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Background The relationship between chemotherapy-related toxicities and prognosis is unclear. Previous studies have examined the association of myelosuppression parameters or neuropathy with survival and reported conflicting results. This study aims to investigate 13 common chemotherapy toxicities and their association with relapse-free survival and breast cancer-specific survival. Methods Chemotherapy-related toxicities were collected prospectively for 6,248 women with early-stage breast cancer from four randomised controlled trials (NEAT; BR9601; tAnGo; Neo-tAnGo). Cox proportional-hazards modelling was used to analyse the association between chemotherapy-related toxicities and both breast cancer-specific survival and relapse-free survival. Models included important prognostic factors and stratified by variables violating the proportional hazards assumption. Results Multivariable analysis identified severe neutropenia (grades ≥3) as an independent predictor of relapse-free survival (hazard ratio (HR) = 0.86; 95 % confidence interval (CI), 0.76–0.97; P = 0.02). A similar trend was seen for breast cancer-specific survival (HR = 0.87; 95 % CI, 0.75–1.01; P = 0.06). Normal/low BMI patients experienced more severe neutropenia (P = 0.008) than patients with higher BMI. Patients with fatigue (grades ≥3) showed a trend towards reduced survival (breast cancer-specific survival: HR = 1.17; 95 % CI, 0.99–1.37; P = 0.06). In the NEAT/BR9601 sub-group analysis by treatment component, this effect was statistically significant (HR = 1.61; 95 % CI, 1.13–2.30; P = 0.009). Conclusions This large study shows a significant association between chemotherapy-induced neutropenia and increased survival. It also identifies a strong relationship between low/normal BMI and increased incidence of severe neutropenia. It provides evidence to support the development of neutropenia-adapted clinical trials to investigate optimal dose calculation and its impact on clinical outcome. This is important in populations where obesity may lead to sub-optimal chemotherapy doses. Electronic supplementary material The online version of this article (doi:10.1186/s12916-015-0547-5) contains supplementary material, which is available to authorized users.

nology is an ideal field for the application of systems approaches, with its detailed descriptions of cell types (over 200 immune cell types are defined in the scope of the Immunological Genome Project (ImmGen)), wealth of reagents and easy access to cells. Thanks to the broad and robust approaches allowed by gene-expression microarrays and related techniques, the transcriptome is probably the only ‘-ome’ that can be reliably tackled in its entirety. Generating a complete perspective of gene expression in the immune system

Natural antibodies produced by CD5+ B-1 B cells include those with specificity for senescent erythrocytes (anti-BrMRBC, anti-PtC) and for thymocytes (anti-thymocyte autoantibody, ATA). Here we describe work from our laboratories studying two prototypic examples, V(H)11Vkappa9-encoded anti-BrMRBC and V(H)3609Vkappa21c-encoded ATA. Using V(H)11-mu transgenic mice, we discovered that certain natural autoantibodies utilize V(H) genes that are selected against in bone marrow B cell development, but not fetal liver, effectively restricting their generation to fetal/neonatal life. Studies with ATA-mu transgenic mice demonstrated a critical requirement for self antigen in the accumulation of B cells with this specificity and for the production of high levels of serum ATA. Finally, analysis of B cell development in ATA-mu kappa transgenic mice revealed two distinct responses by B cells to expression of this B cell receptor (BCR): most developing B cells in spleen of adult mice were blocked at an immature stage and only escaped apoptosis by editing their BCR to eliminate the ATA specificity; nevertheless, high levels of serum ATA were observed, indicating that some B cells differentiated to antibody-forming cells without altering their specificity. Thus, our studies reveal mechanisms for restricting the generation of B cells producing natural autoantibodies, demonstrate a key positive selection step in their development, and show that most developing B cells in adult mice bearing such specificities fail to reach a mature stage.

