10 B-cell development Monday, 23 June 1997 - Oral presentations

10:00-l 2:oo Room M

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.

0.2.02 B-cell development Materlal and Methods: We have introduced functional immunoglobulin ~1 or

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.

0.2.02.1 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 Tokyo Metropofhan Institute of Medical Science, Tokyo, Japan, 2 Dept. 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 CL heavy 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 sig- nal(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 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 transi- tion 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-trans- ducing 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 rearrange- ment (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 pre- vents 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 two ordeiof 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

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, 2 Institute 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 corn- ponent 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 re- sponses 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 im- portant but not essential for mudne, as well as human B-cell development.

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, 2 National Veterinary and Food Research Institute, Regional Laboratory of Kuopio, Finland

Introduction: DHQ52 is one of the 12 D gene segments of the mouse im- munoglobulin 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 se- quences 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 em- brvonic stem (ES) cells bv homdogous recombination with the Cre/loxP sys- tern.. 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