Differential Expression of LLT1, SLAM Receptors CS1 and 2B4 and NCR Receptors NKp46 and NKp30 in Pediatric Acute Lymphoblastic Leukemia (ALL).

Acute lymphoblastic leukemia (ALL) represents the most common pediatric cancer. Most patients (85%) develop B-cell ALL; however, T-cell ALL tends to be more aggressive. We have previously identified 2B4 (SLAMF4), CS1 (SLAMF7) and LLT1 (CLEC2D) that can activate or inhibit NK cells upon the interaction with their ligands. In this study, the expression of 2B4, CS1, LLT1, NKp30 and NKp46 was determined. The expression profiles of these immune receptors were analyzed in the peripheral blood mononuclear cells of B-ALL and T-ALL subjects by single-cell RNA sequencing data obtained from the St. Jude PeCan data portal that showed increased expression of LLT1 in B-ALL and T-ALL subjects. Whole blood was collected from 42 pediatric ALL subjects at diagnosis and post-induction chemotherapy and 20 healthy subjects, and expression was determined at the mRNA and cell surface protein level. A significant increase in cell surface LLT1 expression in T cells, monocytes and NK cells was observed. Increased expression of CS1 and NKp46 was observed on monocytes of ALL subjects at diagnosis. A decrease of LLT1, 2B4, CS1 and NKp46 on T cells of ALL subjects was also observed post-induction chemotherapy. Furthermore, mRNA data showed altered expression of receptors in ALL subjects pre- and post-induction chemotherapy treatment. The results indicate that the differential expression of the receptors/ligand may play a role in the T-cell- and NK-cell-mediated immune surveillance of pediatric ALL.

SLAMF6 compartmentalization enhances T cell functions.

Signaling lymphocyte activation molecule family member 6 (SLAMF6) is a T cell co-receptor. Previously, we showed that SLAMF6 clustering was required for T cell activation. To better understand the relationship between SLAMF6 location and function and to evaluate the role of SLAMF6 as a therapeutic target, we investigated how its compartmentalization on the cell surface affects T cell functions. We used biochemical and co-culture assays to show that T cell activity is enhanced when SLAMF6 colocalizes with the CD3 complex. Mechanistically, co-immunoprecipitation analysis revealed the SLAMF6-interacting proteins to be those essential for signaling downstream of T cell receptor, suggesting the two receptors share downstream signaling pathways. Bispecific anti-CD3/SLAMF6 antibodies, designed to promote SLAMF6 clustering with CD3, enhanced T cell activation. Meanwhile, anti-CD45/SLAMF6 antibodies inhibited SLAMF6 clustering with T cell receptor, likely because of the steric hindrance, but nevertheless enhanced T cell activation. We conclude that SLAMF6 bispecific antibodies have a role in modulating T cell responses, and future work will evaluate the therapeutic potential in tumor models.

Extracellular vesicles derived from PPRV-infected cells enhance signaling lymphocyte activation molecular (SLAM) receptor expression and facilitate virus infection.

Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. PPRV is lymphotropic in nature and SLAM was identified as the primary receptor for PPRV and other Morbilliviruses. Many viruses have been demonstrated to engage extracellular vesicles (EVs) to facilitate their replication and pathogenesis. Here, we provide evidence that PPRV infection significantly induced the secretion levels of EVs from goat PBMC, and that PPRV-H protein carried in EVs can enhance SLAM receptor expression in the recipient cells via suppressing miR-218, a negative miRNA directly targeting SLAM gene. Importantly, EVs-mediated increased SLAM expression enhances PPRV infectivity as well as the expression of various cytokines related to SLAM signaling pathway in the recipient cells. Moreover, our data reveal that PPRV associate EVs rapidly entry into the recipient cells mainly through macropinocytosis pathway and cooperated with caveolin- and clathrin-mediated endocytosis. Taken together, our findings identify a new strategy by PPRV to enhance virus infection and escape innate immunity by engaging EVs pathway.