Cd79a (called mb-1 here) encodes the Ig-alpha signaling component of the B cell receptor. The early B cell-specific mb-1 promoter was hypermethylated at CpG dinucleotides in hematopoietic stem cells but became progressively unmethylated as B cell development proceeded. The transcription factor Pax5 activated endogenous mb-1 transcription in a plasmacytoma cell line, but could not when the promoter was methylated. In this context, early B cell factor (EBF), a transcription factor required for B lymphopoiesis, potentiated activation of mb-1 by Pax5. EBF and the basic helix-loop-helix transcription factor E47 each contributed to epigenetic modifications of the mb-1 promoter, including CpG demethylation and nucleosomal remodeling. EBF function was enhanced by interaction with the transcription factor Runx1. These data suggest a molecular basis for the hierarchical dependence of Pax5 function on EBF and E2A in B lymphocyte development.

ANTIBODIES to DNA and nucleoproteins are found in sera of individuals with systemic autoimmune disease. In the population (and in the autoimmune mouse strain MRL/lpr) there is a great variety of such antinuclear antibodies, but individuals with systemic lupus erythematosus or single MRL mice express a subset only of the antinuclear specificities found in the population. These observations have been interpreted to mean that these antibodies arise by immunization1. The oligoclonal nature of the autoantibody response and the evidence of selection acting on somatically mutated autoantibodies favour this interpretation2,3. Specific activation of autoantibodies in disease implies either that autoantibodies are regulated in non-diseased individuals or that autoantigen availability is variable. The former has been demonstrated in anti-DNA transgenic mice. In normal mice, transgene-encoded antibodies against double-stranded (ds) DNA are not expressed in serum or on B cells4–6. Here we describe modified anti-dsDNA transgenic mice which allow us to study the site and developmental stage at which such B-cell regulation occurs. This model shows that in normal mice B cells expressing anti-DNA specificity are deleted in the bone marrow at a pre-B to immature B transitional stage.

Self-reactive B cells can be regulated by either deletion or inactivation. These manifestations of self-tolerance have been dramatically shown in transgenic mice in which the number of self-reactive cells has been artificially expanded. We have now extended these models to ask if B-cell tolerance as described for non-disease-associated antigens also operates for the targets of autoimmunity. The target we have chosen is DNA. Anti-DNA antibodies are diagnostic of certain autoimmune syndromes in humans and are a characteristic of the murine model of systemic autoimmunity, the MRl/lpr mouse. Antibodies to both single-stranded and double-stranded DNA have been implicated in disease. By generating anti-DNA transgenic mice, we have addressed the question of whether DNA-specific B cells are regulated in normal (non-autoimmune) mice. We indeed found that most transgenic B cells bind DNA, yet we failed to detect secreted anti-DNA. We suggest that as a consequence of their self-reactivity these B cells are developmentally arrested.

Interleukin-2 (IL-2) has a key role in the antigen-specific clonal growth of T lymphocytes, by virtue of its interaction with a specific cell-surface receptor (IL-2R). The growth signal seems to be delivered by IL-2 bound to the high-affinity, but not the low-affinity, receptor. Genes encoding IL-2 and its receptor (that is, Tac-antigen) have been cloned and analysed in detail. We have now achieved cell-type-specific reconstitution of the high-affinity human IL-2R by expressing the complementary DNA cloned from normal lymphocytes. A mouse T-lymphocytic line, EL-4, expressed human IL-2R with high (dissociation constant (Kd) = 160-220 pM) and low (Kd = 2.1-2.2 nM) affinity for recombinant human IL-2, while mouse L929 cells expressed only a single class of the IL-2R with lower affinity (Kd = 34.5 nM) for the ligand. We also show that the human IL-2R expressed in EL-4 cells responds to IL-2 and mediates reversed signal transduction: growth of the EL-4 cells harbouring the IL-2R is inhibited specifically by human recombinant IL-2. The approach described here may provide a general experimental framework for elucidating the molecular basis of signal transduction mediated by specific receptor-ligand interaction.

