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Selective alterations in the antibody repertoire in DHQ52 deficient mice
10
Monday, 23 June 1997 - Oral presentations
B-cell development
10:00-l 2:oo
0.2.02
0.2.02.1
Room M
B-cell development
Cross-linking of igp on proB ceils induces cellular differentiation through activation of tyrosine kinases and MAP kinase cascade
K. Nagata’ , S. Kuramachi ‘, T. Nakamura *, F. Kitamura ‘, K. Kuida’ , H. Karasuyama ’ . ’ Dept.Immunolog)! The TokyoMetropofhan Institute of Medical Science, Tokyo,Japan, 2Dept. infectious Diseases and Applied Immunofog~ The institute of Medical Science, University of Tokyo,Japan Introduction: The preB cell receptor (preBCR) is expressed at the large preB cell stage and plays critical roles for early B cell development. It has been demonstrated that preBCR drives cell cycle and induces allelic exclusion as well as cellular differentiation. However, it is not well defined yet how the signals from preBCR are transduced inside of preB cells. Cultured preB cell lines do not seem to be adequate for studying the preBCR signaling. They are relatively resistant to the stimulation by cross-linking of preBCR. Moreover, no study so far has succeeded in inducing preB cell lines to differentiate further by cross-linking preBCR. In this study, we established a new system to analyze preBCR signaling, in which the cross-linking of IgcrAgg heterodimer on bone marrow pros cells induces differentiation of proB cells to the small preB cell stage in a manner comparable with that through the preBCR. Materials and Methods: mAbs specific for mouse lgb were produced by immunizing hamsters with IgM/lga/fg~ complexes prepared from B cell lines. The antibodies were used for cell surface staining, immunoprecipitation and cross-linking of Igo on proB cells in vivoand in vitro. RAG-deficient mice, where B cell development is blocked at the proB cell stage, were ip injected with anti-lga mAb, and their bone marrow cells were analyzed a week later. Bone marrow cells from RAG-deficient mice were incubated with anti-lga mAb in vitro, followed by immunoprecipitation and Western blotting with anti-phosphotyrosine Ab or by MAP kinase assay. Resufts: lgp was detected on the surface of proB cell lines even in the absence of preBCR and BCR. It constituted disulftde-linked heterodimer with lgcl and associated with an unknown 95 kD protein. The lg/3 immunoprecipttates from proB cell lines were found to contain kinase activity, suggesting that the lgo/lgfi complex on proB cells has potential to transduce signals. When RAG-deficient mice were treated with anti-lgfi mAb, bone marrow proB cells were induced to differentiate to the small preB cell stage as observed when the CLheavy chain transgene was expressed in RAG-deficient mice. Cross-linking of lgp on bone marrow proB cells from RAG-deficient mice in vitro induced a rapid tyrosine-phosphorylation of several proteins as well as the activation of MAP kinase (ERK). Conclusion: The cross-linking of lg/3 on proB cells appears to elicit a signal(s) equivalent to that delivered by preBCR, suggesting the importance of lgolllg~ heterodimer in signaling through pmBCR. Our new system should be a powerful tool to analyze the preBCR signaling pathway in detail. We will also discuss a novel SerfThr kinase expressed in preB cells.
0.2.02.2
other components of the pre-B cell receptor complex. The question therefore arises whether the defect in p chain andlor lga synthesis could explain the block at the pro-8 cell stage in Pax-&deficient mice. Materlal and Methods: We have introduced functional immunoglobulin ~1or p-lgb transgenes into PaxQdeficient mice and analyzed their effect on B cell development by flow cytometry and analysis of in vitro cell clonability. Rwufts: Neither expression of a functionally rearranged p transgene nor expression of a p-lgp fusion gene were able to advance B cell development to the pre-B cell stage in Pax-5 mutant mice. Bone marrow pro-B cell lines dertved from PaxC(-l-)xp mice express a pre-B cell receptor complex and are able to proliferate in vitro. Conclusion: The p-lgj3 fusion protein is known to signal the transition from the pro-B to pre-B cell stage even in the absence of p, Iga and lgp proteins. The inability of the F-lgp and the p transgenes to complement the Pax-5 defect therefore demonstrates that neither the absence Of Vu-to-DHJH rearrangement nor the reduced lgo expression are responsible for the developmental block in Pax-5 mutant mice. The expression of the pre-B cell receptor at the surface of the Pax5(-/-) pro-B cells does not induce a loss of pro-B cell compartment nor a down regulation of pre-B cell receptor in vitm, suggesting that signalling through these pre-B cell receptor in these cells is impaired. We conclude therefore that Pax-5 exerts its effect on early B cell development through (so far unknown) target genes which are not involved in the synthesis of the pm-B cell receptor complex.
