Noncanonical effector functions of the T-memory-like T-PLL cell are shaped by cooperative TCL1A and TCR signaling.

T-cell prolymphocytic leukemia (T-PLL) is a poor-prognostic neoplasm. Differentiation stage and immune-effector functions of the underlying tumor cell are insufficiently characterized. Constitutive activation of the T-cell leukemia 1A (TCL1A) oncogene distinguishes the (pre)leukemic cell from regular postthymic T cells. We assessed activation-response patterns of the T-PLL lymphocyte and interrogated the modulatory impact by TCL1A. Immunophenotypic and gene expression profiles revealed a unique spectrum of memory-type differentiation of T-PLL with predominant central-memory stages and frequent noncanonical patterns. Virtually all T-PLL expressed a T-cell receptor (TCR) and/or CD28-coreceptor without overrepresentation of specific TCR clonotypes. The highly activated leukemic cells also revealed losses of negative-regulatory TCR coreceptors (eg, CTLA4). TCR stimulation of T-PLL cells evoked higher-than-normal cell-cycle transition and profiles of cytokine release that resembled those of normal memory T cells. More activated phenotypes and higher TCL1A correlated with inferior clinical outcomes. TCL1A was linked to the marked resistance of T-PLL to activation- and FAS-induced cell death. Enforced TCL1A enhanced phospho-activation of TCR kinases, second-messenger generation, and JAK/STAT or NFAT transcriptional responses. This reduced the input thresholds for IL-2 secretion in a sensitizer-like fashion. Mice of TCL1A-initiated protracted T-PLL development resembled such features. When equipped with epitope-defined TCRs or chimeric antigen receptors, these Lckpr-hTCL1Atg T cells gained a leukemogenic growth advantage in scenarios of receptor stimulation. Overall, we propose a model of T-PLL pathogenesis in which TCL1A enhances TCR signals and drives the accumulation of death-resistant memory-type cells that use amplified low-level stimulatory input, and whose loss of negative coregulators additionally maintains their activated state. Treatment rationales are provided by combined interception in TCR and survival signaling.

Capability of CI-Orbitrap for Gas-Phase Analysis in Atmospheric Chemistry: A Comparison with the CI-APi-TOF Technique.

Chemical ionization Orbitrap mass spectrometry (CI-Orbitrap) represents a promising new technique for gas-phase analysis in analytical and atmospheric chemistry mainly due to its very high mass resolving power. In this work, we performed the first side-by-side comparison between a CI-Orbitrap and the widely used atmospheric pressure interface time-of-flight mass spectrometry (CI-APi-TOF) using two different chemical ionization methods, i.e., acetate-ion-based (CH3COO-) and aminium-ion-based (n-C3H7NH3+) schemes. The capability of the CI-Orbitrap at accurately measuring low concentrations of gaseous species formed from the oxidation of α-pinene was explored. Although this study reveals a lack of linearity of the CI-Orbitrap when measuring product ions at very low concentrations (<1 × 106 molecules cm-3), very good agreement between both techniques can be achieved by applying a newly developed linearity correction. It is experimentally shown that the correction function is independent of the reagent ion used. Thus, accurate quantification of organic compounds at concentrations as low as 1 × 105 molecules cm-3 by the CI-Orbitrap can be achieved. Finally, by means of tandem mass spectrometry, the unique capability of the Orbitrap allows the direct determination of the binding energy of cluster ions between analyte and reagent ions, that is needed for the assessment of a chosen ionization scheme.

Detection and imaging of atmospheric radio flashes from cosmic ray air showers.

The nature of ultrahigh-energy cosmic rays (UHECRs) at energies >10(20) eV remains a mystery. They are likely to be of extragalactic origin, but should be absorbed within approximately 50 Mpc through interactions with the cosmic microwave background. As there are no sufficiently powerful accelerators within this distance from the Galaxy, explanations for UHECRs range from unusual astrophysical sources to exotic string physics. Also unclear is whether UHECRs consist of protons, heavy nuclei, neutrinos or gamma-rays. To resolve these questions, larger detectors with higher duty cycles and which combine multiple detection techniques are needed. Radio emission from UHECRs, on the other hand, is unaffected by attenuation, has a high duty cycle, gives calorimetric measurements and provides high directional accuracy. Here we report the detection of radio flashes from cosmic-ray air showers using low-cost digital radio receivers. We show that the radiation can be understood in terms of the geosynchrotron effect. Our results show that it should be possible to determine the nature and composition of UHECRs with combined radio and particle detectors, and to detect the ultrahigh-energy neutrinos expected from flavour mixing.