Mouse monoclonal anti-dansyl antibodies with the same antigen-binding sites but different heavy chain constant regions were generated. The extent of segmental flexibility in times of nanoseconds and the capacity to fix complement were greatest for IgG2b, intermediate for IgG2a, and least for IgG1 and IgE. Hence, the effector functions of immunoglobulin isotypes may be controlled in part by the freedom of movement of their Fab arms.

The early stages of murine B-cell differentiation are characterized by a series of immunoglobulin gene rearrangements which are required for the assembly of heavy(H) and light(L)-chain variable regions from germline gene segments. Rearrangement at the heavy-chain locus is initiated first and consists of the joining of a diversity (DH) gene segment to a joining (JH) gene segment. This forms a DJH intermediate to which a variable (VH) gene segment is subsequently added. Light-chain gene rearrangement follows and consists of the joining of a VL gene segment to a JL gene segment: once a productive light-chain gene has been formed the cell initiates synthesis of surface immunoglobulin M (sIgM) receptors (reviewed in ref. 1). These receptors are clonally distributed and may undergo further diversification either by somatic mutation or possibly by continued recombinational events. Such recombinational events have been detected in the Ly 1+ B-cell lymphoma NFS-5, which has been shown to rearrange both lambda and H-chain genes subsequent to the formation of sIgM (mu kappa) molecules. Here we have analysed a rearrangement of the productive allele of NFS-5 and found that it is due to a novel recombination event between VH genes which results in the replacement of most or all of the coding sequence of the initial VHQ52 rearrangement by a germline VH7183 gene. Embedded in the VH coding sequence close to the site of the cross-over is the sequence 5' TACTGTG 3', which is identical to the signal heptamer found 5' of many DH gene segments. This embedded heptamer is conserved in over 70% of known VH genes. We suggest that this heptamer mediates VH gene replacement and may play an important part in the development of the antibody repertoire.

The simultaneous and rapid measurement of the amounts of two different fluorochrome-coupled antibodies bound to single cells (two-colour immunofluorescence) provides a very powerful means for the identification of lymphocyte subpopulations1. Using a dual-laser fluorescence-activated cell sorter (FACS) we show that two monoclonal antibodies, anti-IgM and anti-IgD, labelled respectively with fluorescent and ‘Texas red’ (a new red-fluorescent dye) reveal several previously unrecognized B-cell subpopulations in mouse spleen and lymph nodes. Measured individually, these surface markers (IgM and IgD) show only that B cells are broadly heterogeneous with respect to the amount of surface immunoglobulin expressed2; however, measured simultaneously, they clearly define at least two B-cell subsets. One of these populations, which is predominant in spleen and constitutes the overwhelming majority of B cells in lymph nodes, is missing in CBA/N (Xid) mice known to be deficient with respect to their B-cell immune responses3.

CBA/N mice carrying the X-linked immune deficiency gene (xid) have fewer splenic B cells than normal CBA mice and are unresponsive to a certain class of antigens. Studies of B-cell surface-marker expression and immune responsiveness have led to the commonly accepted idea that the B cells in adult xid mice are immature and resemble the B cells of young (1-3 week old) normal mice. That is, like young animals, xid mice lack cells in the most numerous of three IgM/IgD B-cell subpopulations (designated I in Fig. 1a, b) present in adult spleen. We now report, however, that this picture is an oversimplification and that in fact the B cells in adult xid mice differ from those present in either adult or young normal mice. Using quantitative three-colour fluorescence-activated cell sorter (FACS) analyses, we have compared the correlated expression of IgM, IgD and a newly discovered B-lymphocyte antigen (BLA-1) on splenic B cells in normal and xid mice. We show here (1) that most B cells in adult xid mice (as in normals) are BLA-1- whereas all B cells in young animals are BLA-1+; (2) that the major difference in the IgM/IgD B-cell subpopulations found between xid and normal mice is limited to the BLA-1- cells; and (3) that xid mice have increased numbers of BLA-1+ population III B cells.