10.2.02.3
1 Unveiling the difference in B ceil development between man and mice deficient in common cytokine receptor y chain
Lama I. Sharara', Christian A.J. VoBhenrich *, Werner Mliller’, Alain Fischer I, James P. DiSanto ’ ’ /fVSE/Wf U429,HSpitaf Neckec Paris, France, 2Institute for Genetics, Cologne, Germany Introduction: X-linked severe combined immunodeficiency disease (SCID-Xl) results from mutations in the common cytokfne receptor y chain (y& a cornponent of the receptors for interteukins -2, -4, -7, -9, and -15. SCID-Xl patients are characterized by the complete absence of T and NK cells while B cells are present in normal numbers. In contrast, yc- mice lack NK cells and have reduced numbers of mature T and B cells. However ye- mice lose their B cells with age such that antigen dependent B-cell responses cannot be elicited. The difference in B-cell development was originally attributed to species-specific cytoklne/receptor function differences between man and mice. Materials & Methods: B-cell phenotype, Ig production and antigen responses were compared between yc- mice, nukru and double mutant mice. Results: We noticed that murtne yc- B-cell loss was concomitant with the predominance of activated TCRaS_T cells in the spleen and the bone marrow suggesting a cause-and-effect relationship between yc- T cells and B-cell loss. Elimination of residual T-cells from yc- mice (in nu’ye- double mutant mice) prevented age-related B-cell loss. B cells in ndy,- mice were slightly reduced in number, were phenotypicatly mature and responded normally to mitogenic stimulation. Importantly, nu’y,- mice were able to mount a specific T-independent antigen response in viva following antigenic challenge. Conclusion: These results rule out the species-specific divergence in B-cell development between man and mice and demonstrate that murtne yc- T-cells are responsible for the difference in B cell development between ycdeficient humans and mice. We suggest that yc-dependent signaling pathways are important but not essential for mudne, as well as human B-cell development.
Functional immunogiobuiin transgenes cannot induce pre B ceil transition in Pax-5 deficient mice
Claire Thevenin, Meinrad Busslinger. Research institute of Molecular Pathology. Dr Bohr-gasse 7, A- 1030 Vienna, Austria Introduction: An important checkpoint in B cell development is the transition from the pro-B to the pre-B cell stage. Productive rearrangement of the immunoglobulin heavy-chain (/gw gene initiates this transition by signaling through the pre-B cell receptor complex which is composed of the rearranged p chain, the surrogate light chain proteins A5 and VpreB and the signal-transducing proteins Iga and Igp. The pro-8 to pre-B cell transition is abrogated by targeted inactivation of genes which either code for components of the pre-B cell receptor (NMT, 15, IDLY,Is@)or prevent immunoglobulin gene rearrangement (RAG-1 and RAG-2). Moreover, expression of a functionally rearranged LC. transgene can complement the recombination defect of RAG-deficient mice. Expression of the pre-B cell receptor induces the progression to the pre-B cell stage and a reduction of the pro-B cell compartment resulting in a loss of in vitro clonability of bone marrow cells. Inactivation of the Pax-5 gene also prevents transition from the pro-B to the pre-B cell stage due to a developmental block at the pro-B cell stage in bone marrow of Pax&deficient mice. Moreover, Pax-5 has been imolicated in the reoulation of V(DU recombination at the IoH locus, as the efficiency of Vn-to-DnJl rearrangement is reduced by twoordeiof magnitude in Pax-B-deficient pro-B cells. In addition to the absence of @chain synthesis, these pro-B cells express lower levels of I~cz,but normal levels of all
10.2.02.4
1 Selective alterations in the antlbody repertoire in DHQ52 deficient mice
J. Pelkonen, J. Kestler ‘, S. Pelkonen 2, L. Nitschke’. Department of Clinical Microbiolo~ University of Kuopio, PO. 6. 1527, FIN-702 t 1 Kuopio, Finland, ’ Max-Plan&Institute for Immunobiology, Freiburg, Germany, 2National Veterinary and Food Research Institute, Regional Laboratory of Kuopio, Finland Introduction: DHQ52 is one of the 12 D gene segments of the mouse immunoglobulin heavy chain locus. However, several features make the DHQ52 gene particularty interesting: 1) The J proximal localization of the DHQ52 gene is conselved within species 2) DHQ52 gene and its closely neighbourtng sequences are highly conserved within species 3) DHQ52 gene is one of the most frequently used D gene in the initial DJ rearrangements. In order to study the biological role of the DHQ52 gene and the surrounding cis-acting elements in VDJ-recombination and in B-cell differentation we have generated DHQ52 deficient mice. Materials and Methods: The DHQ52 gene segment was deleted in embrvonic stem (ES) cells bv homdogous recombination with the Cre/loxP systern..Targeted‘E&ells were used to establish heterozygous and homozygous DHQ52-deficient mouse lines. Simultaneously with the DHQ52 deletion we were able to introduce a small modification at the JH region which allowed comparison
Monday, 23 June 1997 - Oral presentations
of the recombination frequency at the mutated and wildtype alleles by PCR. In addiion. the JH modification allowed isolation of the rearranged gene segments separately from mutated and wildtype alleles. Reau~ Deletion of the DHQ52 gene caused a general reduction in the recombination frequency in the mutated allele and general reduction on B cells expressing the antigen receptor originating from the mutated allele. In addition, several Interesting selective changes in the antibody repertoire were found. The significance of these findings will be discussed.