Human monoclonal IgG isotypes differ in complement activating function at the level of C4 as well as C1q.

Humanized antibodies are likely to have a major role in therapy and it is important to define their interaction with physiological effectors. By comparing a matched series of chimeric human mAbs we found that igG1 was most efficient in complement lysis, although IgG3 bound more C1q. To resolve this paradox we compared the ability of human IgG1, IgG2, IgG3, IgG4, and IgE and rat IgG2b to cause C1q binding, C1 binding and activation, C4 activation, C4b binding, and C3b binding. Rat IgG2b was included because this isotype has already successfully been used for therapy. Human IgG1 was less efficient than IgG3 and fixing C1q and C1 on the cell surface, but the number of C4 molecules bound per C1 was 10-fold greater for IgG1 than for IgG3. This difference, amplified through later stages of the complement cascade, can account for the superiority of IgG1 for cell lysis. The efficiency of IgG1 in fixing C4 was not due to a favored binding site on the antibody molecule, since virtually all of the bound C4b was attached to the cells. Rather, it appeared that the activation of C4 by C1s was greatly favored by IgG1 compared with IgG3. It should be possible to combine the optimal properties of IgG1 and IgG3 antibodies to produce an improved therapeutic reagent.

The immunogenicity of chimeric antibodies.

Mice were immunized with model xenogeneic (both the VH frameworks and the CH domains of human origin), chimeric (just VH frameworks human), or self antibodies, and the antiantibody responses were dissected. Only the self antibody did not elicit a response. A strong response was elicited by the most xenogeneic antibody with approximately 90% against the C and approximately 10% against the V. The anti-V response was not attenuated in the chimeric antibody, demonstrating that foreign VH frameworks can be sufficient to lead to a strong antiantibody response. The magnitude of this xenogeneic anti-VH response was similar to that of the allotypic response elicited by immunizing mice of the Igha allotype with an Ighb antibody. Thus, although chimerization can diminish antiantibody responses, attention should be paid both to V region immunogenicity and to polymorphism.

A human immunoglobulin lambda locus is similarly well expressed in mice and humans.

Transgenic mice carrying a 380-kb region of the human immunoglobulin (Ig) lambda light (L) chain locus in germline configuration were created. The introduced translocus on a yeast artificial chromosome (YAC) accommodates the most proximal Iglambda variable region (V) gene cluster, including 15 Vlambda genes that contribute to >60% of lambda L chains in humans, all Jlambda-Clambda segments, and the 3' enhancer. HuIglambdaYAC mice were bred with animals in which mouse Igkappa production was silenced by gene targeting. In the kappa-/- background, human Iglambda was expressed by approximately 84% of splenic B cells. A striking result was that human Iglambda was also produced at high levels in mice with normal kappa locus. Analysis of bone marrow cells showed that human Iglambda and mouse Igkappa were expressed at similar levels throughout B cell development, suggesting that the Iglambda translocus and the endogenous kappa locus rearrange independently and with equal efficiency at the same developmental stage. This is further supported by the finding that in hybridomas expressing human Iglambda the endogenous L chain loci were in germline configuration. The presence of somatic hypermutation in the human Vlambda genes indicated that the Iglambda-expressing cells function normally. The finding that human lambda genes can be utilized with similar efficiency in mice and humans implies that L chain expression is critically dependent on the configuration of the locus.

Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies.

Cell lines have been established that secrete a matched set of human chimeric IgM, IgG1, IgG2, IgG3, IgG4, IgE, and IgA2 antibodies that are directed against the hapten 4-hydroxy-3-nitrophenacetyl. These chimeric antibodies secreted from mouse plasmacytoma cells behave exactly like their authentic human counterparts in SDS-PAGE analysis, binding to protein A and in a wide range of serological assays. The antibodies have been compared in their ability to bind human C1q as well as in their efficacy in mediating lysis of human erythrocytes in the presence of human complement. A major conclusion to emerge is that whereas IgG3 bound C1q better than did IgG1, the chimeric IgG1 was much more effective than all the other IgG subclasses in complement-dependent hemolysis. The IgG1 antibody was also the most effective in mediating antibody-dependent cell-mediated cytotoxicity using both human effector and human target cells. These results suggest that IgG1 might be the favoured IgG subclass for therapeutic applications.

