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A 150 bp element can silence rearrangement of the chicken immunoglobulin lambda locus in transgenic mice
98
Unconventional lymphocyte subsets
23 June 1997 - Poster presentations
clones were selected and one of them was able to induce the exoression of 58 kDa glycoprotein on Cos 7-trans#ectedcells as demonstrated b; staining with NIM-R7 mAb. The clone was named pNV7. The sequencing of pNV7 is under progress. Conclusion: The 58 kDa glycoprotein is present in all stages of maturation on the surlace of B cells, but staining is higher wlth the most mature cell lines. The clone pNV7 has an insert of 1700 bp likely to encode ~58 antigen. The data of the sequence will be presented at the meeting.
P.2.02.25
A 150 bp element can silence rearrangement of the chicken immunoglobulin lambda locus in tranaaenic mice
L. Cocea, A. Dahan, L. Ferradini, C.-A. Reynaud, J.-C. Weill. lnstifuf National Sante et de la RechercheMBdicale,Ftmce
de la
Introduction: The regulation of rearrangement and allelic exclusion of immunoglobulin loci is complex and could require both positive and negative elements. We have previously shown in mice transgenic for the chicken immunoglobulin lambda locus that the 1.8 kb fragment to be deleted during the VA1J1 rearrangement process contains a rearrangement silencer and adjacent anti-silencer sites [Lauster, R. etal. (1993) EMBOJ. 12: 4815-231. This fragment is also a transcriptional silencer in CAT-assays perfoned on mouse pre-B cells. Materialsand Methods: We have reduced the silencer to a 150 bp minimal element in CAT-assay and tested its effect on rearrangement in transgenic mice. The 150 bp minimal element has been further divided into smaller fragments and their respective specific contributions to the silencer effect has been analysed in CAT- and gel-retardation assays. RaaultszThe minimal silencer reduces rearrangement in transgenics to levels 10 times lower that those obtained with a control neutral globin fragment. The 150 bp fragment can be divided into 4 specific fragments by gel retardation assay using extracts from cells positive for silencing. The deletion of a 29 bp fragment completely abolishes the transcriptional silencing. Conclusion: Mouse pre-B cells contain highly consenred factors which can regulate rearrangement of a chicken light chain locus. Moreover, a putative anti-silencer Site has alS0 been identified in the mouse VkJk Segment Upstream of the Jk locus [Ferradini L. et al. (1998) Science 271: 1418-20], thus strongly suggesting that such regulation of the rearrangement process may be general.
P2.02.26
CD1 1b is an activation marker expressed on a laroe woulation of SheeD DeriDheral blood B cells “urn
.
.
.
.
N. Chevallier, M. Berthelemy, D. Le Rhun, V. Lain& F. FbmBnia, B. Polack, D. Levy, I. Schwartz-Comil. UFIA INRA IPCM, 7 avenue du G6ntW de Gaulle, 94704 Maisons Alfvrt cedex, France Introduction: Restricted usage of the V genes is the hallmark of human, mouse and rabbit CD5+ B cells also called Bl cells. BI cells are differently represented among species and make 1% of the circulating B lymphocytes in mice, 10 to 30% in humans and 90% in rabbits. Strikingly, the sheep VH and Vi repertoire usage is limited to few genes but the circulating sheep B cells have been reported not to express the CD5 molecule. Results: We show here that a large proportion of sheep peripheral blood B cells (40 to 80%) express the LeuCAM CD11b molecule which is usually found on Bi cells in the mouse. Similarly to the mouse Bl cells, the sheep CD11b+ B cells were shown to express CDllc, high levels of IgM and low levels of CD5. Light scattering properties and cell cycle analyses demonstrated that the CD11b+ B lvmohocvtes are larger and show a higher proliferative activity as compared to the ‘CDli b- B lymphocytes, suggesting that CD11b expression is ass&iated to a pre-activation or to an activation stage. The activation/pre-actfvation stage of the CD11b+ B cells was also illustrated-by their higher pr&ensy to spontan&us apoptosis. FurthemxJre, CDIIb expression appeared and/or increased on B cells following in v&activation with ionomycin and not with PMA or anti p chain antibodies and the modulation of CD11b expression on B cells was dependent on monocytes. Conclusion: These data suggest that CD11b expression defines a differentiation stage of the sheep B cells that may be majoritarely composed of the mouse equivalent Bl cells.
P.2.02.27
Phenotypic anaiyaia cord blood B lymphocytes
R. Ezsi I, S. Valent*, F. Paulin2, P. Farkas3, Pettinyi G. Gy’ , K. Paloczi ‘. ’ National Institute of Hematology and Immunology, kemm&weis Universify of Medicine, Budapest, Hungary; *2nd Dept. of obstetrics and Gynecolcg~ Semmelweis University of Medicine, Budapest, Hungary, 3 3rd Dept. of internal Medicine, Semmelweis University of Medicine, Budapest, Hungary
Introduction: The phenotypic analysis of human umbilical cord blood (CB) mononuclear cells is important to study their maturity and differentiation regarding their transplantable capacity.
