute tubular injury, inflammation, edema, renal fibrosis, and rejection. Histological results were correlated with MRI parameters. RESULTS: In the renal medulla FA (0.25 ± 0.06 vs. 0.29 ± 0.06, P < 0.01) and ADCmono (1.73 ± 0.13*10(-3) vs. 1.93 ± 0.16*10(-3) mm(2) /s, P < 0.001) were significantly reduced in DGF patients compared with patients with initial function. For ADCdiff and Fp similar reductions were observed. FA and ADCmono significantly correlated with renal function (r = 0.53 and r = 0.57, P < 0.001) and were inversely correlated with the amount of renal fibrosis (r = -0.63 and r = -0.65, P < 0.05). CONCLUSION: Combined DTI and DWI detected allograft dysfunction early after kidney transplantation and correlated with allograft fibrosis. J. Magn. Reson. Imaging 2016;44:112-121.

The role of leukemia-derived B7-H1 (PD-L1) in tumor-T-cell interactions in humans.

OBJECTIVE: Expression of the B7 homolog B7-H1 (PD1-Ligand) has been proposed to enable tumor cells to evade immune surveillance. Recently, B7-H1 on murine leukemia cells was reported to mediate resistance to cytolytic T-cell destruction. We here investigate the expression and function of the B7 homolog B7-H1 in human leukemia. PATIENTS AND METHODS: Leukemia cells from 30 patients and 9 human leukemia cell lines were investigated for B7-H1 expression by flow cytometry. Functional relevance of B7-H1 for tumor-immune interactions was assessed by coculture experiments using purified, alloreactive CD4 and CD8 T cells in the presence of a neutralizing anti-B7-H1 antibody. RESULTS: Significant B7-H1 expression levels on leukemia cells were detected in 17 of 30 patients and in eight of nine cell lines. In contrast to various other tumor entities and the data reported from a murine leukemia system, no significant inhibitory effect of leukemia-derived B7-H1 on CD4 and CD8 cytokine production (IFN-gamma, IL-2), proliferation or expression of T-cell activation markers (ICOS, CD69) was observed. Furthermore, in the presence of neutralizing B7-H1 antibody (mAb 5H1) occurred no significant changes in T cell IFN-gamma or IL-2 production or proliferation. CONCLUSIONS: Our data demonstrate that leukemia-derived B7-H1 seems to have no direct influence on T-cell activation, proliferation, and cytokine production in humans. Further experiments are warranted to delineate factors and characterize yet-unidentified B7-H1 receptor(s) that determine inhibitory and stimulatory functions of B7-H1 in human leukemia.

Expression of the B7-related molecule B7-H1 by glioma cells: a potential mechanism of immune paralysis.

Human glioblastoma is a highly lethal tumor that is known for its immune inhibitory capabilities. B7-homologue 1 (B7-H1), a recently identified homologue of B7.1/2 (CD80/86), has been described to exert costimulatory and immune regulatory functions. We investigated the expression and the functional activity of B7-H1 in human glioma cells in vitro and in vivo. Although lacking B7.1/2 (CD80/86), all 12 glioma cell lines constitutively expressed B7-H1 mRNA and protein. Exposure to IFN-gamma strongly enhanced B7-H1 expression. Immunohistochemical analysis of malignant glioma specimens revealed strong B7-H1 expression in all 10 samples examined, whereas no B7-H1 expression could be detected on normal brain tissues. To elucidate the functional significance of glioma cell-related B7-H1 expression, we performed coculture experiments of glioma cells with alloreactive CD4+ and CD8+ T cells. Glioma-related B7-H1 was identified as a strong inhibitor of CD4+ as well as CD8+ T-cell activation as assessed by increased cytokine production (IFN-gamma, interleukin-2, and interleukin-10) and expression levels of the T-cell activation marker (CD69) in the presence of a neutralizing antibody against B7-H1 (mAb 5H1). B7-H1 expression may thus significantly influence the outcome of T-cell tumor cell interactions and represents a novel mechanism by which glioma cells evade immune recognition and destruction.

Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance.

Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.

The CD28 related molecule ICOS: T cell modulation in the presence and absence of B7.1/2 and regulational expression in multiple sclerosis.

Costimulatory signals play a key role in regulating T cell activation and are believed to have decisive influence in the inciting and perpetuating cellular effector mechanisms in autoimmune diseases such as multiple sclerosis (MS). Inducible costimulator protein (ICOS), a recently identified member of the CD28-family, presumably affects the differentiation of Th1/Th2 cells after primary activation and modulates the immune response of effector/memory T cells. This study examines the expression and functional role of ICOS costimulation in healthy donors and patients with MS. After nonspecific or antigen-specific stimulation, ICOS is preferentially expressed on CD4 Th2-T cells. ICOS-costimulation affects the production of Th1 and Th2 cytokines both in the absence and presence of B7/CD28 costimulation, thus suggesting that ICOS costimulation can modulate cytokine secretion also in a CD28-independent manner. Levels of constitutive and inducible ICOS expression on human T cell subsets from peripheral blood were quantified in healthy donors and patients with MS. Constitutive expression of ICOS on T cells varies between 0.1% and 42.3%. There were no significant differences between both groups in the baseline expression or inducibility of ICOS on CD4 or CD8 T cells. ICOS expression could be demonstrated on CSF T lymphocytes in patients with acute MS relapses but was not elevated compared with peripheral blood. In essence we show that ICOS is upregulated on human T cells after stimulation and can modulate both Th1 and Th2 cytokine production in the absence and presence of B7-costimulation. In MS patients we demonstrate the functionality of the ICOS costimulatory pathway. Potential implications of ICOSL/ICOS interactions for MS immunopathogenesis are discussed.

Human muscle cells express the costimulatory molecule B7-H3, which modulates muscle-immune interactions.

OBJECTIVE: Interactions between the family of B7 ligands and their receptors are increasingly recognized as crucial for stimulation and/or inhibition of immune responses. The present study was undertaken to examine the expression and functional relevance of B7 homolog 3 (B7-H3), a novel B7 homolog attributed significant immunoregulatory functions, in human muscle cells in vivo and in vitro. METHODS: Thirty-five muscle biopsy specimens obtained from patients with polymyositis, dermatomyositis, inclusion body myositis, or noninflammatory myopathies and normal controls were analyzed by immunohistochemistry for B7-H3 expression. The expression of B7-H3 protein on primary human myoblasts and TE671 muscle rhabdomyosarcoma cells was studied by flow cytometry and Western blot analysis. B7-H3 small interfering RNA (siRNA) was used to study the impact of knockdown of B7-H3 on CD8+ cell-mediated lysis in skeletal muscle cells. RESULTS: B7-H3 was not detectable on normal muscle fibers. In contrast, its expression was markedly increased on muscle fibers from patients with inflammatory myopathies. Cell-surface staining was most prominent in the contact areas between muscle fibers and inflammatory cells. B7-H3 protein was detected on myoblasts cultured from control and myositis patient muscle tissue as well as in TE671 muscle rhabdomyosarcoma cells. Knockdown of B7-H3 by siRNA in TE671 cells enhanced CD8+ T cell-specific lysis, indicating a functional role of B7-H3 in the protection of skeletal muscle from immune-mediated lysis. CONCLUSION: Our results demonstrate that human muscle cells express B7-H3, a functional coinhibitory molecule of the B7 family. B7-H3 may play an important role in muscle-immune interactions, providing further evidence of the active role of muscle cells in local immunoregulatory processes.