High dimensional analysis reveals distinct NK cell subsets but conserved response to stimulation in umbilical cord blood and adult peripheral blood

Growing interest surrounds adoptive cellular therapies utilizing Natural Killer (NK) cells, which can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding NK cell receptor expression and diversity in such cellular sources will guide future therapeutic designs. We used a 20-color flow cytometry panel to compare unstimulated and cytokine-activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD-1, TIGIT, and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and dimensionality reduction analyses revealed enrichment in CD56^neg as well as mature NKp46^neg and CD56⁺CD16⁺ NK cell populations in UCB whereas CD57⁺ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following stimulation with IL-12, IL-15, and IL-18. Cytokine stimulation was associated with the downregulation of TIGIT and CD16 on multiple NK cell subsets in UCB and APB. Among UCB CD16⁻ NK cell populations, TIGIT⁺ NK cells produced more IFN-γ than their TIGIT⁻ counterparts. Our data demonstrate higher immune checkpoint expression on UCB NK cells compared to APB. However, the expression of TIGIT immune checkpoint is not indicative of NK cell exhaustion.     

Immunohistochemical analysis of ageing human B and T cell populations reveals an age-related decline of CD8 T cells in spleen but not gut-associated lymphoid tissue (GALT)

It is thought that senescence of the immune system is responsible, at least in part, for many health problems associated with ageing. Previous studies on changes in lymphocyte composition have used flow cytometry to study peripheral blood lymphocytes (PBL's), or cells isolated from rodent tissue, and have yielded conflicting results. We have used immunohistochemistry to determine whether the B and T cells in human tissue from spleen and gut are affected by age. Areas of germinal centre, mantle zone and marginal zone of B cell follicles were measured. In addition, CD4 and CD8 T cells in T cell areas and in B cell follicles were counted. We observed a striking age-related decrease in the proportion of CD8+ T cells in the T cell zones of the spleen. This decrease was not apparent in the T cell population that occupies splenic B cell areas, or in GALT. Further differences, in CD4+ cells, were seen between T cell populations in the T cell zones and those in B cell areas. These findings highlight differences between lymphocyte populations in different lymphoid tissues, and different compartments within each tissue, which may be of importance in future studies of the ageing immune system.     

Quantitative analysis of cell-free Epstein-Barr virus DNA in the plasma of patients with peripheral T-cell and NK-cell lymphomas and peripheral T-cell proliferative diseases

BACKGROUND: The level of circulating EBV DNA is a prognostic marker in patients with some EBV-associated malignant diseases. OBJECTIVES: To investigate the presence and nature of Epstein-Barr virus (EBV) DNA in the plasma and to evaluate the correlation of plasma concentrations of EBV DNA with the EBV genomic status in peripheral blood T-cells and neoplastic cells and with the clinical outcome of patients with peripheral T-cell and NK-cell lymphomas (PTCL) and peripheral T-cell proliferative diseases (PTPD). STUDY DESIGN: EBV DNA in the plasma of 45 patients and 45 controls was measured using real-time PCR. The presence of the EBV genome in the isolated peripheral blood lymphocytes (CD3+ and CD3- cells) was analysed by PCR. Detection of EBV-encoded early RNA (EBER) in corresponding tumor tissues was carried out using in situ hybridization. DNase I digestion was applied to plasma samples to detect naked EBV DNA. RESULTS: Cell-free EBV DNA was detected in 32/38 (84%) of PTCL patients and 5/7 (71%) of PTPD patients, but not in the controls. Patients with EBV genome in peripheral blood CD3+ cells and EBV genome (EBER) in the tumor cells, compared to those without these findings, had significantly higher plasma EBV DNA levels. The majority of circulating EBV DNA molecules was naked form. The plasma EBV DNA levels were not related to survival. CONCLUSIONS: The concentration of EBV DNA in the plasma was not a prognostic marker in PTCL and PTPD patients.     