Materiala and Methods: In this work we have studied the differential exoression of Bcell antigens on CDC-/HlA-DR+ B cells (BI b, 82) and CD5+RILA:DR+ cells (Bla) from the CB (n = 8) and adult peripheral blood (PB) (n = 8). CDCPE, HlA-DR-PetCP and FITC labeled anti-8 cell MoAb panel of the 8th International Workshop on Human Differentiation Antigens were used for detection of B cell subpopulations. FacsCalibur (B-D) flow cytometer was used for evaluation of triple-labeled cells. ReauRa: CB Bla (CDmlA-DR++) cells proved to be positive with CD9, CD19, CD20, CD21, CD22, CD23, CD24, CD32, CD37, CD39, CD45RA. CD78. CD79, MHC-II, IgM and anti-lg light chains MoAbs. CB Bib (CDB-/HLA-DR+) cells reacted with CD9, CD19, CD20, CD21, CD22, CD32, CD45RA, CD79. MHC-II, IgM and K or A MoAbs. PB B cells (B2) expressed CDIQ, CD20, CD21, CD22, CD24, CD32, CD37, Class-II, CD78, CD79 antigens. Conclusion: Unlike to the PB the CB B lymphocytes proved to be predominantly BI cells representing a new-born B cell repertoire. Besideclexpressing many B cell antigens both the CB Bla and Blb cells showed CD9+, CD45RA+, IgM+ immature, “naive” B cell phenotype. Functionally, Bla cells are capable producing polyreactfve IgM and natural autoantibodies but not IgG. This antibody profil might be insufficient regarding the recipient humoral immune defense result in more severe immunodeficiency after CB transplantation.
P.2.02.28
Fetal B ceil development
J. Keamey, F. Martin, C. Benedict, A. Oliver. Division of Developmental and Clinical Immunology, Department of Microbiology Univezsify of Alabama at Birmingham, Birmingham, AL, USA
To study the role of restricted diversity in the early repertoire, we produced tranagenic mice with a rearranged VHI~X-@ heavy chain gene isolated from the pre-immune repertoire in fetal liver and also transgenic mice in which Tdt is expressed during fetal B cell development. There are developmental stages during which VHBlX-expressing B cell progenitors expand or reduce comparIment sizes in fetal liver and bone marrow. These include a reduction in the early B22O+CD43+compartments which is dependent on the association of the transgene with 15. In contrast, pm-B and immature B cell compartments are expanded in the fetus but not in the adult. In the periphery of these mice, 295% of B cells express the transgene in association with a variety of kappa light chains. Lambda bearing B cells do not develop in these mice and appear to be blocked at the immature B cell stage. However, there are populations of self-reactive B cells with a VKICJK~ rearrangement which have ihe phenotype and localization pattern of long-lived marginal zone B cells. In the second transoenic line. where oremature Tdt exoression occurs in fetal liver, a large population of ire-B ceils and B cells is absent in perlnatal liver and spleen until some time after birth. These results suggest that N region addition is not tolerated in fetal B cells which are deleted. The disappearance of this subset of B cells has the functional consequem that the T15 Id portion of anti-PC response is absent in adults. This finding supports previoussvidence that B cell precursors for certain clonally-restricted responses are generated early in development and suggests that a reason for the absence of Tdt activity in fetal life is to pemlt the generation of a restricted germline-encoded repertoire. (Supported by NIH grant Al14782)
09:00-18:30/12:0&14:00
Forum lounges
P.2.03
Unconventional lymphocyte subsets
P.2.03.01
A distinct and persistent CD4dUkD8MgM double positive Flymphocyta subpopulation In a renal transplant patient with cytomegalovirua reactivation under immunoauppreaaion
R.J. Rentenaar I.*, P.C. Wever 1,2,F.N.J. van Diepen 2, P.Th.A. Schellekens *, T.A. Out”, S. Surachno’, R.J.M. ten Berge1.2. ‘Rena/ Transplant Unit, Academic Medical Center; Amsterdam, The Netherlands, 2Clinkal and Labrarory Immunology Unir, Academic Medical Center; Amsterdam, The Netherlands
double positive (DP) T-lymIntroduction: The presence of a CD4brigh*CD8dU” ohccvte suboooulation is freouentlv observed in healthv individuals as well as in &&iation &ith various diseases.-In contrast, a CD4di”CD8b*ht DP T-lymphocyte subpopulation has been described in association with acute viral infection only (C. Ortolani et al. 1993). These cells disappeared from the peripheral blood
Unconventional lymphocyte subsets
23 June 1997 - Poster presentations
clones were selected and one of them was able to induce the exoression of 58 kDa glycoprotein on Cos 7-trans#ectedcells as demonstrated b; staining with NIM-R7 mAb. The clone was named pNV7. The sequencing of pNV7 is under progress. Conclusion: The 58 kDa glycoprotein is present in all stages of maturation on the surlace of B cells, but staining is higher wlth the most mature cell lines. The clone pNV7 has an insert of 1700 bp likely to encode ~58 antigen. The data of the sequence will be presented at the meeting.
