Davina Opstelten

RT7-defined alloantigens in rats are part of the leucocyte common antigen family.

Haemopoietic cells carry a variety of cell‐surface molecules, some of which are known to have allotypic variation. In rats, the RT7 alloantigenic system has been well documented using alloantisera. We have produced the first mouse hybridomn cell line secreting an antibody. H1S41, which binds to leucocytes of rat strains carrying the RT7.2 but not the RT7.1 determinant. An lgG2b isotype switch variant (HIS4l.2b) of the original HIS41 (IgG1 isotype) was also made. HIS41 showed a clear and discrete binding in immunofluorescenl and histological experiments and has already been used in several studies on haemopoietic cell turnover and differentiation employing PVG rats congenic for RT7. The present study addresses the question of whether the RT7 gene products are members of the L‐CA family, which has been a matter of controversy over the last decade. When using HIS41 for the analysis of tissue distribution and molecular weight of RT7 gene products, a strong similarity was evident with the data reported for the L‐CA detected by MRC OX‐1 and MRC OX‐30. These two MoAb have been reported to bind to all members of the L‐CA family. All haemopoietic cells, excluding erythrocytes and the more mature stages of erythropoiesis, stained with HIS41. The molecular weights of HIS41 binding molecules on thymocytes and peripheral T cells were comparable to the L‐CA precipitated by MRC OX‐1. Capping and sequential immunoprecipitation studies indicated that HIS41 and MRC OX‐30‐ binding molecules were identical. MRC OX‐1. however, appeared to bind only a subset of these molecules. Thus, our study confirms the identity of RT7.2 gene products and L‐CA. lt also revealed a difference between MRC OX‐1 and MRC OX‐30 not noticed previously.

Monoclonal Antibodies to Rat B Lymphocyte (Sub-)Populations

The study of B lymphocyte differentiation is greatly facilitated by the use of suitable monoclonal antibodies (MAb’s). For mice and men a variety of MAb’s binding to cell surface determinants of B lineage cells has been described (1). However, as yet almost no B lineage specific MAb’s have been described for the rat. Since this animal appears very appropiate for studies of early phases of B lymphocyte differentiation (2) as well as late phases, in particular with respect to marginal zone cells (3), we produced MAb’s against surface antigens of rat B lineage cells.

In situ visualization of hemopoietic cell subsets and stromal elements in rat and mouse bone marrow by immunostaining of frozen sections.

We have developed a method to section frozen long bones of rat and mouse and stained bone marrow (BM) by (double) immunofluorescence and immunoperoxidase. Here we report this method and reveal the location of early hemopoietic progenitors (Thy-1) and myeloid cells (Mac-1) in mouse BM, and early hemopoietic progenitors and lymphoid cells (Thy-1), erythroid cells (HIS49), and macrophages (ED2) in rat BM. In mouse BM our new findings include (a) the scattered localization of early hemopoietic progenitors (Thy-1low) all over the marrow, and (b) the presence of Thy-1+ stromal cells, mainly subendosteally. In rat BM an important finding is that of (a) a subendosteal region of 12-14 hemopoietic cell layers characterized by an abundance of Thy-1 and the virtual absence of erythroid cells, and (b) the scattering of Thy-1very bright cells which are candidates for the earliest hemopoietic progenitors in this species. The results illustrate that the technique is an excellent tool for studying the topology of BM as an organ of hemopoiesis.

Germinal centres and the B cell system

SummaryFollowing appendectomy and sublethal (450 rads) whole body X-irradiation, reconstitution with autologous appendix germinal centre cells led to excellent germinal centre formation in the recipient’s spleen. Heat killing of the cells to be injected completely abolished this phenomenon. Apparently a suspension of appendix germinal centre cells contains a (sub)populations of cells capable of germinal centre formation (Germinal CentrePrecursor Cells).Following whole body X-irradiation while shielding the appendix, germinal centre activity could only be detected after antigenic stimulation in otherwise normally regenerated splenic follicular structures. This suggests that the cell population recentlyderived from appendix germinal centres is able to function once again as a population of germinal centreprecursors.