IO.2.02.5
1 Analysis of VH/VL combinations in single peripheral IgG-producing B cells in the normal and autolmmune repertoire
R.M.T. de Wildt, I.M. Tomlinson’, F.H.J. van den Hoogen I, W.J. van Venrooij, G. Wtnter*, R.M.A. Hoet. L?epartmentof Biochemistry, University of Nvmagen, The Netherlands, ’ Department of Rheumatol~ University Hospital Nlmegen, Nijmegen, The Netherlands, * Centre for Protein Engineering, MRC, Cambridge, UK
Introduction:The phage combinatorial library approach has proven to be a major step forward in studies on the human autoimmune B cell repertoire. However it remains doubtful whether the heavy (VH) and light (VL) chains of antibodies obtained from these libraries resemble original in vivo pairings. Little is known if preferential VH/VL family or segment pairings exist in viw and if these pairings differ in autoimmune patients. To study this we determined the otfginal VWVLcombinations present in single peripheral IgG-positive B cells in a healthy individual and two patients suffering from systemic lupus etythematosus (SLE). Methods: We developed a simple method for the immottilization of VH and VL regions originating from individual B cells. Our method is based on the clonal expansion of B cells in which cell-cell interactions (CD40-CD4OL) as well as soluble factors were shown to be essential. This B cell culture system combined with single B cell sorting (flow cytometer) was used to analyze in an unbiased way individual IgG-positive B cells. Resutte/Concluslon: In this way VH/VL pairings present in about 200 single B cells from a healthy individual were analyzed. The individual VH and VL utilization showed a non-stochastic usage of V genes. The combinations of VlUVL paitfngs present in individual 6 cells appear to be completely random causing no limitations on the diversity of the repertoire. Due to the high growth efficiency of single cultured B cells (50 to 70% outgrowth in these cultures) most likely no bias based on the capadty of the B cells to be stimulated, is introduced by this system. Using the same approach we are currently analyzing Vl-WL pairings originating from about 200 single peripheral IgG-positive B cells from SLE patients. Results of this study will be presented. 0.2.02.6
Age-related impaired affinity maturation and differential D-JH aene usaae in human VH&containing B’lympho&tes from healthy individuals
I.F. van Dijk-H&d', I. Sdderstrbm 2, S. Feld’, D. Holmberg *, I. Lundkvist ‘. ’ Division for Clinical Immunolog)r Kamlinska institute at Huddinge Hospital, Sweden, *LIapartment for Applied Cell and Molecular Biology, Urned Universit) Sweden Introduction: Immune function is of pm-eminent importance for those surviving to old age. Yet little is known about B cell development and repertoire composition at the molecular level in elderly individuals. Materlale and Methods: To elucidate the basic molecular events underlying humoral immunity during ontogeny and senescence, we have analyzed a panel of 179 PCR derived VHG-DJH rearrangements from cord blood, peripheral blood and spleen. Reauk Our data show a difference in D and JH gene usage between lymphocytes from peripheral blood and spleen. The mutational frequencies in the VH6 gene rises from 0.61% in cord blood to 1.96% in peripheral blood lymphocytes derived from young adults, and decreases to 0.60% in samples from individuals older than 50 years. The number of rearrangements carrying mutations follows a similar pattern: 22% in cord blood, 73% in the age group 20-49 years and 57% in the age group over 50 years. The mutational frequencies among the mutated genes are, however, similar for cord blood and young adults, 2.76% and 2.51%, respectively, and 1.3% in older adults. Conduclon: Differential D- and JH usage is observed only among functional rearrangements, suggesting selection of repertoires rather than restricted rearrangement possibilities as the underlying mechanism. The age-related impaired affinity maturation might partly explain the decrease in immunological responsiveness among elderly and should therefore have implications for immunotherapeutic approaches for longevity and health improvement.
