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B cell activation and peripheral lymphocyte renewal
96
B-cell development
23 June 1997 - Poster presentations
an enhancer that together confer pre-6 cell-specifii expression on a reporter gene in transformed cell lines. We have generated and analysed three lines of transgenic mice which express human CD25 under the control of 5’)is.In all three strains precursor B cells, but not mature B lymphocytes or other blood cell lineages, express human CD25 in parallel to endogenous 15. High expression of human CD25 on B-lineage cells of transgenic mice has allowed the identification of a new B220+ CDIQA5+ precursor of the B220+ CDlC A5+ c-kit+ preB-I cells. The B220+ CDIQA5+ cells are clonabte on stromal cells in the presence of interleukin-7 with a frequency similar to the pm-81 cells. The CDIQ- precursors have a sizeabte part of their immunoglobulin heavy chain gene loci in germline configuration, while the CDlQ+ preB-I cells are predominantly DJu rearranged. The results indicate that random integration of the 722 bp long 5’ region of the A5 gene into the mouse genome confers tissue and differentiation stage specific expression of a transgene.
P.2.02.16
No&A B cell development In VpreBl deficient
A. MBrtensson ‘, Y. Argon 2, F. Melchers 3, J.L. Dul*, I-L. MArtensson ‘. 1tmmunofogy Unit, University of Lund, Sdlvegatan 21, S-223 62 Lund, Sweden, zDepartment of Pathology and the Committee on Immunology The Untversity of Chicago, Chicago, IL 63637, USA, 3Sasel institute for lmmunolog)r Grenzachemtrasse 467, CH-4665 Base/, Switzerland The surrogate light chain (SL) is composed of two polypeptides, VpreB and 15. In the mouse there are two VpreB genes which are QQ%identical within the coding regions. The two genes are co-expressed at the RNA level and encode functional proteins that can assemble with 15. However, it is not known whether both gene products serve the same function in vivo. We have established mice that are deficient in VpreBl (VpBl-‘-) by homologous recombination in ES cells. Lymphoid organs from the mice have been analysed by Southern blotting, RT-PCR, immunisations and FACS. B cell development in the VpBl-‘- mice is normal as judged by: (i) the number of B lymphocytes in bone marrow (BM), spleen and pentoneum, (ii) the presence of pre-BI, pre-811,immature and mature B cells in BM and (iii) the production of a normal immune response against Tdependent antigens. At the RNA level the VpBl -I- mice express VpreBP and A.5 but not VpreBl. Analysis of BM cells for cell surface expression of SL chain detected a complex using antibodies recognising A5 as well as VpreB (VP245). Since VpreBl is not expressed and VP245 mcognises both VpreBl and VpreBP this demonstrates that VpreB2 is expressed on the cell surface. Thus, this suggests that VpreBP is functional in vivo and associates with A5 and @to form a functional pre-B receptor.
P.2.02.17
;,cA;Avation
and peripheral lymphocyte
F. Agenes, MM. Rosado, A.R. MC Lean, A. Freitas. LDL, lnstitut Pasteur Paris, France We have studied the fate of mature lymph node B cells transfered into B6 syngeneic immunodeficient mice (RAG-/- or u MT mice). After an initial decline following transfer, the number of donor derived B cells rapidly increased in the hosts spleen. The phenotype of these lymphocytes shifted from “mature” to “activated” e.g. increased size, high levels of IgM & HSA and low levels of IgD 8 CD23 Although, the total number of B cells recovered is very low (l-2.106 cell&PL), the fraction of IgM secreting cells is increased (10% compare to 1% in normal spleens) and, as a consequence, serum IgM level is the same as in nomal mice. This phenomenon is T cell independent, since identical results were obtained when RAG-/- mice were in]ected with lymph node cells from T cell deficient CD3e-/- donnom. These acttvated B cells represent a self-renewing population that persist in the host for at least 4 months. An increased fraction of these B cells incorporate BrdU, compared to mature B cells from normal mice, and they continuously produce plasma cells during this period. To study the rate of replacement of this cell population, we performed reconstitution experiments where peripheral cells where co-injected with bone marrow precursors. Preliminary results suggest that the pool of activated B cells is very slowly replaced by newly formed BM derived B cells. These results suggest that B cell selection may follow the rule of “first come, first served” and that plasma cells may be generated from a stable pool of activated B cells which has a slow turnover.