GERMINAL-CENTERS AND THE B-CELL SYSTEM .4. FUNCTIONAL-CHARACTERISTICS OF RABBIT APPENDIX GERMINAL CENTER (-DERIVED) CELLS

SummaryFunctional capacities of rabbit appendix germinal centre cells were tested in appendectomized, 450 rads X-irradiated rabbits, reconstituted with autologous appendix germinal centre cells, which are obtained by mechanical separation - “stripping” - of the appendix. For comparison, functional capacities of appendix germinal centre derived cells were tested in rabbits, 450 rads X-irradiated with the appendix shielded. In both types of experimental systems, slow restoration of primary IgM responsiveness to Salmonella Java paratyphi-B (PAR) was observed ; in contrast, the capacity for memory cell production (of both μ and γ-type) to PAR was restored very early in both systems. These results suggest germinal centres to be antigen dependent microenvironments for selective as well as non-selective amplification ofimmature B cells, which after subsequent maturation become part of the pool of either memory or virgin mature B cells respectively.

B lymphocyte development and transcription regulation in vivo.

Publisher Summary The targeted mutation and ectopic or forced expression assays in vivo have helped identify genes that encode factors necessary for normal B cell development. In somewhat more depth, spatial aspects of transcription regulation are outlined in this chapter, as these are crucial to the understanding of how well-balanced specific changes in gene expression are achieved in vivo . The current knowledge of B cell development in the fetal, neonatal, young adult, and older mouse is reviewed, with an emphasis on the changes in gene expression patterns. The advancement in understanding the marrow organ as inductive microenvironment for postnatal B cell development is also highlighted. The chapter also reviews how the experiments on transcription regulation enrich the insight in pathways of B cell genesis. Further identification of genes disturbed in their expression, including those encoding cell cycle and death regulators, will give insight in genetic programs operating in hemopoiesis and B cell development. Novel genetic tools in vivo allows a follow-up of the effect of mutations in healthier animals. Transcription factors necessary for B cell development may be tested further for the ability to drive the differentiation pathways by ectopic expression in suitable cell types, for example, in hemopoietic stem and progenitor cells, by using the control region of the CD34 gene. The current information on the phenotype of mice with mutations in transcription factors indicates that much valuable information may still be retrieved from them. Together with the growing insight in surface receptor engagement and signaling pathways, an integrative vision of B cell development in vivo is emerging.

LYMPHOCYTE MIGRATION ACROSS MAJOR HISTOCOMPATIBILITY BARRIERS IN SPLENECTOMIZED RATS

Localisation and migration patterns of iv injected radio-labelled thoracic duct (TD) lymphocytes were studied in particular with regard to passage through lymph nodes and re-entry into thoracic duct lymph. To avoid unwanted splenic sequestration of migrating lymphocytes presenting alloantigens to the recipient, only splenectomized recipients were used. Donor cells and recipients differed at the MHC (RT-1) locus, either in fully allogeneic (AO -- greater than BN and v.v.) or semi-allogeneic (AO -- greater than AO X BN and v.v.) combinations. In two of these combinations (BN -- greater than AO and AO X BN -- greater than AO) deficient output in TD lymph correlated with deficient localisation in lymph nodes and high amounts of radioactivity in the liver. In the other allogeneic combination (AO -- greater than BN), however, high TD output (i.e. when compared with the syngeneic combination BN -- greater than BN) correlated with good localisation in lymph nodes and low (control) levels of radioactivity in the liver. It was postulated that lymphocyte migration from blood to lymph under these circumstances can only be studied as an artifact secondary to whether or not migrating cells are removed from the circulation before they can reach and cross HEVs. These Allogeneic (or Altered) Lymphocytes Removing Tissues (by definition: Extranodular) may (conceptually) be comprised within one system: ALERT. It is our working hypothesis that the study of lymphocyte migration across (major) histocompatibility barriers is seriously impaired by the functioning of ALERT. It might be worthwhile to try and create conditions in which interference by this system is prevented, e.g. by using tolerant animals or bone-marrow chimeras.