B-cell development 0.2.02.7
11
A novel rat B cell subpopulation as revealed in xenogeneic rat + mouse SCID chimeras
Genft Jan Deenen, Peter M. Dammers, Frans G.M. Kroese. Dept of Histology and Cell Biolm University of Gmningen, Oostersingel 69- 1, 9713 EZ Groningen, The Nethertands Introduction: B cells am continuously generated in the bone marrow from stem cells through a series of well defined events that include rearrangement of the immunoglobulin variable region genes. Newly formed B cells leave the BM to differentiate to mature B cells. In rats, immature and mature B cells can easily distinguished from each other on the basis of Thy-l expression: immature rat B cells express high levels of Thy-l while this is absent from mature cells. When we combine the expression of Thy-l with levels of slgM and slgD we can distinauish four seouentiallv related virgin (i.e. non-memow) B cell subooo~lations: newly formed’B cells $gMbrlgDdUThy~l+),early recitilating folli&ar B cells (IgMdUIgDbrThy-l+). recirculating follicvlar B cells (lgMdulgob’Thy-l-) and marginal z&e B &Is (IgMbrlgDdU~hy-l-). In mice a t&or subset of the B cells are not continuously produced in the bone marrow but are maintained by self-replenishment, express low levels of CD5, are enriched in the petftoneal cavity and have a distinct antibody repertoire. These so-called B-l cells appear to constitute a distinct B cell lineage. In contrast with mice, however, we have not been able to demonstrate so far unequivocally CD5 expressing B cells in adult animals. Detection of B-l cells in rats might be hampered in part by the observation that less than 1% of the peritoneal cavity cells in the rat are B cells. Why B cells are relatively absent from the rat peritoneal cavity is unclear but might e.g. be due to absence of B-l cells in this species or by differences in (microscopic) anatomy between rats and mice. In this study we address this issue by constructing rat + mouse SCID chimeras. Materialsand Methods:Rat fetal liver cells (day 16-17 of gestation) were transferred i.p. into 3 Gy irradiated C.B17-SCID mice. Results: Flowcytometry analysis of chime& spleens shows the presence of all B cell subpopulations found in untreated control rats, at their usual frequency, already within one month after transfer. These findings indicate that rat B cells develop normally in rat -+ mouse SCID chimeras. However, the peritoneal cavity of these xenogeneic chimeras harbors high numbers of B cells of rat origin. Apparently, the accumulation of B cells in the peritoneal cavity is an intrinsic property of the non-lympho-hemopoietic system of the mouse. Flow cytometry analysis of the B cells present in the chimerfc peritoneal cavity reveals a B cell subpopulation with a previously unidentffied phenotype: a relative large proportion of these peritoneal B cells resembles splenic marginal zone (MZ) B cells with respect to their levels of IgM, IgD and CD45WHIS24 and are IgMbrlgDdUHIS24dU, but in contrast these cells express high levels of Thy-l. We have shown recently that these B cells also express the determinant recognized by the newly developed monoclonal antibody HIS57, which reacts strongly with rat splenic MZ B cells, but not (or only weakly) with other B cell subsets. Concluslonr: We have identified a new B cell subset, which resembles to some extent to MZ B cells and is preferentially located in the peritoneal cavity. Together these data may suggest that these calls are a possible candidate for the rat homologue of B-1 cells as identified in the mouse.
10.2.02.8
1 A role for ~~4/31lntegrin In B cell stimulation through homotyplc adhesion
A. Silvy I, P. Altevogt *, P. Mondibm I, C. Bella ‘, T. Defiance l. 1/NSERM U 404, lnstitut Pasteur, Lyon, France, 2 Tumor immunology Pmgram, German Cancer Research Center, Heidelberg. Germany
Introduction:We previously demonstrated that the combination of IL-2 and IL-10 promotes the differentiation of measles virus @#/)-specific human memory B cells into Ab-secmting cells in vitro As for most Ag-specific culture systems, the induction of a secondary anti-MV Ab response in vitro required cultums to be performed at high cell densities. In the course of this study, we observed that, under such culture conditions, B cells also produced large amounts of polyclonal antibodies in response to IL-2 and IL-lo, without the need for any exogenous costimulus. This finding argued for a role of adhesion molecules in B cell activation and prompted us to examine the nature of the costimulatory signal provided through homotypic adhesion. Materialsand Methods:Tonsillar B cells were purified both by negative and positive selection procedures to exclude the possibility that B cell activation could be driven by a non-B cell contaminant and cultured at high cell densities to favour close cell to cell contacts. Various monoclonal antibodies directed against molecules involved in adhesive interactions (CDlla, CD29, CD54, CD44, CD49d, CDBO)were tested for their influence on the Ab synthesis promoted by IL-2 and IL-10 in different experimental systems in which B cells were activated or not by engagement of the BCR (anti-lg antibodies, MV) or CD40. Resuk Both positively and negatively selected tonsillar B cells were induced for proliferation and polyclonal lg synthesis upon stimulation with IL-2 and IL-10 in high density cultures. The endogenous costimulatory signal in these cultures was neither provided by CD40 nor by a component of the serum as the