Hepatic cytotoxic T-cell infiltrates in patients with peripheral T-cell proliferative diseases/lymphomas: clinicopathological and molecular analysis

Seventy patients with various types of peripheral T-cell proliferative disease/lymphoma who manifested with prolonged fever, weight loss, anemia, lymphadenopathy, hepatosplenomegaly and elevated serum levels of alkaline phosphatase and/or lactate dehydrogenase were evaluated. Histopathological examination of the livers revealed T-cell infiltration into the hepatic sinusoids and portal tracts. The morphology of the infiltrated T cells varied from mature small lymphocytes to malignant lymphoid cells. The liver pathology was classified into four groups on the basis of cellular atypia. Group A and group B showed mature lymphoid cell infiltration; however, only group B had multiple large areas of hepatocellular necrosis. Group C showed atypical lymphoid cell infiltration and in group D malignant lymphoid cell infiltrates were demonstrated. The majority of the antigenic phenotypes of these T-cell infiltrates were CD3+, CD4-, CD8+, CD20-, CD45RO+, CD56-, CD57-, TIA-1+ and betaF1-. Epstein-Barr virus RNA in the nuclei of the infiltrated T cells was recorded in 38.6% of the patients and was more common in groups C and D. Patients in groups B, C and D had a very poor prognosis, median survival was only 1 month, whereas median survival in group A patients was 36 months. Chemotherapy was not effective in improving survival. Monoclonal band/s of T-cell receptors (TCR) beta and/or gamma gene rearrangements were detected in 88.6% of patients, and DNA-sequence analysis showed high identity to the human TCR germline gene.     

Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast DEAD-box RNA helicases Ded1 and eIF4A

DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5′ end or subsequent scanning of the 5′ UTR, but whether they perform unique or overlapping functions in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in budding yeast Saccharomyces cerevisiae by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A variant encoded by tif1-A79V (in a strain lacking the ortholog TIF2) yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5′ UTR length and propensity for secondary structure, implicating Ded1 in scanning through structured 5′ UTRs. Reporter assays confirmed that cap-distal stem–loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs shows a heightened requirement for eIF4A, dependence on eIF4A is correlated with requirements for Ded1 and 5′ UTR features characteristic of Ded1-dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5′ UTRs, and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs.In translation of most eukaryotic mRNAs, the initiation codon is selected by the scanning mechanism, wherein a 43S preinitiation complex (PIC) containing methionyl initiator tRNA (tRNA_i) attaches to the capped 5′ end of mRNA and scans the 5′ UTR for an AUG codon. PIC attachment to the mRNA is stimulated by eIF4F, comprised of cap-binding protein eIF4E, DExD/H-box helicase eIF4A, and scaffold subunit eIF4G, which stimulates eIF4A''s ATP-dependent RNA helicase activity (Rogers et al. 2002; Schutz et al. 2008). Activation of eIF4A is expected to generate single-stranded RNA to facilitate ribosome binding, and mRNAs with more structured 5′ UTRs display a greater requirement for eIF4A/eIF4F (Blum et al. 1992; Svitkin et al. 2001; Pestova and Kolupaeva 2002; Mitchell et al. 2010). Interaction of eIF4G with components of the 43S complex, eIF3, eIF5, or eIF1, is thought to facilitate recruitment of the PIC to cap-bound eIF4F (for review, see Hinnebusch 2014).As might be expected, eIF4F and ATP hydrolysis are dispensable in reconstituted systems for assembly of 48S PICs at the AUG codon on synthetic mRNAs with unstructured 5′ UTRs but are required with native mRNAs (Pestova et al. 1998; Pestova and Kolupaeva 2002; Mitchell et al. 2010). Moreover, inactivating eIF4A in mammalian extracts decreased translation of reporter mRNAs with cap-proximal structures by impairing eIF4F binding to mRNA and reducing 43S-mRNA attachment. However, translation of mRNAs devoid of structures was also reduced (Svitkin et al. 2001); similarly, translation of reporter mRNAs with only 8-nt-long 5′ UTRs was impaired in yeast extracts containing defective eIF4A variants (Blum et al. 1992). Thus, eIF4A helicase activity might be needed to dissociate RNA-RNA interactions other than stable stem–loops (SLs) or resolve mRNA-protein interactions in the 5′ UTR for efficient PIC attachment. Indeed, eIF4A is essential in yeast even though the 5′ UTRs of most yeast mRNAs are relatively short and devoid of highly stable, well-defined SLs (Ringner and Krogh 2005; Lawless et al. 2009; Kertesz et al. 2010; Wan et al. 2012; Rouskin et al. 2014).In mammalian reconstituted systems, eIF4A can facilitate scanning through a SL of moderate stability distal from the cap (Pestova et al. 1996). Other DExD/H helicases, DHX29 and yeast Ded1 (the ortholog of mammalian DDX3X), were required with SLs of higher stability (Pisareva et al. 2008; Abaeva et al. 2011), although none (DHX29, DDX3X, or yeast Ded1) could substitute for eIF4F for 48S PIC assembly on beta-globin mRNA. Thus, it appeared that DHX29 and DDX3X/Ded1 specifically stimulate scanning through secondary structures, whereas eIF4F enhances both PIC attachment and scanning but is less effective in resolving strong SLs (Abaeva et al. 2011). In yeast cells, a ded1 mutation had a stronger effect than an eIF4A mutation (tif1-A79V in a strain lacking TIF2) on translation of a reporter harboring a long 5′ UTR (Berthelot et al. 2004), and a ded1 mutation impaired scanning through a SL located distal to the 5′ cap (Chiu et al. 2010). On the other hand, inactivation of Ded1 in yeast (Chuang et al. 1997) or knock-down of DHX29 in mammalian cells (Parsyan et al. 2009) impairs bulk translation, suggesting that DHX29 and Ded1 are not dedicated solely to mRNAs with strong, cap-distal secondary structures that impede scanning. The role of mammalian DDX3X in translation is not well understood (Lai et al. 2008; Lee et al. 2008), although recent work indicates that it resolves cap-proximal structures, possibly to facilitate eIF4F binding to the mRNA (Soto-Rifo et al. 2012), but it also can promote joining of 60S subunits to 48S PICs independent of helicase activity (Geissler et al. 2012).DDX3X and Ded1 can function as translational repressors when present at abnormally high concentrations. Overexpressed DDX3X functioned as an inhibitory eIF4E-binding protein that competes with eIF4G for eIF4F assembly (Shih et al. 2008). Ded1 overexpression impairs yeast cell growth and induces assembly of stress granules; and excess Ded1 inhibits translation in cell extracts at the step of 48S PIC assembly. These inhibitory activities do not require Ded1 helicase activity. It was suggested that Ded1 interacts with eIF4G to assemble messenger ribonucleoprotein particles (mRNPs) that accumulate in stress granules and subsequently activates repressed mRNPs under favorable growth conditions through its helicase activity (Hilliker et al. 2011). Whereas Ded1 function in stimulating bulk translation is well established (Chuang et al. 1997; de la Cruz et al. 1997), it has not been shown that inactivating yeast Ded1 de-represses translation of mRNAs in unfavorable growth conditions. Clearly, additional work is needed to define the in vivo functions of Ded1 and DDX3X.In this study, we sought to determine whether eIF4A and Ded1 perform largely overlapping functions or, rather, have distinct activities in stimulating translation initiation in living yeast cells. To this end, we conducted genome-wide ribosomal profiling (Ingolia et al. 2009) of Saccharomyces cerevisiae mutants defective for one or the other helicase under conditions where bulk translation is reduced, but not abolished, and identified mRNAs whose translational efficiencies are impaired to the greatest extent in each mutant.     

Combined analysis of intracellular signalling and immunophenotype of human peripheral blood basophils by flow cytometry: a proof of concept