Identification of SLAMF1 as an immune-related key gene associated with rheumatoid arthritis and verified in mice collagen-induced arthritis model.

Background: Rheumatoid arthritis (RA) is the most common inflammatory arthropathy. Immune dysregulation was implicated in the pathogenesis of RA. Thus, the aim of the research was to determine the immune related biomarkers in RA. Methods: We downloaded the gene expression data of RA in GSE89408 and GSE45291 from Gene Expression Omnibus public database (GEO). Differentially expressed genes (DEGs) were identified between RA and control groups. Infiltrating immune cells related genes were obtained by ssGSEA and weighted gene co-expression network analysis (WGCNA). We performed functional enrichment analysis of differentially expressed immunity-related genes (DEIRGs) by "clusterProfiler" R package, key genes screening by protein-protein interaction (PPI) network of DEIRGs. And mice collagen-induced arthritis (CIA) model was employed to verify these key genes. Results: A total of 1,885 up-regulated and 1,899 down-regulated DEGs were identified in RA samples. The ssGSEA analysis showed that the infiltration of 25 cells was significantly different. 603 immune related genes were obtained by WGCNA, and 270 DEIRGs were obtained by taking the intersection of DEGs and immune related genes. Enrichment analyses indicated that DEIRGs were associated with immunity related biological processes. 4 candidate biomarkers (CCR7, KLRK1, TIGIT and SLAMF1) were identified from the PPI network of DEIRGs and literature research.In mice CIA model, the immunohistochemical stain showed SLAMF1 has a significantly high expression in diseased joints. And flow cytometry analysis shows the expression of SLAMF1 on CIA mice-derived CTL cells, Th, NK cells, NKT cells, classical dendritic cell (cDCs) and monocytes/macrophages was also significantly higher than corresponding immune cells from HC mice. Conclusion: Our study identified SMLAF1 as a key biomarker in the development and progression of RA, which might provide new insight for exploring the pathogenesis of RA.

SLAMF1 is expressed and secreted by hepatocytes and the liver in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent forms of chronic liver disease in the United States and worldwide. Nonalcoholic steatohepatitis (NASH), the most advanced form of NAFLD, is characterized by hepatic steatosis associated with inflammation and hepatocyte death. No treatments are currently available for NASH other than lifestyle changes, and the disease lacks specific biomarkers. The signaling lymphocytic activation molecule family 1 (SLAMF1) protein is a self-ligand receptor that plays a role in orchestrating an immune response to some pathogens and cancers. We found that livers from humans and mice with NASH showed a more prominent immunohistochemistry staining for SLAMF1 than non-NASH controls. Furthermore, SLAMF1 levels are significantly increased in NASH plasma samples from mice and humans compared with their respective controls. In mice, the levels of SLAMF1 correlated significantly with the severity of the NASH phenotype. To test whether SLAMF 1 is expressed by hepatocytes, HepG2 cells and primary murine hepatocytes were treated with palmitic acid (PA) to induce a state of lipotoxicity mimicking NASH. We found that PA treatments of HepG2 cells and primary hepatocytes lead to significant increases in SLAMF1 levels. The downregulation of SLAMF1 in HepG2 cells improved the cell viability and reduced cytotoxicity. The in vivo data using mouse and human NASH samples suggests a potential role for this protein as a noninvasive biomarker for NASH. The in vitro data suggest a role for SLAMF1 as a potential therapeutic target to prevent hepatocyte death in response to lipotoxicity.NEW & NOTEWORTHY This study identified for the first time SLAMF1 as a mediator of hepatocyte death in nonalcoholic fatty liver disease (NASH) and as a marker of NASH in humans. There are no pharmacological treatments available for NASH, and diagnostic tools are limited to invasive liver biopsies. Therefore, since SLAMF1 levels correlate with disease progression and SLAMF1 mediates cytotoxic effects, this protein can be used as a therapeutic target and a clinical biomarker of NASH.

Isolation of Murine Myeloid Progenitor Populations by CD34/CD150 Surface Markers.