Monday, 23 June 1997 - Oral presentations
B-cell development
10:00-l 2:oo
0.2.02
0.2.02.1
Room M
B-cell development
Cross-linking of igp on proB ceils induces cellular differentiation through activation of tyrosine kinases and MAP kinase cascade
K. Nagata’ , S. Kuramachi ‘, T. Nakamura *, F. Kitamura ‘, K. Kuida’ , H. Karasuyama ’ . ’ Dept.Immunolog)! The TokyoMetropofhan Institute of Medical Science, Tokyo,Japan, 2Dept. infectious Diseases and Applied Immunofog~ The institute of Medical Science, University of Tokyo,Japan Introduction: The preB cell receptor (preBCR) is expressed at the large preB cell stage and plays critical roles for early B cell development. It has been demonstrated that preBCR drives cell cycle and induces allelic exclusion as well as cellular differentiation. However, it is not well defined yet how the signals from preBCR are transduced inside of preB cells. Cultured preB cell lines do not seem to be adequate for studying the preBCR signaling. They are relatively resistant to the stimulation by cross-linking of preBCR. Moreover, no study so far has succeeded in inducing preB cell lines to differentiate further by cross-linking preBCR. In this study, we established a new system to analyze preBCR signaling, in which the cross-linking of IgcrAgg heterodimer on bone marrow pros cells induces differentiation of proB cells to the small preB cell stage in a manner comparable with that through the preBCR. Materials and Methods: mAbs specific for mouse lgb were produced by immunizing hamsters with IgM/lga/fg~ complexes prepared from B cell lines. The antibodies were used for cell surface staining, immunoprecipitation and cross-linking of Igo on proB cells in vivoand in vitro. RAG-deficient mice, where B cell development is blocked at the proB cell stage, were ip injected with anti-lga mAb, and their bone marrow cells were analyzed a week later. Bone marrow cells from RAG-deficient mice were incubated with anti-lga mAb in vitro, followed by immunoprecipitation and Western blotting with anti-phosphotyrosine Ab or by MAP kinase assay. Resufts: lgp was detected on the surface of proB cell lines even in the absence of preBCR and BCR. It constituted disulftde-linked heterodimer with lgcl and associated with an unknown 95 kD protein. The lg/3 immunoprecipttates from proB cell lines were found to contain kinase activity, suggesting that the lgo/lgfi complex on proB cells has potential to transduce signals. When RAG-deficient mice were treated with anti-lgfi mAb, bone marrow proB cells were induced to differentiate to the small preB cell stage as observed when the CLheavy chain transgene was expressed in RAG-deficient mice. Cross-linking of lgp on bone marrow proB cells from RAG-deficient mice in vitro induced a rapid tyrosine-phosphorylation of several proteins as well as the activation of MAP kinase (ERK). Conclusion: The cross-linking of lg/3 on proB cells appears to elicit a signal(s) equivalent to that delivered by preBCR, suggesting the importance of lgolllg~ heterodimer in signaling through pmBCR. Our new system should be a powerful tool to analyze the preBCR signaling pathway in detail. We will also discuss a novel SerfThr kinase expressed in preB cells.
0.2.02.2
other components of the pre-B cell receptor complex. The question therefore arises whether the defect in p chain andlor lga synthesis could explain the block at the pro-8 cell stage in Pax-&deficient mice. Materlal and Methods: We have introduced functional immunoglobulin ~1or p-lgb transgenes into PaxQdeficient mice and analyzed their effect on B cell development by flow cytometry and analysis of in vitro cell clonability. Rwufts: Neither expression of a functionally rearranged p transgene nor expression of a p-lgp fusion gene were able to advance B cell development to the pre-B cell stage in Pax-5 mutant mice. Bone marrow pro-B cell lines dertved from PaxC(-l-)xp mice express a pre-B cell receptor complex and are able to proliferate in vitro. Conclusion: The p-lgj3 fusion protein is known to signal the transition from the pro-B to pre-B cell stage even in the absence of p, Iga and lgp proteins. The inability of the F-lgp and the p transgenes to complement the Pax-5 defect therefore demonstrates that neither the absence Of Vu-to-DHJH rearrangement nor the reduced lgo expression are responsible for the developmental block in Pax-5 mutant mice. The expression of the pre-B cell receptor at the surface of the Pax5(-/-) pro-B cells does not induce a loss of pro-B cell compartment nor a down regulation of pre-B cell receptor in vitm, suggesting that signalling through these pre-B cell receptor in these cells is impaired. We conclude therefore that Pax-5 exerts its effect on early B cell development through (so far unknown) target genes which are not involved in the synthesis of the pm-B cell receptor complex.