BACKGROUND: The signal transduction pathways and control mechanisms involved in IgE-mediated basophil activation remain incompletely understood. OBJECTIVES: To investigate whether basophilic intracellular signal transduction and immunophenotype can be analysed simultaneously by flow cytometry. METHODS: Basophils in whole blood were stimulated with anti-IgE and latex antigen at various concentrations and during different time courses. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) as a representative of the intracellular signal transduction pathway and surface expression of CD63 was assessed simultaneously flow cytometrically. The effect of pre-incubation with IL-3 was assessed. RESULTS: Stimulation of the basophils with anti-IgE and allergen induces a rapid phosphorylation of p38 MAPK that peaks between 1 and 5 min and returns to baseline levels after 60 min. In contrast, CD63 up-regulation demonstrates a maximal but more continuous expression that peaks approximately 5 min later than phosphorylation of p38 MAPK. Specific inhibition of p38 MAPK reduced or almost completely abrogated up-regulation of CD63. Pre-incubation of the basophils with IL-3 produces a rapid p38 MAPK phosphorylation over basal levels, but this was weaker and shorter than for anti-IgE stimulation. Pre-incubation of the basophils with IL-3 did not potentiate anti-IgE-induced phosphorylation of p38 MAPK and did affect spontaneous or IgE-mediated CD63 up-regulation. CONCLUSIONS: This study provides the proof that the flow cytometer allows an integrated analysis of basophilic intracellular signalling and immunophenotyping. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy. CAPSULE SUMMARY: This study is the first to provide evidence for a combined analysis of basophilic intracellular signalling and immunophenotyping by flow cytometry. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy.     

Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species

The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1–7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340–350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819–825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1–7) for all strains analyzed so far (n=29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3–7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogenous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively. The latter group has not been described previously and is specifically recognized by an OspA-specific monoclonal antibody L32 1F7.     

Genetic control of the immune response to myoglobin. VI. Inter-site influences in T-lymphocyte proliferative response from analysis of cross-reactions of ten myoglobins in terms of substitutions in the antigenic sites and in environmental residues of the sites

The recent determination of the antigenic structure of sperm-whale Mb⁴ and the findings that the same five antigenic sites are recognized by antibodies raised against this protein in several host species, including mouse, enabled us to focus attention on the molecular factors underlying immune recognition and responses. We have also shown that the sites recognized by mouse B-cells (antibodies) are also recognized by T-cells and that the responses to the sites are each under separate genetic control. Furthermore, we recently demonstrated that the binding capacity of an antigenic site in Mb variants is Influenced by substitutions in the antigenic sites and in residues close (within 7.0 Å) to the sites. The genetic exclusion of a response to some antigenic sites in given inbred mouse strains and the knowledge of the sites that suffer decreases or total losses in binding activity In several Mb variants affords a unique system of reduced complexity to study the molecular requirements for T-cell recognition of the Mb antigenic sites. In the present work the cross-reactions of ten myoglobins by proliferation of sperm-whale Mb-primed mouse T-cells were analyzed in terms of substitutions in the sites and in environmental residues of the sites. Help between sites 1 and 2 and between one or both of these and site 3 was observed in several mouse strains. A suppresslve Influence by site 5 on the overall proliferative response to Mb was observed in two strains (D2.GD and A.TTR4). It was concluded that the overall response to Mb is regulated by Inter-site Influences which can either be of a cooperative (help) nature or of a suppresslve nature.     

Limiting dilution analysis of proliferative T cell responses to mycobacterial 65-kDa heat-shock protein fails to show significant frequency differences between synovial fluid and peripheral blood of patients with rheumatoid arthritis.

Recent evidence has pointed to the mycobacterial 65-kDa heat-shock protein (hsp 65) as an antigen that may be important in the pathogenesis of rheumatoid arthritis (RA). Using limiting dilution analysis the frequency of purified protein derivative of tuberculin (PPD) and hsp 65-responsive T cells was measured in paired peripheral blood and synovial fluid samples of patients with RA. There was no increase in the anti-PPD or anti-hsp 65 frequency in synovial fluid compared with peripheral blood. In addition, no difference was found between peripheral blood of RA patients and healthy controls. These results do not support the idea of an important pathogenic role of T cells responding to hsp 65, or a cross-reacting antigen, in RA.     

β2 -Microglobulin-deficient NK cells show increased sensitivity to MHC class I-mediated inhibition,but self tolerance does not depend upon target cell expression of H-2Kb and Db heavy chains

Mice lacking β2 -microglobulin (β₂ m− mice) express greatly reduced levels of MHC class I molecules, and cells from β₂ m− mice are therefore highly sensitive NK cells. However, NK cells from β₂ m− mice fail to kill β₂ m− normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of β₂ m− NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to β₂ m− NK cells, we made the following observations: First, β₂ m− NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to β₂ m− concanavalin A (Con A)-activated T cell blasts. Secondly, β₂ m− NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than β₂ m+ NK cells. Third β₂ m− NK cells were not only tolerant to β₂ m− Con A blasts but also to Con A blasts from H-2Kb − /Db − double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of β₂ m− NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in β₂ m− mice, which may instead operate through mechanisms involving triggering pathways.     