Myeloid progenitors are intermediates between Hematopoietic Stem Cells (HSCs) and Myeloid effector progeny. In mouse bone marrow, they are part of the Lineage- cKit+ Sca1- (LK) compartment. To date, most researchers used CD34 and FcγR surface markers for the dissection of this compartment into various populations. Surprisingly, however, this approach does not provide distinct separation by fluorescence-activated cell sorting (FACS). In this study, we suggest using CD150 instead of FcγR. We re-analyzed published single-cell RNA-Seq data and found that CD34/CD150 provides better sub-populations separation, compared to the "classical" CD34/FcγR-based approach. We confirm our findings by independent FACS analysis. We demonstrate comparable differentiation potential of the newly-obtained LK sub-populations, like previous "classical" ones. Therefore, we suggest the CD34/CD150 gating strategy, utilizing commonly-used surface markers, as a robust and reproducible separation of the LK compartment into distinct sub-populations.

SLAMF1 Is Dispensable for Vaccine-Induced T Cell Development but Required for Resistance to Fungal Infection.

Homotypic signaling lymphocyte activation molecule (SLAM) receptor-ligand cell surface interactions between myeloid and lymphoid cells regulate innate and adaptive immune responses. In this article, we report that SLAMF1 is indispensable for host resistance to primary and vaccine-induced protection against fungal infection. Because vaccine immunity is dependent on cell-mediated immunity, we investigated the development of Ag-specific T cells. We studied the T cell-intrinsic and -extrinsic role of SLAMF1. We generated SLAMF1-/- TCR transgenic mice and analyzed the responses of adoptively transferred T cells. We also tracked endogenous Ag-specific T cells by using a tetramer. Intrinsic and extrinsic SLAMF1 signaling was dispensable for the development of antifungal Th1 and Th17 cells, which are requisite for the acquisition of vaccine-induced immunity. Despite intact T cell development, vaccinated SLAMF1-/- mice failed to control fungal infection. Failed accumulation of Ag-specific T cells in the lung on infection of vaccinated mice was due to uncontrolled early infection and inflammation, revealing a role for SLAMF1 in innate host immunity.

SLAMF1/CD150 expression and topology in prostate and breast cancer cell lines.

BACKGROUND: SLAMF1/CD150 receptor is aberrantly expressed in malignant hematopoietic cells compared to ubiquitous expression in their normal analogues. However, the data about CD150 expression and function outside the hematopoietic system are limited. The aim of this pilot study was to examine the profile of mRNA expression of CD150 isoforms and the protein topology in highly and low malignant breast (BC) and prostate cancer (PC) cell lines. MATERIALS AND METHODS: The study was performed on BC T47D, MDA-MB-231, ВСС/Р and BC/ML cell lines and PC LNCap, Du-145 and PC-3 cell lines. The quantitative polymerase chain reaction was applied for study of CD150 isoforms mRNA expression and flow cytometry was used for determination of protein localization. RESULTS: Analyzed BC cell lines did not express CD150 on the cell surface membrane (csCD150-), but more than 45% of cells were positive for CD150 cytoplasmic reaction (cyCD150+). The cyCD150 expression level in T47D cells of luminal BC subtype was higher than in basal BC cell lines MDA-MB-231, ВСС/Р and BC/ML. The PC cell lines expressed CD150 both on the cell surface and in cytoplasm. The highest number of csCD150+ and cyCD150+ cells was revealed in less aggressive androgen responsive, non-metastatic LNCap cell line. All studied BC and PC cell lines expressed mRNA of canonical transmembrane mCD150 and novel nCD150 isoforms but with different pattern of prevalence. Soluble CD150 isoform was revealed at the low level only in BCC/P BC cell line and LNCap, PC-3 PC cell lines. CONCLUSIONS: We have shown that BC and PC cell lines differentially expressed multifunctional receptor CD150 at the mRNA and protein levels that may indicate its association with the degree of their malignancy.

Determination of the SLAMF1 self-association affinity constant with sedimentation velocity ultracentrifugation.

