No abstract

The distribution of immunoglobulin (Ig) isotypes within specific B cell clones in vivo after immunization is not well defined. Using an IgVH/CDR3- and isotype-specific reverse transcription polymerase chain reaction method, we have carried out a survey of the diversification of the isotype in a splenic response to phenyl-oxazolone (phOx) on a chicken serum albumin carrier. The phOx-specific VH (VHOx-1 with specific CDR3 motif) is associated with all of the heavy chains (μ, δ, α, γ, and ε) after simple immunization with antigen in alum. The kinetics of expression of each isotype are distinct and reproducible. Focusing mainly on the expression of secretory Ig transcripts, IgM, IgG1, and IgE are found after priming, whereas IgD and IgA appear after boosting. Secretory IgD transcripts are found reproducibly at moderate levels and may, therefore, contribute significantly to the secreted Ig response in mice. Most crucially, we find enhanced levels of secretory IgM/VHOx-1 transcripts (with ‘phOx-specific’ CDR3) after boosting, strongly indicating the existence of IgM memory cells that give rise to an enhanced specific IgM secretion in the secondary response.

Pneumococcal serotype‐specific anti‐capsular polysaccharide antibodies protect against invasive pneumococcal disease. Within an individual the diversity of these antibodies is limited. To evaluate the repertoire of antibodies to pneumococcus and determine whether oligoclonality is seen both between serotypes and between individuals, we sampled the B cell repertoire induced by polysaccharide and conjugate vaccine in adult volunteers. Fifteen hybridomas secreting pneumococcus‐specific monoclonal antibodies were generated from five volunteers. Ten were isotype switched, six were IgG2 and four were IgA. These included two isotype switch variants of the same clone. VH3 and Vκ2 were used by 10 / 15 and 7 / 13 of the sequenced clones, respectively, with identical genes, VH3 – 48 and Vκ2‐A17 used by a number of volunteers to a variety of serotypes. VDJ junctional characteristics and complementarity‐determining region (CDR) 3 length were variable. High levels of somatic mutation in CDR1 and 2, inconsistent with a primary response, were found in 10 / 11 of the isotype‐switched antibodies, including those induced by plain polysaccharide antigens. These data suggest that wild‐type infection or nasopharyngeal carriage of Streptococcus pneumoniae in adults may induce memory and the response to subsequent immunization with plain polysaccharide or conjugate pneumococcal vaccines may have the characteristics of a secondary response.

Summary. Immune reconstitution after conventional allogeneic transplantation is a major determinant of survival. We conducted a detailed investigation of T-and B-cell immune reconstitution and clinical outcome in 19 patients with multiple myeloma undergoing reduced-intensity stem cell transplantation using in vivo T-cell depletion with alemtuzumab. These patients experienced delayed T-cell recovery, particularly in the naïve (CD45 RA + ) CD4 compartment. T-cell receptor spectratype analysis showed a reduced repertoire diversity, which improved rapidly after the administration of donor leucocyte infusions and subsequent conversion to full donor T-cell chimaerism. Posttransplant recovery of CD19 + B cells was also delayed for up to 18 months. Spectratype analysis of IgH CDR3 repertoire revealed a gradual normalization in IgM spectratype complexity by 6-12 months after transplant. There was a high incidence of viral infection, particularly cytomegalovirus reactivation, but the regimen-related mortality was low, perhaps because of the very low incidence of acute graftversus-host disease (GVHD; grade I-II skin GVHD was seen in 5/19 patients). Over 80% of all patients have relapsed at a median of 283 (range 153-895) d after transplant, suggesting that the initially low rate of GVHD comes at a high price with regard to the desired graft-versus-myeloma effect.

Vaccines induce memory B-cells that provide high affinity secondary antibody responses to identical antigens. Memory B-cells can also re-instigate affinity maturation, but how this happens against antigenic variants is poorly understood despite its potential impact on driving broadly protective immunity against pathogens such as Influenza and Dengue. We immunised mice sequentially with identical or variant Dengue-virus envelope proteins and analysed antibody and germinal-centre (GC) responses. Variant protein boosts induced GCs with a higher proportion of IgM+ B cells. The most variant protein re-stimulated GCs with the highest proportion of IgM+ cells with the most diverse, least mutated V-genes and with a slower but efficient serum antibody response. Recombinant antibodies from GC B-cells showed a higher affinity for the variant antigen than antibodies from a primary response, confirming a memory origin. This reveals a new process of antibody memory, that IgM memory cells with fewer mutations participate in secondary responses to variant antigens, demonstrating how the hierarchical structure of B-cell memory is used and indicating the potential and limits of cross-reactive antibody based immunity.

The cytokine receptor common gamma chain mutation in X‐linked SCID results in a failure of T and NK cell development and an as yet undefined defect of B cells. Using immunoglobulin isotype‐specific reverse transcription‐PCR we show that although hematopoietic stem cell transplantation restores a diverse repertoire of class‐switched B cell clones, on further analysis these are almost all of donor origin. This suggests that host B cells, which predominate after unconditioned transplantation, are still defective even in the presence of normal T cells. These studies imply that effective humoral reconstitution can only be achieved by the engraftment of normal donor B cells.

We have studied immune reconstitution in a patient with paediatric-onset polyarteritis nodosa treated with high-dose immunosuppressive agents followed by stem cell rescue. The patient developed several new autoimmune phenomena over the 18 months after immunosuppression and stem cell rescue. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) heteroduplex and isotype-specific RT-PCR analysis of immunoglobulin expression showed that the T- and B-cell repertoires were highly restricted in the first few months after treatment. The dominant T-cell clones seen after reconstitution were persistently expanded, were different from those which could be demonstrated before autologous stem cell transplantation, and were in the CD8(+) population. Our data also show that 12 months after treatment these expanded T-cell clones were within the CD45RA(+) population, suggesting that reversion from the CD45RO(+) to the CD45RA(+) phenotype had occurred in vivo.

Development and maintenance of cells in the murine follicular and marginal zone compartments is thought to involve differing levels of stimulation of the BCR, although it is still not clear which BCR ligands mediate these events. How the delineation between naïve and antigen experienced B-cell populations relates to cell phenotype and how precise or blurred this delineation is, is also not well understood. Here we show, using PCR to analyse the antibody response to phenyl-oxazolone in the mouse, that the antibody repertoire of CD21hi/CD23− marginal zone B-cells shows persistent increase in levels of particular IgM after immunization with foreign antigen. Further, we show that these IgM have different but related VH/CDR3 sequences from those seen in the class-switched response to oxazolone that we have also analysed. We also detect an effect of antigen on the follicular B-cell repertoire that is less persisting. These results provide evidence consistent with the signal strength model of mature B-cell development being extended to include stimulation by foreign antigen, and also further the known zone of influence of foreign antigen on the B-cell compartment.