Phenotypic convergence and divergence of surface immunoglobulin and CD40 signals

23 June 1997 - Poster presentations

Concfueion: The loss of subcellular particle containing certain adhesion molecules seems to be an early and specific event in the GC B cell apoptosis.

P.3.02.04

CDS6 (67-2) on human B ceils: A tunctionnai role in proliferation and seiective differentiation in IgE and igG4-producing ceils

Pascale Jeannin, Yves Delneste, Jonathan Ellis’, Jean-Yves Bonnefoy. Geneva Biomedical Research Institute, lmmunolcgy Department, GlaxoWekome R & D, CH-7228 Plan les Ouates, Geneva, Switzerland, 1GlaxoWekome Medicines Research Centre, lmmunopathology Unit, Stevenage, Hertbtdshire, SGI 2NX UK

Introduction:lmmunoglobulin (Ig) E production by B cells requires two primary signals provided by T cells, IL-4 or IL-13 and CD40 ligand (CD4OL). In addition, costimulatory signals, such as CD23CD21 interaction, contribute further ensuring at a selective control over this production. Recently, CD28, expressed on T cells, has been reported to be involved in this process. Since the CD26 ligands, CD80 (87-l) and CD66 (87-2). are expressed on human tonsillar B cells, we have investigated whether signaling via the B7 molecules affects IgE synthesis. Material and Metbods: Purffied human tonsillar B cells were stimulated by IL-4 plus anti-CD40 mAb in the presence of different anti-B7 mAbs. Results: Cross-linking of CD86 with IT2.2 potentiates: (a) IgE and 1964 production, without affecting the poduction of the others isotypes, (b) E transcripts expression, (c) B cell proliferation, (d) cell surface density of CD23 and (e) CD23-binding to CD21-expressing B cells. In contrast, the expression of other B cell surface molecules such as CD1la, CD30 and CD68 remained unaffected. The neutralizing anti-CD23 mAb, caused a dose-dependent inhibition of the effect of IT2.2 on IgE synthesis. Finally, IT2.2 alone or in combination with only one of these stimuli did not show any effect on B cells. Conclusion:This study is the first demonstration of a signaling role for CD86. Together with IL-4 or IL-13 and CD4OL,CD86 favors CD23CD21 pairing and consequently functions as a selective and potent costimulus for human IgE and IgO4 synthesis.

1P.3.02.05 ] Antigen receptor-induced apoptosis of human g3;rn$i center B ceils Is targeted to a centrocytic G. Billian, P. Mondiere, M. Berard, C. Bella, T. Defiance. INSERM U 404. lnstitut Pasteur, Lyon, France

Introduction:Selftolerance may be compromised during the shaping of the mature B cell immune repertoire because somatic mutations of Ig V genes in the oem-rinalcenter (GC) may generate B cell clones with anti-self reactivity. In the course of prevfous studies, we had reproducibly observed that surface lg inhibits the CD4Odependent proliferation of GC B cells. Here, we - ligation _ provide evidence for the existence, in activated GC B cells, of a death-signaling pathway distinct from the Fas/Fas ligand system and directly connected to the B cell antigen receptor @CR). Materisisend Methods: GC, virgin and memory B cells were purified from human tonsils by negative selection procedures, based on their differential expression of IgD, CD38 and CD44 B cells were activated by a mouse fibroblast cell line stably transfected with the CD40 ligand (CD4@L) in the presence or absence of a surrogate antigen (anti-lg antibodies). Four forms of anti-lg antibodies mediating different degrees of surface lg cross-linking were tested: immobilized, soluble, F(ab’)P and F(ab) fragments. Two read-out systems were used: proliferation and apoptosis (examination of the nuclei morphology with the fluorescent dye Hoechst 33342). The phenotype of CD40-activated GC B cells was followed with respect to the expression of IgG, CD19, CD21, CD35 CD36, CD44 and CD77. Reeults: All the above-mentioned anti-lg reagents suppressed in a dosedependent fashion the proliferative response of GC B cells promoted by the CD40-L transfectant, but did not affect the proliferation of virgin and memory B cells. This demonstrates: i) that the growth-inhibitory effect of the surrogate Ag is not mediated by co-aggregation of the BCR and Fcg receptors, ii) the susceptibility to the BCR-dependent apoptosis in the mature B cell compartment is restdcted to GC B cells. The negative regulatory effect of the anti-lg antibodies on GC B cell growth was mediated through the induction of apoptosis but did not involve the Fas/Fas-L system as it could not be abrogated by a chimedc soluble Fas-Fc molecule. Interestingly, IL-4 was the sole cytokine able to reverse the BCR-mediated apoptosis in CD40-activated GC B cell cultures. We found that CD40 ligation on GC B cells induces the emergence of a population presenting the phenotypical features of centmcytes (sIghi, CD38+, CD@, CD44”). Condurlon:Basedon these experimental findings, we propose that the GC is the site for both positive and negative selection of the B cell repertoire and that the BCR-mediated apoptosis is instrumental in the clonal deletion of somatically mutated B cell clones with self reactivity. These observations suggest that there is an analogy between the antigen receptor-driven death of immature B cells in the bone marrow and the negative selection process taking place in the GC.