Signaling lymphocytic activating molecule family member 1 (SLAMF1 or CD150) is a cell surface glycoprotein expressed on various immune populations, regulating cell-cell interactions, activation, differentiation, and inflammatory responses and has been suggested as a potential target for inflammatory diseases. Signaling is believed to be mediated by high-affinity homophilic interactions; the recombinant soluble form of SLAMF1 has optimal activity in the range of 20 µg/mL. This contradicts with a rather weak homo-dimerization binding constant (KD) value reported previously; however, the analytical approach and data analysis suffered from various technical limitations at the time and therefore warrants re-examination. To address this apparent discrepancy, we determined the KD of soluble SLAMF1 using sedimentation velocity analytical ultracentrifuge (SV-AUC). A globally fitted monomer-dimer model properly explains the data from a wide concentration range obtained with both UV and fluorescence detection systems. The analysis suggests the dimerization KD value for human SLAMF1 is 0.48 µM. Additionally, our data show that SLAMF1 self-association is not driven by non-specific binding to glycans supporting the view of specific protein-protein interaction. We anticipate antibody biotherapeutics capable of modulating the biological consequences of SLAMF1 interactions will be readily identified.

Single-cell transcriptomic landscape reveals tumor specific innate lymphoid cells associated with colorectal cancer progression.

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes differing from conventional T lymphocytes in having no antigen-specific receptors. ILCs include natural killer (NK) cells, helper-like ILC1s, ILC2s, and ILC3s, and lymphoid tissue-inducer (LTi) cells. Tumor ILCs are frequently found in various cancers, but their roles in cancer immunity and immunotherapy remain largely unclear. We report here the single-cell characterization of blood and gut helper-like ILC subsets in healthy conditions and in colorectal cancer (CRC). The healthy gut contains ILC1s, ILC3s, and ILC3/NKs, but no ILC2s. Additional tumor-specific ILC1-like and ILC2 subsets were identified in CRC patients. Signaling lymphocytic activation molecule family member 1 (SLAMF1) was found to be selectively expressed on tumor-specific ILCs, and higher levels of SLAMF1+ ILCs were observed in the blood of CRC patients. The SLAMF1-high group of CRC patients had a significantly higher survival rate than the SLAMF1-low group, suggesting that SLAMF1 is an anti-tumor biomarker in CRC.

Myeloid-Derived Suppressor Cells in Trypanosoma cruzi Infection.

Myeloid-derived suppressor cells (MDSCs) are immature heterogeneous myeloid cells that expand in pathologic conditions as cancer, trauma, and infection. Although characterization of MDSCs is continuously revisited, the best feature is their suppressor activity. There are many markers for MDSC identification, it is distinctive that they express inducible nitric oxide synthase (iNOS) and arginase 1, which can mediate immune suppression. MDSCs can have a medullary origin as a result of emergency myelopoiesis, but also can have an extramedullary origin. Early studies on Trypanosoma cruzi infection showed severe immunosuppression, and several mechanisms involving parasite antigens and host cell mediators were described as inhibition of IL-2 and IL-2R. Another mechanism of immunosuppression involving tumor necrosis factor/interferon γ-dependent nitric oxide production by inducible nitric oxide synthase was also described. Moreover, other studies showed that nitric oxide was produced by CD11b+ Gr-1+ MDSCs in the spleen, and later iNOS and arginase 1 expressed in CD11b+Ly6C+Ly6Glo monocytic MDSC were found in spleen and heart of T. cruzi infected mice that suppressed T cell proliferation. Uncontrolled expansion of monocytic MDSCs leads to L-arginine depletion which hinders nitric oxide production leading to death. Supplement of L-arginine partially reverts L-arginine depletion and survival, suggesting that L-arginine could be administered along with anti-parasitical drugs. On the other hand, pharmacological inhibition of MDSCs leads to death in mice, suggesting that some expansion of MDSCs is needed for an efficient immune response. The role of signaling molecules mediating immune suppression as reactive oxygen species, reactive nitrogen species, as well as prostaglandin E2, characteristics of MDSCs, in T. cruzi infection is not fully understood. We review and discuss the role of these reactive species mediators produced by MDSCs. Finally, we discuss the latest results that link the SLAMF1 immune receptor with reactive oxygen species. Interaction of the parasite with the SLAMF1 modulates parasite virulence through myeloid cell infectivity and reactive oxygen species production. We discuss the possible strategies for targeting MDSCs and SLAMF1 receptor in acute Trypanosoma cruzi infection in mice, to evaluate a possible translational application in human acute infections.