P.2.02.25
A 150 bp element can silence rearrangement of the chicken immunoglobulin lambda locus in tranaaenic mice
L. Cocea, A. Dahan, L. Ferradini, C.-A. Reynaud, J.-C. Weill. lnstifuf National Sante et de la RechercheMBdicale,Ftmce
de la
Introduction: The regulation of rearrangement and allelic exclusion of immunoglobulin loci is complex and could require both positive and negative elements. We have previously shown in mice transgenic for the chicken immunoglobulin lambda locus that the 1.8 kb fragment to be deleted during the VA1J1 rearrangement process contains a rearrangement silencer and adjacent anti-silencer sites [Lauster, R. etal. (1993) EMBOJ. 12: 4815-231. This fragment is also a transcriptional silencer in CAT-assays perfoned on mouse pre-B cells. Materialsand Methods: We have reduced the silencer to a 150 bp minimal element in CAT-assay and tested its effect on rearrangement in transgenic mice. The 150 bp minimal element has been further divided into smaller fragments and their respective specific contributions to the silencer effect has been analysed in CAT- and gel-retardation assays. RaaultszThe minimal silencer reduces rearrangement in transgenics to levels 10 times lower that those obtained with a control neutral globin fragment. The 150 bp fragment can be divided into 4 specific fragments by gel retardation assay using extracts from cells positive for silencing. The deletion of a 29 bp fragment completely abolishes the transcriptional silencing. Conclusion: Mouse pre-B cells contain highly consenred factors which can regulate rearrangement of a chicken light chain locus. Moreover, a putative anti-silencer Site has alS0 been identified in the mouse VkJk Segment Upstream of the Jk locus [Ferradini L. et al. (1998) Science 271: 1418-20], thus strongly suggesting that such regulation of the rearrangement process may be general.
P2.02.26
CD1 1b is an activation marker expressed on a laroe woulation of SheeD DeriDheral blood B cells “urn
.
.
.
.
N. Chevallier, M. Berthelemy, D. Le Rhun, V. Lain& F. FbmBnia, B. Polack, D. Levy, I. Schwartz-Comil. UFIA INRA IPCM, 7 avenue du G6ntW de Gaulle, 94704 Maisons Alfvrt cedex, France Introduction: Restricted usage of the V genes is the hallmark of human, mouse and rabbit CD5+ B cells also called Bl cells. BI cells are differently represented among species and make 1% of the circulating B lymphocytes in mice, 10 to 30% in humans and 90% in rabbits. Strikingly, the sheep VH and Vi repertoire usage is limited to few genes but the circulating sheep B cells have been reported not to express the CD5 molecule. Results: We show here that a large proportion of sheep peripheral blood B cells (40 to 80%) express the LeuCAM CD11b molecule which is usually found on Bi cells in the mouse. Similarly to the mouse Bl cells, the sheep CD11b+ B cells were shown to express CDllc, high levels of IgM and low levels of CD5. Light scattering properties and cell cycle analyses demonstrated that the CD11b+ B lvmohocvtes are larger and show a higher proliferative activity as compared to the ‘CDli b- B lymphocytes, suggesting that CD11b expression is ass&iated to a pre-activation or to an activation stage. The activation/pre-actfvation stage of the CD11b+ B cells was also illustrated-by their higher pr&ensy to spontan&us apoptosis. FurthemxJre, CDIIb expression appeared and/or increased on B cells following in v&activation with ionomycin and not with PMA or anti p chain antibodies and the modulation of CD11b expression on B cells was dependent on monocytes. Conclusion: These data suggest that CD11b expression defines a differentiation stage of the sheep B cells that may be majoritarely composed of the mouse equivalent Bl cells.
P.2.02.27
Phenotypic anaiyaia cord blood B lymphocytes
R. Ezsi I, S. Valent*, F. Paulin2, P. Farkas3, Pettinyi G. Gy’ , K. Paloczi ‘. ’ National Institute of Hematology and Immunology, kemm&weis Universify of Medicine, Budapest, Hungary; *2nd Dept. of obstetrics and Gynecolcg~ Semmelweis University of Medicine, Budapest, Hungary, 3 3rd Dept. of internal Medicine, Semmelweis University of Medicine, Budapest, Hungary
Introduction: The phenotypic analysis of human umbilical cord blood (CB) mononuclear cells is important to study their maturity and differentiation regarding their transplantable capacity.