10.2.02.3
1 Unveiling the difference in B ceil development between man and mice deficient in common cytokine receptor y chain
Lama I. Sharara', Christian A.J. VoBhenrich *, Werner Mliller’, Alain Fischer I, James P. DiSanto ’ ’ /fVSE/Wf U429,HSpitaf Neckec Paris, France, 2Institute for Genetics, Cologne, Germany Introduction: X-linked severe combined immunodeficiency disease (SCID-Xl) results from mutations in the common cytokfne receptor y chain (y& a cornponent of the receptors for interteukins -2, -4, -7, -9, and -15. SCID-Xl patients are characterized by the complete absence of T and NK cells while B cells are present in normal numbers. In contrast, yc- mice lack NK cells and have reduced numbers of mature T and B cells. However ye- mice lose their B cells with age such that antigen dependent B-cell responses cannot be elicited. The difference in B-cell development was originally attributed to species-specific cytoklne/receptor function differences between man and mice. Materials & Methods: B-cell phenotype, Ig production and antigen responses were compared between yc- mice, nukru and double mutant mice. Results: We noticed that murtne yc- B-cell loss was concomitant with the predominance of activated TCRaS_T cells in the spleen and the bone marrow suggesting a cause-and-effect relationship between yc- T cells and B-cell loss. Elimination of residual T-cells from yc- mice (in nu’ye- double mutant mice) prevented age-related B-cell loss. B cells in ndy,- mice were slightly reduced in number, were phenotypicatly mature and responded normally to mitogenic stimulation. Importantly, nu’y,- mice were able to mount a specific T-independent antigen response in viva following antigenic challenge. Conclusion: These results rule out the species-specific divergence in B-cell development between man and mice and demonstrate that murtne yc- T-cells are responsible for the difference in B cell development between ycdeficient humans and mice. We suggest that yc-dependent signaling pathways are important but not essential for mudne, as well as human B-cell development.
Functional immunogiobuiin transgenes cannot induce pre B ceil transition in Pax-5 deficient mice
Claire Thevenin, Meinrad Busslinger. Research institute of Molecular Pathology. Dr Bohr-gasse 7, A- 1030 Vienna, Austria Introduction: An important checkpoint in B cell development is the transition from the pro-B to the pre-B cell stage. Productive rearrangement of the immunoglobulin heavy-chain (/gw gene initiates this transition by signaling through the pre-B cell receptor complex which is composed of the rearranged p chain, the surrogate light chain proteins A5 and VpreB and the signal-transducing proteins Iga and Igp. The pro-8 to pre-B cell transition is abrogated by targeted inactivation of genes which either code for components of the pre-B cell receptor (NMT, 15, IDLY,Is@)or prevent immunoglobulin gene rearrangement (RAG-1 and RAG-2). Moreover, expression of a functionally rearranged LC. transgene can complement the recombination defect of RAG-deficient mice. Expression of the pre-B cell receptor induces the progression to the pre-B cell stage and a reduction of the pro-B cell compartment resulting in a loss of in vitro clonability of bone marrow cells. Inactivation of the Pax-5 gene also prevents transition from the pro-B to the pre-B cell stage due to a developmental block at the pro-B cell stage in bone marrow of Pax&deficient mice. Moreover, Pax-5 has been imolicated in the reoulation of V(DU recombination at the IoH locus, as the efficiency of Vn-to-DnJl rearrangement is reduced by twoordeiof magnitude in Pax-B-deficient pro-B cells. In addition to the absence of @chain synthesis, these pro-B cells express lower levels of I~cz,but normal levels of all
10.2.02.4
1 Selective alterations in the antlbody repertoire in DHQ52 deficient mice
J. Pelkonen, J. Kestler ‘, S. Pelkonen 2, L. Nitschke’. Department of Clinical Microbiolo~ University of Kuopio, PO. 6. 1527, FIN-702 t 1 Kuopio, Finland, ’ Max-Plan&Institute for Immunobiology, Freiburg, Germany, 2National Veterinary and Food Research Institute, Regional Laboratory of Kuopio, Finland Introduction: DHQ52 is one of the 12 D gene segments of the mouse immunoglobulin heavy chain locus. However, several features make the DHQ52 gene particularty interesting: 1) The J proximal localization of the DHQ52 gene is conselved within species 2) DHQ52 gene and its closely neighbourtng sequences are highly conserved within species 3) DHQ52 gene is one of the most frequently used D gene in the initial DJ rearrangements. In order to study the biological role of the DHQ52 gene and the surrounding cis-acting elements in VDJ-recombination and in B-cell differentation we have generated DHQ52 deficient mice. Materials and Methods: The DHQ52 gene segment was deleted in embrvonic stem (ES) cells bv homdogous recombination with the Cre/loxP systern..Targeted‘E&ells were used to establish heterozygous and homozygous DHQ52-deficient mouse lines. Simultaneously with the DHQ52 deletion we were able to introduce a small modification at the JH region which allowed comparison
Monday, 23 June 1997 - Oral presentations
of the recombination frequency at the mutated and wildtype alleles by PCR. In addiion. the JH modification allowed isolation of the rearranged gene segments separately from mutated and wildtype alleles. Reau~ Deletion of the DHQ52 gene caused a general reduction in the recombination frequency in the mutated allele and general reduction on B cells expressing the antigen receptor originating from the mutated allele. In addition, several Interesting selective changes in the antibody repertoire were found. The significance of these findings will be discussed.