Library preparation for next generation sequencing: A review of automation strategies.

Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed.

Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.

Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.

Minimal residual disease (MRD) monitoring via quantitative PCR (qPCR) detection of Ag receptor gene rearrangements has been the most sensitive method for predicting prognosis and making post-transplant treatment decisions for patients with ALL. Despite the broad clinical usefulness and standardization of this method, we and others have repeatedly reported the possibility of false-positive MRD results caused by massive B-lymphocyte regeneration after stem cell transplantation (SCT). Next-generation sequencing (NGS) enables precise and sensitive detection of multiple Ag receptor rearrangements, thus providing a more specific readout compared to qPCR. We investigated two cohorts of children with ALL who underwent SCT (30 patients and 228 samples). The first cohort consisted of 17 patients who remained in long-term CR after SCT despite having low MRD positivity (<0.01%) at least once during post-SCT monitoring using qPCR. Only one of 27 qPCR-positive samples was confirmed to be positive by NGS. Conversely, 10 of 15 samples with low qPCR-detected MRD positivity from 13 patients who subsequently relapsed were also confirmed to be positive by NGS (P=0.002). These data show that NGS has a better specificity in post-SCT ALL management and indicate that treatment interventions aimed at reverting impending relapse should not be based on qPCR only.

GvL effects in T-prolymphocytic leukemia: evidence from MRD kinetics and TCR repertoire analyses.

Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.

B-cell tumorigenesis in mice carrying a yeast artificial chromosome-based immunoglobulin heavy/c-myc translocus is independent of the heavy chain intron enhancer (Emu).

We have used YAC (yeast artificial chromosome) technology to create large translocation regions where the c-myc proto-oncogene is coupled to the core region of the human immunoglobulin heavy chain (IgH) locus (from VH2-5 through to Cdelta). Chimeric mice were obtained from embryonic stem cells carrying a single copy of the 240-kb IgH/c-myc translocation region. B-cell tumorigenesis occurs in the translocus mice, even when the entire Emu intron enhancer region between the joining segments and switch mu is deleted. This demonstrates that as yet unidentified regulatory elements in the IgH locus, independent from the known enhancers, are sufficient to cause B-cell specific activation of c-myc after translocation. The phenotype of tumors from IgH/c-myc YAC transgenic mice with or without Emu (B220+, IgM+/IgD+) is reminiscent of Burkitt's lymphoma. A rapidly expanding abnormal B-cell population is present at birth and accumulates in bone marrow, periphery, and spleen, well before discrete tumor establishment. Molecular analysis identified a clonal origin, with rearrangement of one mouse heavy chain allele retained in tumor cells from different sites, whereas subsequent rearrangements of heavy or light chain loci can be diverse. These mice routinely develop mature B-cell tumors early in life and may provide an invaluable resource of a B-cell lymphoma model.

Rapid induction of B-cell lymphomas in mice carrying a human IgH/c-mycYAC.

Activation of the c-myc proto-oncogene by one of the immunoglobulin (Ig) loci after chromosomal translocation is a consistent feature of Burkitt's lymphoma. Different subtypes of this tumor vary in the molecular architecture of the translocation region. In most cases there are no known regulatory elements of the Ig locus neighboring the oncogene and this considerably obscures the mechanism of its deregulation. In order to assess possible oncogene activation signals, we produced an experimental translocation region by insertion of a c-myc gene about 50 kb from the IgH intron enhancer in a yeast artificial chromosome (YAC) containing a 220 kb region of the human Ig heavy chain (IgH) locus. Single copy integration of this YAC into the genome of mouse embryonic stem (ES) cells was achieved by spheroplast fusion. Chimeric mice derived from these ES cells developed monoclonal B-cell lymphomas expressing surface IgM by 8-16 weeks of age. The IgH/c-myc translocus showed different V(h)DJ(H) rearrangement in almost all tumors without any alterations of the distance between c-myc and the IgH intron enhancer. This mouse model can be used for the in vivo analysis of c-myc deregulation and the tumor formation capacity of the IgH locus in aberrant rearrangements.

Dominance of intrinsic genetic factors in shaping the human immunoglobulin Vlambda repertoire.