Germinal centers, isotype switching and hypemutation

107

Our data imply that, at the centrocytic stage, the BCR might mediate a dominant negative regulatory signal which would only he overcome if appropriate T cell help is available.

P.3.02.06

Different isotype expression by cionaiiy related post-germinal center B ceils in muitipie myeioma patients

J.E.J.Guikema l, E. Vellenga2, N.A. Bos’. ’ Dqartment of Histology and cell Biology; Faculty of Medicine, State University of Groningen, the Netherfanda, 2Cepa&ent of Hematotw University Hospital Gmningen, the Netherlands Introduction: Multiple Myeloma (MM) is a B-cell neoplasm which is characterized by the accumulation of plasma cells in the bone marrow and the appearance of a monoclonal immunoglobulin (paraprotein) in the serum of patients. The accumulation of somatic mutations in the Vh genes suggests a post-germinal center origin of these malignant B cells. Autologous stem cell transplantation (ASCT) has enabled the use of high-dose alkylating agents and leads to an increased rate of complete remissions. A frequently observed phenomenon after ASCT is the reappearance of monoclonal immunoglobulins in the serum which express in some cases different isotypes compared to the immunoglobulin isotype produced by the original malignant plasma cells. Oligoclonality (i.e. multiple monoclonal immunglobulins) and light chain variation have a strikingly high incidence. Materialsand Methods: We used a tumorlsotype specific RT-PCR-based strategy using CDR3-based allele specific oligonucleotfde (ASO) primers in combination with isotype specific pnmem to asses the clonality of the cells producing the variant isotypes after ASCT. FlesultszIn 8/7 patients we detected clonal myeloma cells in the bone marrow after ASCT. In 2 cases these cells produce valiant upstream heavy chain (IgH) isotypes, but have the same IgH VDJ-rearrangement. In 3i7 cases we detected clonal myeloma cells in patients that expressed a different light chain isotype. In one of these cases we sorted the post-transplantation bone marrow cells in a K and a A positive population and obtained the same IgH-gene sequence as found in the original pre-transplantation malignant B cells in both the K and A expressing populations. Conclusion: The Vh-genes expressed by these cells are somatically mutated and identical to the sequence obtained from the malignant plasma cells isolated before treatment, suggesting a post-germinal center B cell, which is still capable of expressing both light chain isotypes. The finding of upstream heavy chain isotype variants suggests a pre-switch origin and/or the presence of double/multiple isotype producing cells which have passed through the germinal center.