1P.2.02.18 1 Ontogeny of CD2 expression on porcine B cells J. Sinkoral, M. Sinkoral, Z. Rehakova’ , B. Cukrowska*, I. Spllchal‘, H. Tlaskalov&Hogenova*, B. de Geus 3. ‘Dept.knmwtoL and Gnotobi’, inst. Mbobiot., Acad. Sci., Novy Hmdek, Czech Republic, 2Dept. /mmuno/. and Gnotobiol..,Inst. Micmbiol., Acad. Sci., Prague, Czech Rep&tic, 31D-DL0, Lefystad, the Netherkinds Introduction: Before anti-pig CD3 monodonal antibodies become available, the CD2 antigen had been used to distinguish between B lymphocytes (slg+, CD2-), classical T cells (slgCD2+) and “Null” lymphocytes (slg-CDP-) in pigs. In contrast with such an approach we show here that a prominent subset of CD2+ B cells exists in pigs and that the CD2 molecule can be a differentiation marker of porcineB lymphocvtes. Mat&Is and M&hods:~Minnesotaderived conventtonal (CV), germ-free (GF) and E. coli monoassociated minipigs bred in Novy Hradek, their fetuses, SLA d/d inbred minipigs and Dutch landrace SPF pigs (ID-DLO. Lelystad) were used in our experiments. For flow cytometry lymphocytes were stained with FITC labelled anti-IgM (LIG4) monoclonal antibody (moAb) and anti-CD2 (MSA4,lgG2a) or anti-CD3 (76-2-ll,lgG2a, B cell irrelevant control) moAbs (as neat supematants). PE labelled anti-mouse IgG2a polyclonal antibodies were used as a secondary reagent. Rae&a: Since our finding that almost all B cells in pig fetuses bear the CD2 molecule we have been looking for CD2 expression on B lymphocytes in pigs of different origin during pre- and postnatal ontogeny. CD2+ B cells in all pigs examined have less CD2 on their surface than T cells. We have shown that CD2- B cells are almost absent in fetuses and newborn piglets and appear in the periphery early after birth. Interestingly, OF and E. coli monoassoclated piglets had only minute proportions of CD2- B cells in the periphery even at the age of 2 weeks, when a prominent subset of slgM + CD2- lymphocytes was detected in the periphery of their CV counterparts. The enrichment for CD2- B lymphocytes in CV pigs with increasing age resulted in about the same percentage of both B cell subsets in peripheral blood in adult pigs, in the spleen the slgM+CD2- lymphocytes even prevailed. Conolustons: In contrast with other species pigs have at least two peripheral B cell subsets differing in CD2 expression. In fetuses and newborns mostly virgin, antigen unexperienced lymphocytes are present in the periphery. After microbial colonization antigen activated recirculating lymphocytes appear, a process which is delayed and diminished in GF animals. We hypothesize that slgM+CDP+ lymphocytes represent virgin cells while slgM+CD2- cells form an activated/memory B cell recirculating pool in pigs.
P.2.02.19
The transcription factor Eglcl Is involved In B cell development
Adelheid Dinkel 1,2,Wilhelm K. Aicher‘, Christian Haas’, Kurt BPrki 3, Birgit Ledermann 3, Hermann Eibel’. ’ Clinicat Research Unit Rheumatof~, UnivwsityofFreiburg, Med. Center D-79106 Fmiburg, Germany sDepartment of Biology University of Freibufg,P79104 Freibug, Germany 3Preclinicai Research, Novartis Phanna Ltd. CH-4002 Sasel, Germany Introduction: Egr-1 is a zinc finger transcription factor, which is transiently activated in mature B cell upon B cell receptor crosslinking. To analyze the regulatory potential of Egr-1 in B cells we generated Egr-1 transgenic mice. Materials and Methods: Expression of the Egr-1 transgene is controlled by the immunoglobulin heavy chain enhancer and a VH promoter. The Egr-1 transgenic mice were generated by transfection of embryonal stem cells from BalWc mice using the Egr-1 construct. Reeuh Succesfull integration of the complete Egr-1 construct into the genome of the Egr-1 transgenic mice was confirmed by Southern blot analysis. Transgenic Egr-1 mRNA and the recombinant Egr-1 protein was found in spleen and bone marrow B cells. Analyzing the bone marrow of the Egr-1 transgenic mice we could detect a significantly enlarged population of mature B cells in comparison to non-transgenic liiennates. In addiion we found an upregulation of BP-l and nur77 expression in transgenic B cells. Conclusion: We therefore conclude that the transcription factor Egr-1 promotes B cell maturation in vivo.