Systemic Inflammation in Preclinical Ulcerative Colitis.

BACKGROUND & AIMS: Preclinical ulcerative colitis is poorly defined. We aimed to characterize the preclinical systemic inflammation in ulcerative colitis, using a comprehensive set of proteins. METHODS: We obtained plasma samples biobanked from individuals who developed ulcerative colitis later in life (n = 72) and matched healthy controls (n = 140) within a population-based screening cohort. We measured 92 proteins related to inflammation using a proximity extension assay. The biologic relevance of these findings was validated in an inception cohort of patients with ulcerative colitis (n = 101) and healthy controls (n = 50). To examine the influence of genetic and environmental factors on these markers, a cohort of healthy twin siblings of patients with ulcerative colitis (n = 41) and matched healthy controls (n = 37) were explored. RESULTS: Six proteins (MMP10, CXCL9, CCL11, SLAMF1, CXCL11 and MCP-1) were up-regulated (P < .05) in preclinical ulcerative colitis compared with controls based on both univariate and multivariable models. Ingenuity Pathway Analyses identified several potential key regulators, including interleukin-1ß, tumor necrosis factor, interferon-gamma, oncostatin M, nuclear factor-κB, interleukin-6, and interleukin-4. For validation, we built a multivariable model to predict disease in the inception cohort. The model discriminated treatment-naïve patients with ulcerative colitis from controls with leave-one-out cross-validation (area under the curve = 0.92). Consistently, MMP10, CXCL9, CXCL11, and MCP-1, but not CCL11 and SLAMF1, were significantly up-regulated among the healthy twin siblings, even though their relative abundances seemed higher in incident ulcerative colitis. CONCLUSIONS: A set of inflammatory proteins are up-regulated several years before a diagnosis of ulcerative colitis. These proteins were highly predictive of an ulcerative colitis diagnosis, and some seemed to be up-regulated already at exposure to genetic and environmental risk factors.

Hematopoietic stem cells retain functional potential and molecular identity in hibernation cultures.

Advances in the isolation and gene expression profiling of single hematopoietic stem cells (HSCs) have permitted in-depth resolution of their molecular program. However, long-term HSCs can only be isolated to near purity from adult mouse bone marrow, thereby precluding studies of their molecular program in different physiological states. Here, we describe a powerful 7-day HSC hibernation culture system that maintains HSCs as single cells in the absence of a physical niche. Single hibernating HSCs retain full functional potential compared with freshly isolated HSCs with respect to colony-forming capacity and transplantation into primary and secondary recipients. Comparison of hibernating HSC molecular profiles to their freshly isolated counterparts showed a striking degree of molecular similarity, further resolving the core molecular machinery of HSC self-renewal while also identifying key factors that are potentially dispensable for HSC function, including members of the AP1 complex (Jun, Fos, and Ncor2), Sult1a1 and Cish. Finally, we provide evidence that hibernating mouse HSCs can be transduced without compromising their self-renewal activity and demonstrate the applicability of hibernation cultures to human HSCs.

Replication competence of canine distemper virus in cell lines expressing signaling lymphocyte activation molecule (SLAM) of goat, sheep and dog origin.