The expressed human immunoglobulin Vlambda repertoire demonstrates a strong bias in the use of individual Vlambda segments. Mechanisms that underlie such biases can be divided into two categories: intrinsic genetic processes that lead to the preferential rearrangement and/or expression of certain segments; and selection following light chain expression. Here, we have used two approaches to investigate the factors that shape the human Vlambda repertoire. Firstly, we characterised 136 Vlambda rearrangements (59 productive and 77 non-productive) amplified from the human genomic DNA of peripheral blood cells. Secondly, we analysed Vlambda segment use in a library of 2000 cDNA clones from a transgenic mouse containing a 380 kb region (including 15 functional Vlambda segments) from the human immunoglobulin lambda locus. By hybridisation and sequencing we found that the patterns of use of human Vlambda segments in the transgenic mouse were similar to those found in the expressed human peripheral blood repertoire and in productive and non-productive genomic DNA rearrangements. These data indicate the importance of intrinsic genetic factors in shaping the human Vlambda repertoire and highlight the remarkable conservation of the molecular mechanisms involved in the production of the antibody repertoire in mouse and man. Therefore, transgenic mice represent a good model for analysis of the human antibody repertoire and for the production of human antibodies.

What accounts for vertigo one year after neuritis vestibularis - anxiety or a dysfunctional vestibular organ?

One year after neuritis vestibularis, 29% from a sample of 75 patients still complained of vertigo. The objective of this investigation was to study why patients suffer from persisting vertigo. The alternative hypotheses were that the vertigo experienced could be explained either by a persisting vestibular dysfunction or by psychopathological changes. To elucidate this question, patients were examined with dynamic posturography, the symptom check list (SCL 90 R), the state-trait anxiety inventory (STAI), the agoraphobic cognitions questionnaire (ALQ) and the body sensations questionnaire (BSQ). After one year, only two patients had overall pathological results in the posturography. Following the assumption that sub-clinical dysfunction of the organs of balance can also lead to an altered body perception in combination with anxiety, the pathological results of posturographic sub-tests were related to experiences of vertigo. Here also there were no significant associations. However, vertigo correlated highly significantly with body-related anxiety and anxiety-related apprehension. In conclusion, chronic vertigo after an acute vestibular disorder could be regarded as a somatopsychic process. Persisting experience of vertigo is not explained by sub-clinical organic changes. Anxiety seems to be the crucial factor in persisting vertigo.

Production of human antibody repertoires in transgenic mice.

Transgenic mice have been created that carry human immunoglobulin heavy and light chain genes in germline configuration and that have the corresponding endogenous genes silenced. The transgenes are either minigene constructs or large, almost authentic, transloci on yeast artificial chromosomes and undergo B-cell-specific DNA rearrangement and hypermutation in the mouse lymphoid tissue. Monoclonal antibodies with good affinities for human antigens have been obtained after immunisation. These mice may be a future source of human antibodies for therapy.

Improved assessment of minimal residual disease in B cell malignancies using fluorogenic consensus probes for real-time quantitative PCR.

PCR of clonally rearranged immunoglobulin heavy chain (IgH) gene sequences is increasingly used for detection of minimal residual disease (MRD) in lymphoid malignancies. Inherent quantitating problems are the main drawbacks of traditional PCR technologies. These limitations have been overcome by the recently developed real-time quantitative PCR (RQ PCR) technology. However, clinical application of the few published RQ PCR assays targeting immune gene rearrangements is hampered by the expensive and time-consuming need for individual hybridization probes for each patient. We have developed a new RQ PCR strategy targeting clonally rearranged IgH sequences that solves this problem. The method uses only two different JH hybridization probes and four downstream JH primers homologous to consensus germline JH gene segments. In combination with an allele-specific upstream (ASO) primer the consensus JH probes and primers allow quantitation of about 90% of possible IgH rearrangements. In a series of 22 B-lineage ALL the new assay allowed the detection of one to 10 blasts in a background of 10(5) normal cells. To prove the clinical utility we quantified MRD in 23 follow-up samples of six ALL patients with the new assay in comparison with a published RQ PCR technique that used individually designed primer/probe sets. We showed that the sensitivity of the new RQ PCR assay was slightly higher for four of the six cases and about 100-fold higher for one case, enabling detection of an increasing MRD level as an indicator of subsequent relapse 44 weeks earlier compared to the ASO probe assay in this particular patient. The results suggest, that the novel RQ PCR assay is a rapid, technically simple, reliable, and sensitive alternative to traditional quantification assays and simplifies current approaches of monitoring MRD in clinical trials.