1P.2.02.20 1 Dual action of cyclohexlmide on splenic B cell apoptosls C. Lemaire, K. Andreau, V. Souvannavong, A. Adam. Instkut de biochimie, CNRS-URA 1116, Univemite Paris-Surf, 91405 Orsay France The aim of this study was to investigate requirements for de nova protein synthesis in apoptotic process, which is a controversial topic. We analysed the effects of cycloheximide (CHX), an inhibitor of macromolecular synthesis, on B lymphocytes apoptosis. B cells were purified from murine spleen cells. Cell viability was determined by morphological examination after FDA and BET staining. Apoptosis was char-
B-cell development
23 June 1997 - Poster presentations
an enhancer that together confer pre-6 cell-specifii expression on a reporter gene in transformed cell lines. We have generated and analysed three lines of transgenic mice which express human CD25 under the control of 5’)is.In all three strains precursor B cells, but not mature B lymphocytes or other blood cell lineages, express human CD25 in parallel to endogenous 15. High expression of human CD25 on B-lineage cells of transgenic mice has allowed the identification of a new B220+ CDIQA5+ precursor of the B220+ CDlC A5+ c-kit+ preB-I cells. The B220+ CDIQA5+ cells are clonabte on stromal cells in the presence of interleukin-7 with a frequency similar to the pm-81 cells. The CDIQ- precursors have a sizeabte part of their immunoglobulin heavy chain gene loci in germline configuration, while the CDlQ+ preB-I cells are predominantly DJu rearranged. The results indicate that random integration of the 722 bp long 5’ region of the A5 gene into the mouse genome confers tissue and differentiation stage specific expression of a transgene.
P.2.02.16
No&A B cell development In VpreBl deficient
A. MBrtensson ‘, Y. Argon 2, F. Melchers 3, J.L. Dul*, I-L. MArtensson ‘. 1tmmunofogy Unit, University of Lund, Sdlvegatan 21, S-223 62 Lund, Sweden, zDepartment of Pathology and the Committee on Immunology The Untversity of Chicago, Chicago, IL 63637, USA, 3Sasel institute for lmmunolog)r Grenzachemtrasse 467, CH-4665 Base/, Switzerland The surrogate light chain (SL) is composed of two polypeptides, VpreB and 15. In the mouse there are two VpreB genes which are QQ%identical within the coding regions. The two genes are co-expressed at the RNA level and encode functional proteins that can assemble with 15. However, it is not known whether both gene products serve the same function in vivo. We have established mice that are deficient in VpreBl (VpBl-‘-) by homologous recombination in ES cells. Lymphoid organs from the mice have been analysed by Southern blotting, RT-PCR, immunisations and FACS. B cell development in the VpBl-‘- mice is normal as judged by: (i) the number of B lymphocytes in bone marrow (BM), spleen and pentoneum, (ii) the presence of pre-BI, pre-811,immature and mature B cells in BM and (iii) the production of a normal immune response against Tdependent antigens. At the RNA level the VpBl -I- mice express VpreBP and A.5 but not VpreBl. Analysis of BM cells for cell surface expression of SL chain detected a complex using antibodies recognising A5 as well as VpreB (VP245). Since VpreBl is not expressed and VP245 mcognises both VpreBl and VpreBP this demonstrates that VpreB2 is expressed on the cell surface. Thus, this suggests that VpreBP is functional in vivo and associates with A5 and @to form a functional pre-B receptor.
P.2.02.17
;,cA;Avation
and peripheral lymphocyte
F. Agenes, MM. Rosado, A.R. MC Lean, A. Freitas. LDL, lnstitut Pasteur Paris, France We have studied the fate of mature lymph node B cells transfered into B6 syngeneic immunodeficient mice (RAG-/- or u MT mice). After an initial decline following transfer, the number of donor derived B cells rapidly increased in the hosts spleen. The phenotype of these lymphocytes shifted from “mature” to “activated” e.g. increased size, high levels of IgM & HSA and low levels of IgD 8 CD23 Although, the total number of B cells recovered is very low (l-2.106 cell&PL), the fraction of IgM secreting cells is increased (10% compare to 1% in normal spleens) and, as a consequence, serum IgM level is the same as in nomal mice. This phenomenon is T cell independent, since identical results were obtained when RAG-/- mice were in]ected with lymph node cells from T cell deficient CD3e-/- donnom. These acttvated B cells represent a self-renewing population that persist in the host for at least 4 months. An increased fraction of these B cells incorporate BrdU, compared to mature B cells from normal mice, and they continuously produce plasma cells during this period. To study the rate of replacement of this cell population, we performed reconstitution experiments where peripheral cells where co-injected with bone marrow precursors. Preliminary results suggest that the pool of activated B cells is very slowly replaced by newly formed BM derived B cells. These results suggest that B cell selection may follow the rule of “first come, first served” and that plasma cells may be generated from a stable pool of activated B cells which has a slow turnover.