P.3.02.07

Phenotypic convergence and divergence of surface immunogiobuiin and CD40 signals

K. Axcmna, T. Leanderson. Immunology Group, Dept. of Cell and Molecular Bidogy; Lund Universi& Scilvegatan33, S-223 62 Lund, Sweden

Intrcduction:In response to antigenic stimulation in viw B cells undergo a process of lg selection, lg switch, affinity maturation of the immune response, and somatic mutation in the hypewarfable regions of the lg genes. The preferred anatomical site of these processes is the germinal center. The CD4OCD40 ligand interaction plays a pivotal role in T dependent B cell memory, Ig-switch, and formation of germinal centers as illustrated in the Hyper IgM syndrome and animals lacking either CD40 or its ligand. Another mode of B cell activation is mediated via crosslinking of slg. When B cells are pmstimulated with Ig in vitrc they cannot be restimulated by lg crosslinking, but they respond to restimulation through CD46 as well as to irradiated allo-reactive T helper cells. If cytokines are added together with CD40 the restimulated B cells do not differentiate to plasmacells as they do if restimulated with T helper cells. Rather, stimulation through CD40 down-regulates differentiation of LPS stimulated B cells. MaterlaIr and Methods: B cells were prepared from mouse spleens and pmactivated with either a mitogenic anti-CD40 mAb or anti-lg coupled to Sephamse for 48 hours. B cell blasts were isolated and restimulated for another 48 hours, whereafter cells were pulsed for the last 4 hours with thymidine. Expression of Ig, CD40, MHCII and B-7.2 after 48 hour in vitro stimulation was analyzed with flow cytometry. B cell differentiation was analyzed with the protein A plaque assay. Reeuh Concomitant stimulation of B cells with LPS and CD40 downregulates B cell dterentiation to the same level as occured with Ig cmsslinking. Stimulation of B celfs with an alloreactlve T helper cell and an anti-CD40 m Ab downregulates differentiation with 80% compared to stimulation with T cells only. When B cells are prestimulated through CD40 or anti-lg Sepharose they do not respond to restimulation with anti-lg Sephamse, although slg is still expressed on CD40 prestimulated B cells as determined with flow cytometry. lg crosslinking when compared to activation via CD40 is a dominant negative modulator for the surface expression of most activation antigens, including MHCII, Ig, and CD40, but acts as a positive modulator for expression of B-7.2.

108

Getminal centers, isotype switching and hypennutation

Condusion: In our in vifro model B cells do not respond to restimulation with anti-lg independent of initial preactfvation signal while CD40-mediated restimulation is always permitted. Since lg is still expressed on anti-CD40 preactivated B cells unresponsiveness to restimulation with Ig must be due to changes in the intracellular signalling pathways. Thus, CD40 and slg signals mediate common as well as distinct phenotypic outcomes in peripheral B cells.

P.3.02.08

lgG4 and IgE productlon by B cells co-cultured wlth human Thl and Th2 clones

Bauke A. de Boer, Yvonne C.M. Kruize, Maria Yazdanbakhsh. Depsrtmenf of Pan&o/log Leiden University.Leiden, The Netherlands Introduotfon: Human lymphatic filariasis is characterized by elevated antigenspecific lgG4. Specific IgE, however, remains relatively low, suggesting that lgG4 and IgE production is uncoupled. In search for a mechanism that leads to lgG4 switch without IgE, we studied T and B cell co-cultures consisting of naive, IgD+ B cells and T cell clones with a Thl or Th2 phenotype. To compare lgG4 and IgE production by commkted B cells, IgD‘ B cells were co-cultured with stimulated Thl or Th2 clones. Materials and Hods: Antigen-specific Th clones were co-cultured with purtfied B cells and stimulated with anti-CD2 + 11-2. Supematants of these cultures were harvested after 12 days of incubation and evaluated for immunoglobulins. Detemrination of lgG4 and IgE was done using highly specific ELlSAs. IL-I and IL-13 activities were abrogated using neutralizing antibodies. Results: Filaria-specific Th2-type clones differed in their ability to stimulate lgG4 and IgE production, although similar levels of IL-4 and IL-13 were secreted. Thl -type clones that produced no detectable 11-4,were able to induce switch to lgG4 in the absence of any IgE. Moreover neutralizing antibodies to IL-4 and IL-13 could not inhibit lgG4 production. In order to evaluate lgG4 production by memory B cells, we studied the IgD- B cell compartment. IgE production was again completely dependent on the presence of IL-4 whereas IgO4 was detectable in cultures without any IL-4 Interestingly IL-4 even inhibited lgG4 production by the IgD- B cells. Conclusion:Switch to lgG4 may occur independently of IL-4 and 11-13. Currently we are investigating whether CWO-L alone or in combination with other T helper cell factors can induce IgD+ B cells to switch to lgG4. The differential regulation of lgG4 and IgE production may also occur at the level of committed B cells.