Materiala and Methods: In this work we have studied the differential exoression of Bcell antigens on CDC-/HlA-DR+ B cells (BI b, 82) and CD5+RILA:DR+ cells (Bla) from the CB (n = 8) and adult peripheral blood (PB) (n = 8). CDCPE, HlA-DR-PetCP and FITC labeled anti-8 cell MoAb panel of the 8th International Workshop on Human Differentiation Antigens were used for detection of B cell subpopulations. FacsCalibur (B-D) flow cytometer was used for evaluation of triple-labeled cells. ReauRa: CB Bla (CDmlA-DR++) cells proved to be positive with CD9, CD19, CD20, CD21, CD22, CD23, CD24, CD32, CD37, CD39, CD45RA. CD78. CD79, MHC-II, IgM and anti-lg light chains MoAbs. CB Bib (CDB-/HLA-DR+) cells reacted with CD9, CD19, CD20, CD21, CD22, CD32, CD45RA, CD79. MHC-II, IgM and K or A MoAbs. PB B cells (B2) expressed CDIQ, CD20, CD21, CD22, CD24, CD32, CD37, Class-II, CD78, CD79 antigens. Conclusion: Unlike to the PB the CB B lymphocytes proved to be predominantly BI cells representing a new-born B cell repertoire. Besideclexpressing many B cell antigens both the CB Bla and Blb cells showed CD9+, CD45RA+, IgM+ immature, “naive” B cell phenotype. Functionally, Bla cells are capable producing polyreactfve IgM and natural autoantibodies but not IgG. This antibody profil might be insufficient regarding the recipient humoral immune defense result in more severe immunodeficiency after CB transplantation.
P.2.02.28
Fetal B ceil development
J. Keamey, F. Martin, C. Benedict, A. Oliver. Division of Developmental and Clinical Immunology, Department of Microbiology Univezsify of Alabama at Birmingham, Birmingham, AL, USA
To study the role of restricted diversity in the early repertoire, we produced tranagenic mice with a rearranged VHI~X-@ heavy chain gene isolated from the pre-immune repertoire in fetal liver and also transgenic mice in which Tdt is expressed during fetal B cell development. There are developmental stages during which VHBlX-expressing B cell progenitors expand or reduce comparIment sizes in fetal liver and bone marrow. These include a reduction in the early B22O+CD43+compartments which is dependent on the association of the transgene with 15. In contrast, pm-B and immature B cell compartments are expanded in the fetus but not in the adult. In the periphery of these mice, 295% of B cells express the transgene in association with a variety of kappa light chains. Lambda bearing B cells do not develop in these mice and appear to be blocked at the immature B cell stage. However, there are populations of self-reactive B cells with a VKICJK~ rearrangement which have ihe phenotype and localization pattern of long-lived marginal zone B cells. In the second transoenic line. where oremature Tdt exoression occurs in fetal liver, a large population of ire-B ceils and B cells is absent in perlnatal liver and spleen until some time after birth. These results suggest that N region addition is not tolerated in fetal B cells which are deleted. The disappearance of this subset of B cells has the functional consequem that the T15 Id portion of anti-PC response is absent in adults. This finding supports previoussvidence that B cell precursors for certain clonally-restricted responses are generated early in development and suggests that a reason for the absence of Tdt activity in fetal life is to pemlt the generation of a restricted germline-encoded repertoire. (Supported by NIH grant Al14782)
09:00-18:30/12:0&14:00
Forum lounges
P.2.03
Unconventional lymphocyte subsets
P.2.03.01
A distinct and persistent CD4dUkD8MgM double positive Flymphocyta subpopulation In a renal transplant patient with cytomegalovirua reactivation under immunoauppreaaion
R.J. Rentenaar I.*, P.C. Wever 1,2,F.N.J. van Diepen 2, P.Th.A. Schellekens *, T.A. Out”, S. Surachno’, R.J.M. ten Berge1.2. ‘Rena/ Transplant Unit, Academic Medical Center; Amsterdam, The Netherlands, 2Clinkal and Labrarory Immunology Unir, Academic Medical Center; Amsterdam, The Netherlands
double positive (DP) T-lymIntroduction: The presence of a CD4brigh*CD8dU” ohccvte suboooulation is freouentlv observed in healthv individuals as well as in &&iation &ith various diseases.-In contrast, a CD4di”CD8b*ht DP T-lymphocyte subpopulation has been described in association with acute viral infection only (C. Ortolani et al. 1993). These cells disappeared from the peripheral blood