Cellular receptors play an important role in entry and cell to cell spread of morbillivirus infections. The cells expressing SLAM and Nectin-4 have been used for successful and efficient isolation of canine distemper virus (CDV) in high titre. There are several methods for generation of cells expressing receptor molecules. Here, we have used a comparatively cheaper and easily available method, pcDNA 3.1 (+) for engineering Vero cells to express SLAM gene of goat, sheep and dog origin (Vero/Goat/SLAM (VGS), Vero/Sheep/SLAM (VSS) and Vero/Dog/SLAM (VDS), respectively). The generated cell lines were then compared to test their efficacy to support CDV replication. CDV could be grown in high titre in the cells expressing SLAM and a difference of log two could be recorded in virus titre between VDS and native Vero cells. Also, CDV could be grown in a higher titre in VDS as compared to VGS and VSS. The finding of this study supports the preferential use of SLAM expressing cells over the native Vero cells by CDV. Further, the higher titre of CDV in cells expressing dog-SLAM as compared to the cells expressing SLAM of non-CDV hosts (i.e. goat and sheep) points towards the preferential use of dog SLAM by the CDV and may be a plausible reason for differential susceptibility of small ruminants and Canines to CDV.

SLAM/SAP Decreased Follicular Regulatory T Cells in Patients with Graves' Disease.

Signaling lymphocytic activation molecule (SLAM) and SLAM-associated protein (SAP) play important role in inflammatory and autoimmune diseases. Our study is aimed at detecting the expression of SLAM and SAP in patients with Graves' disease (GD) and analyzing the effect of SLAM/SAP on circulating blood CD4+CXCR5+Foxp3+ follicular regulatory T (Tfr) cells. The level of SAP in CD4+CXCR5+ T cells and the level of SLAM on CD19+ B cells were significantly increased in the patients with GD, but no significant difference in the level of SLAM on CD4+CXCR5+ T cells was observed between the patients with GD and the healthy controls. A decrease in the percentage of Foxp3+ cells in CD4+CXCR5+ T cells was observed following anti-SLAM treatment, but the percentages of IFN-γ + cells, IL-4+ cells, and IL-17+ cells showed no obvious differences. The proportion of circulating Tfr cells was decreased in the patients with GD, and the proportion of circulating Tfr cells had a negative correlation with the level of SAP in CD4+CXCR5+ T cells and the levels of autoantibodies in the serum of the patients with GD. Our results suggested that the SLAM/SAP signaling pathway is involved in the decrease of circulating Tfr cells in Graves' disease.

Mutated Measles Virus Matrix and Fusion Protein Influence Viral Titer In Vitro and Neuro-Invasion in Lewis Rat Brain Slice Cultures.

Measles virus (MV) can cause severe acute diseases as well as long-lasting clinical deteriorations due to viral-induced immunosuppression and neuronal manifestation. How the virus enters the brain and manages to persist in neuronal tissue is not fully understood. Various mutations in the viral genes were found in MV strains isolated from patient brains. In this study, reverse genetics was used to introduce mutations in the fusion, matrix and polymerase genes of MV. The generated virus clones were characterized in cell culture and used to infect rat brain slice cultures. A mutation in the carboxy-terminal domain of the matrix protein (R293Q) promoted the production of progeny virions. This effect was observed in Vero cells irrespective of the expression of the signaling lymphocyte activation molecule (SLAM). Furthermore, a mutation in the fusion protein (I225M) induced syncytia formation on Vero cells in the absence of SLAM and promoted viral spread throughout the rat brain slices. In this study, a solid ex vivo model was established to elucidate the MV mutations contributing to neural manifestation.