Germinal Centers and the B Cell System VIII. Functional Characteristics and Cell Surface Markers of Germinal Center Cell Subsets Differing in Density and in Sedimentation Velocity

Germinal center cells from the rabbit appendix were fractionated by velocity sedimentation and isopycnic gradient centrifugation. Subsets were analysed with respect to cell size and surface markers, and were functionally characterized by testing the capacities for primary antibody synthesis, memory cell production, and formation of new germinal centers in an autologous transfer system. The migratory behaviour of the germinal center cell subsets within the spleen of homologous recipients was also studied using autoradiography. Both cell fractionation methods yielded a separation of large and small cells. Surface immunoglobulin and C3 receptors were equally expressed on germinal center cells differing in size and density. The different subsets were also equally capable in giving rise to IgM-antibody-forming cells and memory cells upon antigenic stimulation. Furthermore, large germinal centers were newly formed in the spleen of the recipients, irrespective of the cell subset injected. It was concluded that the results do not support the hypothesis that, inside germinal centers, the differentiation of large lymphoid cells (centro-blasts) into small centrocytes also implies a maturation process. Subsets of germinal center cells, however, showed a different and characteristic migratory behaviour; while small cells migrated preferentially to the corona of lymphocytes in spleen follicles, large, light cells showed an affinity for the germinal center area. We postulate that, upon stimulation, immature B cells develop an affinity for the germinal center microenvironment, to participate in a germinal center reaction.

Expression of HIS50 Ag: a rat homologue of mouse heat-stable antigen and human CD24 on B lymphoid cells in the rat

Recently, a cDNA encoding a newly identified rat antigen (HIS50 Ag) that binds to monoclonal antibody (mAb) HIS50 was cloned and shown to be homologous to cDNA encoding murine heat‐stable antigen (HSA) and human CD24. Here we show that, like CD24 and HSA, at least part of HIS50 Ag is inserted into the plasma membrane by a glycosylphosphatidylinositol (GPI)–lipid linkage and we describe its expression in rat haemolymphopoietic tissues. HIS50 Ag expression was almost exclusively confined to B lymphoid cells, the vast majority of T lymphoid cells, erythroid and myeloid cells were HIS50−. Cell suspension analysis indicated that in bone marrow (BM) almost all Thy‐1+ cells, HIS24+ cells [ HIS24 recognizes the B‐cell form of leucocyte common antigen (LCA)], terminal deoxynucleotidyl transferase‐positive (TdT+) cells and (c+s)κ+ cells expressed HIS50 Ag, and all (c+s)μ+ cells. A presumably early population of B lymphoid cells, expressing HIS24 Ag without HIS50 Ag, TdT or immunoglobulin (HIS24+HIS50−TdT−Ig−), constituted 1·6% of BM nucleated cells. In blood, one‐fifth of mononuclear cells were HIS50+, and about 85% of these expressed μ and/or κ chains. In spleen, flow cytometry analysis and immunohistology demonstrated heterogeneous expression of HIS50 Ag: immunoglobulin M (IgM)bright cells (as found largely in red pulp and marginal zone) were HIS50bright, while IgMdull cells expressed low or undetectable levels of HIS50 Ag. Germinal centre B cells expressed high levels of HIS50 Ag. Germinal centres of lymph nodes and tonsil of man also bound HIS50. We conclude that HIS50 Ag expression in the haemolymphopoietic system of rat is virtually restricted to the B lineage.

PRE-B-CELLS IN RAT BONE-MARROW - IDENTIFICATION, SURFACE-MARKERS AND ISOLATION

In adult rats, as in other species, bone marrow has the highest potential for B-lymphocyte genesis as determined by a long-term repopulation assay for B-lymphocyte stem cells (1). These cells carry the Thy-1 and W3/13 antigens (2, 3). Pre-B cells characterized by the presence of µ heavy chains in the cytoplasm (cµ) but not on the cell surface (sµ) have been described in mice (4), men (5) and rabbits (6) but not in rats, and appear to be the immediate precursors of B-lymphocytes (7–10). The relation between pre-B cells and B-lymphocyte stem cells is not clear, however. Therefore, the aims of the present study were to identify cµ+sµ- pre-B cells in rat bone marrow and to find surface markers that would permit isolation of viable pre-B cells to subsequently assay their functional potential in vivo.