1 -

Somatic mutations and lsotvoe _. switch In folllcular lymphomas

W.M. Aatts, R.J. Bende, E.J. Steenbergen, S.T. Pals, C.J.M. van Noesel. Department of Pathoiom Academicmedical center, Meibergdreef 9, 7705 AZ Amsterdam, The Netherlands Follicular non-Hodgkin’s lymphomas (FLs) resemble normal germinal centers as the tumor cells grow in a network of normal follicular dendrttic cells and are capable of somatic mutation and isotype switch. In normal B cell development these events occur in a coordinated fashion. To investigate to what extent FLs resemble normal germinal center B cells functionally, the vattabte domains (V) of Ighl- and IgG-expressing FLs were amplified by RT-PCR and sequenced. A broad range in the number of somatic mutations was found in both the IgMand IgG-related VH genes, although on average the number of mutations was found to be somewhat higher in the VH genes of IgG-expressing FLs. Intemstingly, some follicular lymphomas contained both IgM- and IgG-expressing tumor cells, which were shown to be derived from the same tumor clone. From one of these patients lymphoma tissue material from two timepoints was available. with an interval of 9 years. At the first timepoint both IgM- and IgG-expressing tumor cells were found. Compared to the germline gene with the closest homology (V3-23), the IgM-related sequence contained 33 somatic mutations. The IgG-related sequence contained 39 mutations, of which 33 were shared with the IgM-related sequence. Nine years later, only @G-expressingcells were found, of which the VH oenes still contained 39 somatic mutations. The maioritv of these mutations (33) were identical to those of the IgG-related sequence &f the first timepoint, whereas 5 were different. Our data suggest that in FLs, like In normal B cells, the majority of somatic mutations occur prior to isotype switching.

P.3.02.10

IgD only expressing germinal center and plasma cells have mutated extensively lgvH genes, deleted Cc1and preferentially expressed IGX light chain

Christophe Arpin, Odette de Bouteiller, Diane Razanajaona, lsabelle Fugier-Vivier, Fmncine BriBm, Jacques Banchemau, Serge Lebecque, Yong-Jun Liu. Scherfng-Plough, Laboratory for lmmunobgica~ Reseanzh, DanMy; France Introduction: We have recently Identified a subset of normal slgM- lgD+CD39+

23 June 1997 - Poster presentations

centroblasts that: i) have accumulated an very high number of somatic mutations; ii) seem sequestred within germinal centers; iii) appear not to differentiate into memory B cells. We have now further analyzed the Ig heavy and light chain isotype expression of these cells, as well as their ability to differentiate into IgD secreting plasma cells. Materfal and Methods: slgM_lgD+CD39+ centmblasts and CD39hi/intracvtopIasmicIgD+ olasma cells were ~utified bv FACS-sortina. Genomic DNA ias’isolated-and ‘analyzed by PCR and sequeicing, to look ibr recombination events leading to IgD isotype switch. lmmunohistology were used to assess Ig light chain isotype expression and to identify IgD secreting plasma cells on tonsil tissue sections. Reeults: We have found that slgM_lgD+CD39+ centroblasts and IgD only secreting plasma cells: i) have undergone Cp to C6 switch; ii) have over-mutated their IgVH genes; Iii) preferentially express I@ light chain. Interestingly, Cr to C6 switch and biased IgS expression have been described for IgD secreting myeloma. Conduslon: Our data illustrate a novel differentiation pathway for normal human mature B cells, characterized by Cr to C6 switch, extensive somatic mutation of Ig variable heavy chain genes and biased expresskm of IgA liiht chain. This pathway, which appears to give rise to plasma cells rather than to memory B cells, might be prone to malignant transformation.