Southern hybridisation analysis of HBV DNA in peripheral blood leucocytes and of different cell types: Changes during the natural history and with interferon-α therapy in patients with hepatitis B virus infection

Hepatitis B virus (HBV) DNA was evaluated in peripheral blood mononuclear cells (PBMC) from 50 individuals utilising Southern hybridisation analysis. HBV DNA sequences were detected in PBMC from 16/29 (55 percent) of chronic hepatitis B virus (HBV) carriers with serum HBeAg and HBV DNA, compared with 1/8 (13%) of carriers with anti-HBe and HBV DNA negative (P = NS). Two of 7 patients with previous HBV infection and chronic liver disease had detectable HBV DNA in PBMC. Of the 19 patients with HBV DNA in PBMC, 18 had high molecular weight species. In addition, five of these had free, monomeric HBV DNA and six patients had low molecular weight bands. For nine of the above patients, total peripheral blood leucocytes were separated into PBMC and polymorphonuclear cells. Four had HBV DNA in PBMC only, two only in polymorphonuclear cells and three in both types of cell. Eleven patients with chronic HBV infection were studied at monthly intervals for 6 months. Six were untreated and five received IFN-alpha. Three patients who responded to IFN-a had HBV DNA present in PBMC before therapy, and two became negative. Two of 3 untreated patients had intermittent HBV DNA in PBMC and the other remained persistently negative. Of the patients positive on more than one occasion, the pattern of HBV DNA was similar. Peripheral blood leucocytes often contain multimers of free HBV DNA, more commonly in patients with serum HBeAg and HBV DNA and may occur even in the absence of serum HBsAg. These findings have implications for recurrence of disease after hepatic transplantation. HBV DNA in PBMC is detected intermittently over time and IFN-α therapy may be associated with eradication. © 1994 Wiley-Liss, Inc.     

Chemokine and chemokine receptor analysis reveals elevated interferon-inducible protein-10 (IP)-10/CXCL10 levels and increased number of CCR5+ and CXCR3+ CD4 T cells in synovial fluid of patients with enthesitis-related arthritis (ERA)

Chemokines and chemokine receptors play a major role in homing of cells to the site of inflammation. Enthesitis-related arthritis (ERA) is a chronic inflammatory arthritis and no data are available on chemokines and their receptors in ERA. Blood (20) and synovial fluid (SF) (11) was collected from patients with ERA, and peripheral blood (PB) was collected from 12 patients with polyarticular juvenile idiopathic arthritis (JIA), nine patients with systemic onset and 18 healthy controls. Chemokines [interleukin (IL)-10/CXCL10, thymus and activation-regulated chemokine (TARC)/CCL17 and regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5] were measured in serum and SF. Chemokine receptor expression was measured by flow cytometry. There was no difference in blood CD4(+) T cells bearing CCR5, CCR4 and CXCR3 in ERA and healthy controls. In paired samples the median frequency of CCR5(+) CD4(+) T cells was higher in SF compared to PB (15.8 versus 3.9%, P < 0.005), as was the frequency of CXCR3(+) T cells (21.61% versus 12.46%, P < 0.05). Median serum interferon-inducible protein-10 (IP-10)/CXCL10 levels were higher in patients with ERA compared to controls (139 versus 93 pg/ml; P < 0.05). Further median SF IP-10/CXCL10 levels were higher than the serum levels (2300 pg/ml versus 139 pg/ml; P < 0.01). Serum levels of RANTES/CCL5 were higher in patients (150 ng/ml) compared to control (99 ng/ml; P < 0.01). The SF levels were significantly lower compared to serum (P < 0.05). TARC/CCL17 levels in SF were lower than serum. There is increased homing of CCR5 and CXCR3(+) CD4 cells to the SF. Increased SF levels of IP-10/CXCL10 may be responsible for this migration in patients with ERA.