IO.2.02.5
1 Analysis of VH/VL combinations in single peripheral IgG-producing B cells in the normal and autolmmune repertoire
R.M.T. de Wildt, I.M. Tomlinson’, F.H.J. van den Hoogen I, W.J. van Venrooij, G. Wtnter*, R.M.A. Hoet. L?epartmentof Biochemistry, University of Nvmagen, The Netherlands, ’ Department of Rheumatol~ University Hospital Nlmegen, Nijmegen, The Netherlands, * Centre for Protein Engineering, MRC, Cambridge, UK
Introduction:The phage combinatorial library approach has proven to be a major step forward in studies on the human autoimmune B cell repertoire. However it remains doubtful whether the heavy (VH) and light (VL) chains of antibodies obtained from these libraries resemble original in vivo pairings. Little is known if preferential VH/VL family or segment pairings exist in viw and if these pairings differ in autoimmune patients. To study this we determined the otfginal VWVLcombinations present in single peripheral IgG-positive B cells in a healthy individual and two patients suffering from systemic lupus etythematosus (SLE). Methods: We developed a simple method for the immottilization of VH and VL regions originating from individual B cells. Our method is based on the clonal expansion of B cells in which cell-cell interactions (CD40-CD4OL) as well as soluble factors were shown to be essential. This B cell culture system combined with single B cell sorting (flow cytometer) was used to analyze in an unbiased way individual IgG-positive B cells. Resutte/Concluslon: In this way VH/VL pairings present in about 200 single B cells from a healthy individual were analyzed. The individual VH and VL utilization showed a non-stochastic usage of V genes. The combinations of VlUVL paitfngs present in individual 6 cells appear to be completely random causing no limitations on the diversity of the repertoire. Due to the high growth efficiency of single cultured B cells (50 to 70% outgrowth in these cultures) most likely no bias based on the capadty of the B cells to be stimulated, is introduced by this system. Using the same approach we are currently analyzing Vl-WL pairings originating from about 200 single peripheral IgG-positive B cells from SLE patients. Results of this study will be presented. 0.2.02.6
Age-related impaired affinity maturation and differential D-JH aene usaae in human VH&containing B’lympho&tes from healthy individuals
I.F. van Dijk-H&d', I. Sdderstrbm 2, S. Feld’, D. Holmberg *, I. Lundkvist ‘. ’ Division for Clinical Immunolog)r Kamlinska institute at Huddinge Hospital, Sweden, *LIapartment for Applied Cell and Molecular Biology, Urned Universit) Sweden Introduction: Immune function is of pm-eminent importance for those surviving to old age. Yet little is known about B cell development and repertoire composition at the molecular level in elderly individuals. Materlale and Methods: To elucidate the basic molecular events underlying humoral immunity during ontogeny and senescence, we have analyzed a panel of 179 PCR derived VHG-DJH rearrangements from cord blood, peripheral blood and spleen. Reauk Our data show a difference in D and JH gene usage between lymphocytes from peripheral blood and spleen. The mutational frequencies in the VH6 gene rises from 0.61% in cord blood to 1.96% in peripheral blood lymphocytes derived from young adults, and decreases to 0.60% in samples from individuals older than 50 years. The number of rearrangements carrying mutations follows a similar pattern: 22% in cord blood, 73% in the age group 20-49 years and 57% in the age group over 50 years. The mutational frequencies among the mutated genes are, however, similar for cord blood and young adults, 2.76% and 2.51%, respectively, and 1.3% in older adults. Conduclon: Differential D- and JH usage is observed only among functional rearrangements, suggesting selection of repertoires rather than restricted rearrangement possibilities as the underlying mechanism. The age-related impaired affinity maturation might partly explain the decrease in immunological responsiveness among elderly and should therefore have implications for immunotherapeutic approaches for longevity and health improvement.