1P.2.02.18 1 Ontogeny of CD2 expression on porcine B cells J. Sinkoral, M. Sinkoral, Z. Rehakova’ , B. Cukrowska*, I. Spllchal‘, H. Tlaskalov&Hogenova*, B. de Geus 3. ‘Dept.knmwtoL and Gnotobi’, inst. Mbobiot., Acad. Sci., Novy Hmdek, Czech Republic, 2Dept. /mmuno/. and Gnotobiol..,Inst. Micmbiol., Acad. Sci., Prague, Czech Rep&tic, 31D-DL0, Lefystad, the Netherkinds Introduction: Before anti-pig CD3 monodonal antibodies become available, the CD2 antigen had been used to distinguish between B lymphocytes (slg+, CD2-), classical T cells (slgCD2+) and “Null” lymphocytes (slg-CDP-) in pigs. In contrast with such an approach we show here that a prominent subset of CD2+ B cells exists in pigs and that the CD2 molecule can be a differentiation marker of porcineB lymphocvtes. Mat&Is and M&hods:~Minnesotaderived conventtonal (CV), germ-free (GF) and E. coli monoassociated minipigs bred in Novy Hradek, their fetuses, SLA d/d inbred minipigs and Dutch landrace SPF pigs (ID-DLO. Lelystad) were used in our experiments. For flow cytometry lymphocytes were stained with FITC labelled anti-IgM (LIG4) monoclonal antibody (moAb) and anti-CD2 (MSA4,lgG2a) or anti-CD3 (76-2-ll,lgG2a, B cell irrelevant control) moAbs (as neat supematants). PE labelled anti-mouse IgG2a polyclonal antibodies were used as a secondary reagent. Rae&a: Since our finding that almost all B cells in pig fetuses bear the CD2 molecule we have been looking for CD2 expression on B lymphocytes in pigs of different origin during pre- and postnatal ontogeny. CD2+ B cells in all pigs examined have less CD2 on their surface than T cells. We have shown that CD2- B cells are almost absent in fetuses and newborn piglets and appear in the periphery early after birth. Interestingly, OF and E. coli monoassoclated piglets had only minute proportions of CD2- B cells in the periphery even at the age of 2 weeks, when a prominent subset of slgM + CD2- lymphocytes was detected in the periphery of their CV counterparts. The enrichment for CD2- B lymphocytes in CV pigs with increasing age resulted in about the same percentage of both B cell subsets in peripheral blood in adult pigs, in the spleen the slgM+CD2- lymphocytes even prevailed. Conolustons: In contrast with other species pigs have at least two peripheral B cell subsets differing in CD2 expression. In fetuses and newborns mostly virgin, antigen unexperienced lymphocytes are present in the periphery. After microbial colonization antigen activated recirculating lymphocytes appear, a process which is delayed and diminished in GF animals. We hypothesize that slgM+CDP+ lymphocytes represent virgin cells while slgM+CD2- cells form an activated/memory B cell recirculating pool in pigs.
P.2.02.19
The transcription factor Eglcl Is involved In B cell development
Adelheid Dinkel 1,2,Wilhelm K. Aicher‘, Christian Haas’, Kurt BPrki 3, Birgit Ledermann 3, Hermann Eibel’. ’ Clinicat Research Unit Rheumatof~, UnivwsityofFreiburg, Med. Center D-79106 Fmiburg, Germany sDepartment of Biology University of Freibufg,P79104 Freibug, Germany 3Preclinicai Research, Novartis Phanna Ltd. CH-4002 Sasel, Germany Introduction: Egr-1 is a zinc finger transcription factor, which is transiently activated in mature B cell upon B cell receptor crosslinking. To analyze the regulatory potential of Egr-1 in B cells we generated Egr-1 transgenic mice. Materials and Methods: Expression of the Egr-1 transgene is controlled by the immunoglobulin heavy chain enhancer and a VH promoter. The Egr-1 transgenic mice were generated by transfection of embryonal stem cells from BalWc mice using the Egr-1 construct. Reeuh Succesfull integration of the complete Egr-1 construct into the genome of the Egr-1 transgenic mice was confirmed by Southern blot analysis. Transgenic Egr-1 mRNA and the recombinant Egr-1 protein was found in spleen and bone marrow B cells. Analyzing the bone marrow of the Egr-1 transgenic mice we could detect a significantly enlarged population of mature B cells in comparison to non-transgenic liiennates. In addiion we found an upregulation of BP-l and nur77 expression in transgenic B cells. Conclusion: We therefore conclude that the transcription factor Egr-1 promotes B cell maturation in vivo.
1P.2.02.20 1 Dual action of cyclohexlmide on splenic B cell apoptosls C. Lemaire, K. Andreau, V. Souvannavong, A. Adam. Instkut de biochimie, CNRS-URA 1116, Univemite Paris-Surf, 91405 Orsay France The aim of this study was to investigate requirements for de nova protein synthesis in apoptotic process, which is a controversial topic. We analysed the effects of cycloheximide (CHX), an inhibitor of macromolecular synthesis, on B lymphocytes apoptosis. B cells were purified from murine spleen cells. Cell viability was determined by morphological examination after FDA and BET staining. Apoptosis was char-