1P.3.02.11 1 Preference of either direct or sequentlal lsotype switch to IgE antibody productlon depends on the antigen dose used for lmmunlxatlon S. Sudowe, A. Rademaekers, E. KMsch. lnsfi~ufeIbr /mmuno/~, Minster. Domagkstr SA, D-49129 Miinsfer, Germany

Uniwsify of

Pttming of CBA/J mice with minute doses of protein antigens (Ag) leads to high IgE antibody (Ab) titers in the immune sera of these animals. In contrast priming with large doses elicits only a marginal production of IgE Ab. In vim0restimulation of spleen cells from animals primed with large doses and lacking in viva IgE Ab leads to a burst of IgE Ab forming cells (I). This in vitroanamnestlc response is lacking in mice pttmed with minute doses of Ag. In order to trace whether the IgE Ab producing B cells are generated via direct isotype switch from Ighl expressing cells or via sequential isotype switch wtth an IgGl expressing transitional stage, we have extended the analysis of the distribution of Ab isotypes to Ag primed IgGl deficient A5’Syl mice (2). The data presented here must be interpreted as following: Priming of mice with minute doses of Ag leads to a direct switch from IgM to IgE Ab expression in both strains. These animals have high IgE Ab titers without establishina an IaE memorv. The direct switch was verified bv PCR and Southern blot analysis ojswitch circle DNA isolated from Ag specific 6 cells of CBAN mice primed with minute doses of Ag. In contrast to immunization with minute doses, priming with large doses of Ag fails to induce in viva IgE Ab production in CBA/J and A5’Syl mice but establishes a BE memory in CBA/J mice which involves IaGl beartna intermediate B cells. In viw these BE memory cells do not en& the status of IgE Ab producing cells. In vftm they can be released from this energy and presumed suppression and develop in an anamnestic response into a large population of IgE Ab forming B cells. This increase in the number of IgE Ab producing cells after resbimulation in vitro is lacking In AYSyl mice, apparently because of their inability to generate IgGl expressing precursor cells. The notion of a sequential switch and an IgGl intermediate BE memory status is also supported by depletion and inhibition experiments. Elimination of IgGl expressing B cells in CBA/J mice primed with high doses of Ag prevents the IgE Ab burst after in vifro challenge with Ag. The data further suggest that the two switch pathways am not mutually exclusive and that the Ag dose can decide which pathway is preferentially used. (Supported bei DFG Ko 379/20-l). [I] Sudowe, S., Specht, C., Kolbe, L. and Kblsch, E. (1995) Intern. Immunol. 7, 179%1907. [2] Jung, S., Rajewsky, K. and Radbruch, A. (1993) Science 259, 994-997.

1P.3.02.12 1 Paracrine regulatlon of germinal center B cell adheslon through the c-Met-heoatocvte growth factorkcatterfiktor pathway . _ R. van der Voott, T.E.I. Taher, R.M.J. Keehnen, C. Smit, M. Groenink, S.T. Pals. Department of Pathology! Academic Medirxl Center, Uniwsiiy of Amsterdam, 1105 AZ Amsterdam, The Nethedands T cell dependent humoml immune responses am initiated by the activation of naive B cells in the T cell amas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendtttic cells (FCC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell migration and interaction with FDC critically depend on integrin-mediated adhesion. To date, the physiological regulators of this adhesion were unknown. In the present report, we investigated a possible role of the c-met-encoded