CD150 and CD180 are negative regulators of IL-10 expression and secretion in chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) is a strikingly heterogeneous disease both at the molecular level and clinical disease course. The malignant B cells obtain key proliferation and survival signals within lymph nodes or bone marrow. Moreover, CLL B cells and tumor microenvironment dynamically co-evolve organizing inflammatory and immunosuppressive microenvironment by direct contact with surrounding cells and secretion of cytokines, growth factors, or extracellular vesicles. Finding a way to weaken obtaining by malignant B cells supportive signals may improve CLL outcome and optimize treatment strategies. The aim of this study was to evaluate whether CD150 and CD180 cell surface receptors could be involved in the regulation of CLL B cells-derived cytokines (CCL3, CCL4, IL-6, and IL-10). The study was performed on malignant B cells isolated from peripheral blood of primary CLL patients. Flow cytometry, qPCR, ELISA, western blot, ex vivo cell surface ligation assay, ex vivo drug sensitivity assay, and cell viability assay were used in this study. The CCL3, CCL4, IL-6, and IL-10 mRNA expression levels were heterogeneously presented among studied CLL cases. The elevated CCL3/CCL4 and decreased IL-6/IL-10 expression level are specific features of CLL B cells with positive CD150 and CD180 expression status. Ligation of CD150 and CD180 receptors did not affect CCL3/CCL4 mRNA expression level in CLL B cells, while IL-6 and IL-10 were significantly decreased. After malignant B cells stimulation via CD150 and CD180 observed reduced IL-10 but not IL-6 levels in culture supernatants. Ligation of CD150 and CD180 was linked to the classical NF-κB pathway via regulation of phosphorylation level of NF-κB inhibitor IκBα. We found several correlations between basal mRNA expression levels of CCL3, CCL4, IL-6, and IL-10 in CLL B cells and their sensitivity to chemotherapeutic drugs ex vivo. High CCL3/CCL4 and low IL-10 mRNA expression levels are associated with malignant B cells' sensitivity to BEN, while high IL-6 levels are a sign of CLL B cells' resistance to FC. The revealed involvement of CD150 and CD180 in cytokine regulation expands our knowledge of the role of CD150 and CD180 in the pathobiology of CLL and their contribution to a favorable clinical outcome. Determining the cytokines expression levels together with CD150 and CD180 expression status may help to predict the responsiveness of CLL B cells to chemotherapeutic drugs and optimize personalized chemotherapy scheme.

Application of error-prone PCR to functionally probe the morbillivirus Haemagglutinin protein.

The enveloped morbilliviruses utilise conserved proteinaceous receptors to enter host cells: SLAMF1 or Nectin-4. Receptor binding is initiated by the viral attachment protein Haemagglutinin (H), with the viral Fusion protein (F) driving membrane fusion. Crystal structures of the prototypic morbillivirus measles virus H with either SLAMF1 or Nectin-4 are available and have served as the basis for improved understanding of this interaction. However, whether these interactions remain conserved throughout the morbillivirus genus requires further characterisation. Using a random mutagenesis approach, based on error-prone PCR, we targeted the putative receptor binding site for SLAMF1 interaction on peste des petits ruminants virus (PPRV) H, identifying mutations that inhibited virus-induced cell-cell fusion. These data, combined with structural modelling of the PPRV H and ovine SLAMF1 interaction, indicate this region is functionally conserved across all morbilliviruses. Error-prone PCR provides a powerful tool for functionally characterising functional domains within viral proteins.

In chronic lymphocytic leukaemia, SLAMF1 deregulation is associated with genomic complexity and independently predicts a worse outcome.

In a series of 349 patients with chronic lymphocytic leukaemia (CLL), we found lower levels of signalling lymphocytic activation molecule family member 1 (SLAMF1) expression in cases with highly complex karyotypes, as defined by the presence of five or more chromosomal abnormalities (CK5; P < 0·001) and with major chromosomal structural abnormalities (P < 0·001). SLAMF1 downregulation was significantly associated with advanced Binet Stage (P = 0·001), CD38 positivity (P < 0·001), high ß2 -microglobulin levels (P < 0·001), immunoglobulin heavy chain variable region gene (IGHV) unmutated status (P < 0·001), 11q deletion (P < 0·001), tumour protein p53 (TP53) disruption (P = 0·011) and higher risk CLL International Prognostic Index categories (P < 0·001). Multivariate analysis showed that downregulated SLAMF1 levels had independent negative prognostic impact on time-to-first treatment (P < 0·001) and overall survival (P < 0·001).