B-cell development 0.2.02.7
11
A novel rat B cell subpopulation as revealed in xenogeneic rat + mouse SCID chimeras
Genft Jan Deenen, Peter M. Dammers, Frans G.M. Kroese. Dept of Histology and Cell Biolm University of Gmningen, Oostersingel 69- 1, 9713 EZ Groningen, The Nethertands Introduction: B cells am continuously generated in the bone marrow from stem cells through a series of well defined events that include rearrangement of the immunoglobulin variable region genes. Newly formed B cells leave the BM to differentiate to mature B cells. In rats, immature and mature B cells can easily distinguished from each other on the basis of Thy-l expression: immature rat B cells express high levels of Thy-l while this is absent from mature cells. When we combine the expression of Thy-l with levels of slgM and slgD we can distinauish four seouentiallv related virgin (i.e. non-memow) B cell subooo~lations: newly formed’B cells $gMbrlgDdUThy~l+),early recitilating folli&ar B cells (IgMdUIgDbrThy-l+). recirculating follicvlar B cells (lgMdulgob’Thy-l-) and marginal z&e B &Is (IgMbrlgDdU~hy-l-). In mice a t&or subset of the B cells are not continuously produced in the bone marrow but are maintained by self-replenishment, express low levels of CD5, are enriched in the petftoneal cavity and have a distinct antibody repertoire. These so-called B-l cells appear to constitute a distinct B cell lineage. In contrast with mice, however, we have not been able to demonstrate so far unequivocally CD5 expressing B cells in adult animals. Detection of B-l cells in rats might be hampered in part by the observation that less than 1% of the peritoneal cavity cells in the rat are B cells. Why B cells are relatively absent from the rat peritoneal cavity is unclear but might e.g. be due to absence of B-l cells in this species or by differences in (microscopic) anatomy between rats and mice. In this study we address this issue by constructing rat + mouse SCID chimeras. Materialsand Methods:Rat fetal liver cells (day 16-17 of gestation) were transferred i.p. into 3 Gy irradiated C.B17-SCID mice. Results: Flowcytometry analysis of chime& spleens shows the presence of all B cell subpopulations found in untreated control rats, at their usual frequency, already within one month after transfer. These findings indicate that rat B cells develop normally in rat -+ mouse SCID chimeras. However, the peritoneal cavity of these xenogeneic chimeras harbors high numbers of B cells of rat origin. Apparently, the accumulation of B cells in the peritoneal cavity is an intrinsic property of the non-lympho-hemopoietic system of the mouse. Flow cytometry analysis of the B cells present in the chimerfc peritoneal cavity reveals a B cell subpopulation with a previously unidentffied phenotype: a relative large proportion of these peritoneal B cells resembles splenic marginal zone (MZ) B cells with respect to their levels of IgM, IgD and CD45WHIS24 and are IgMbrlgDdUHIS24dU, but in contrast these cells express high levels of Thy-l. We have shown recently that these B cells also express the determinant recognized by the newly developed monoclonal antibody HIS57, which reacts strongly with rat splenic MZ B cells, but not (or only weakly) with other B cell subsets. Concluslonr: We have identified a new B cell subset, which resembles to some extent to MZ B cells and is preferentially located in the peritoneal cavity. Together these data may suggest that these calls are a possible candidate for the rat homologue of B-1 cells as identified in the mouse.
10.2.02.8
1 A role for ~~4/31lntegrin In B cell stimulation through homotyplc adhesion
A. Silvy I, P. Altevogt *, P. Mondibm I, C. Bella ‘, T. Defiance l. 1/NSERM U 404, lnstitut Pasteur, Lyon, France, 2 Tumor immunology Pmgram, German Cancer Research Center, Heidelberg. Germany
Introduction:We previously demonstrated that the combination of IL-2 and IL-10 promotes the differentiation of measles virus @#/)-specific human memory B cells into Ab-secmting cells in vitro As for most Ag-specific culture systems, the induction of a secondary anti-MV Ab response in vitro required cultums to be performed at high cell densities. In the course of this study, we observed that, under such culture conditions, B cells also produced large amounts of polyclonal antibodies in response to IL-2 and IL-lo, without the need for any exogenous costimulus. This finding argued for a role of adhesion molecules in B cell activation and prompted us to examine the nature of the costimulatory signal provided through homotypic adhesion. Materialsand Methods:Tonsillar B cells were purified both by negative and positive selection procedures to exclude the possibility that B cell activation could be driven by a non-B cell contaminant and cultured at high cell densities to favour close cell to cell contacts. Various monoclonal antibodies directed against molecules involved in adhesive interactions (CDlla, CD29, CD54, CD44, CD49d, CDBO)were tested for their influence on the Ab synthesis promoted by IL-2 and IL-10 in different experimental systems in which B cells were activated or not by engagement of the BCR (anti-lg antibodies, MV) or CD40. Resuk Both positively and negatively selected tonsillar B cells were induced for proliferation and polyclonal lg synthesis upon stimulation with IL-2 and IL-10 in high density cultures. The endogenous costimulatory signal in these cultures was neither provided by CD40 nor by a component of the serum as the