Gerrit Jan Deenen

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.

Development of components of the mucosal immune system in SCID recipient mice.

We have used adoptive transfer of congenic lymphoid cells from different tissue sources into severe combined immunodeficient (SCID) mice to: (1) compare the contributions of Bl B cells from the peritoneal cavity (PeC) and B2 B cells from Peyer’s patches (PP) to the pool of splenic (Spl) IgM plasma cells and mesenteric lymph node (MLN) and gut lamina propria (LP) IgA plasma cells, and (2) assess the potential of T cell precursors from bone marrow (BM) and PP to give rise to α/β TCR+, CD8+ T cells in the intraepithelial; leukocyte (IEL) compartment upon oral infection with enteric reovirus.

Germinal centers and the B-cell system

SummaryThe migration pattern of germinal center cells of the rabbit appendix was studied and compared with that of appendix dome cells, spleen cells, thymus cells and thoracic duct lymphocytes. To discriminate T-and B-cell migration pathways, normal or T-cell-depleted rabbits were used as donors. Cell suspensions were labeled in vitro with 3H-leucine followed by intravenous transfer. The migration of labeled cells in lymphoid organs was studied using autoradiography, particular attention being paid to the spleen of the recipient. B-cells from the appendix dome, spleen and thoracic-duct lymph migrate to primary follicles or the corona of secondary follicles via thymus-dependent areas of peripheral lymphoid organs. In contrast, a B-cell subpopulation from the germinal centers of the appendix migrates to the center of splenic primary follicles and into germinal centers. The migration of germinal center cells to splenic follicle centers is not enhanced by specific antigens. The migration properties of B-cells, possibly changing during differentiation, may be instrumental in the two types of immune reactions, i.e., plasma-cell reaction and germinal-center reaction.

A Dual Origin for IgA Plasma Cells in the Murine Small Intestine

More than two decades ago, Craig and Cebra1 showed that Peyer’s patches are an important source of progenitor cells for intestinal IgA plasma cells. The vast majority of B cells in Peyer’s patches are conventional B cells, which are produced throughout the life of the animal and which are responsible for high-affinity antibody responses to a variety of antigens. More recently, we provided evidence that probably also B-l cells (previously called Ly-1 or CD5 B cells2) also contribute significantly to the population of IgA plasma cells in the gut, at least in B lineage chimeras.3,4 B-l cells are almost absent from Peyer’s patches and are enriched in the peritoneal cavity. These cells are largely self-replenishing and have a selected antibody repertoire with specificities frequently directed towards “natural antigens”, autoantigens and bacteria-related antigens.5,6 In studies presented here we provide additional data, both from transfer studies with sorted B-l cells and from analysis of JLI,K transgenic mice, to support our hypothesis that B-l cells can contribute to the IgA response of the gut.

IN VIVO IMMUNOLOGY

P. D. Weinstein,!.2 R. G. Mage,2 and A. O. Anderson! !ARD, USAMRIID, Ft. Detrick, Frederick, MD 21702 and 2LI/NIAID, NIH, Bldg. 10, Rm lIN311, Bethesda, MD 20892 In this paper we present genomic DNA sequence and histological evidence that the appendix is a site of diversification of the rabbits primary antibody repertoire. By 6 weeks after birth, the B cell follicular regions of the rabbit appendix and the distribution of the resident lymphoid cells bear a strong morphological resemblance to similar regions within two primary lymphoid tissues, the chicken bursa and the sheep ileal Peyers patch. However, similarities between the rabbit appendix, chicken bursa and sheep ileal Peyers patch end as these animals reach adulthood. The rabbit appendix undergoes morphological and cellular distribution changes as it matures taking on the appearance of a secondary lymphoid tissue, while the sheep ileal Peyers patch and the chicken bursa both involute. We determined DNA sequences of PCR amplified rearranged variable region genes from germinal center B cells of 6 week old rabbits isolated from several different appendix dark zones and light zones. There was a trend toward a higher degree of diversification from the germ-line VH gene DNA sequence in dark zones than light zones. It is likely that both gene conversion and somatic hypermutation are responsible for the nucleotide changes we observed. Our findings suggest that the rabbit appendix functions as a mammalian bursal equivalent early in development. As the rabbit matures, the appendix appears to evolve into a secondary lymphoid tissue resembling secondary GALT in appearance and possibly in function.

Homing of germinal-center cells into germinal centers of lymph node via afferent lymphatics

SummaryAffinity of lymphoid cells for the microenvironment of germinal centers (GC), as detectable in transfer experiments by rapid homing in spleen GC from the blood, is a capacity expressed by only a subset of lymphoid cells, in particular by those constituting a GC. However, when introduced into the blood stream, these cells do not home into GC of lymph nodes and gut-associated lymphoid tissues. To investigate further this homing inability for high endothelial venule (HEV)-containing lymphoid tissues, GC cells isolated from donor rabbit appendix were labeled in vitro with 3H-leucine and injected into an afferent lymph vessel of recipient popliteal lymph nodes. Draining lymph nodes were removed 15 min to 24 h after cell administration and prepared for radioautography. For reference, the migration of cells isolated from Peyers patches and thoracic duct lymph was also studied. By use of appendix GC cells, large numbers of labeled cells were found to migrate into GCs of the outer cortex centripetally, i.e., from the subcapsular sinus through the lymphocyte corona into the GC proper. The same was observed for cells from Peyers patches, although in smaller numbers. Thoracic duct lymphocytes were only localized in the lymphocyte corona and the deep cortex. Thus, appendix GC cells and a subpopulation of cells from Peyers patches can reach lymph node GC, but only when administered intralymphatically. We conclude that cells expressing affinity for the GC microenvironment do so for both spleen and lymph node GC, but do not have the capacity to interact with the wall of HEV; its implication for the understanding of the dynamics of a GC reaction is discussed.

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.

Identification of a novel rat B cell subset in the peritoneal cavity of xenogeneic rat to mouse scid chimeras

In the mouse B-1 cells represent a small subset of B cells with a unique phenotype, anatomical distribution pattern, function and developmental origin.1 In essence, these B cells are self-replenishing cells that are strongly enriched in the peritoneal cavity (PerC) as IgMhighIgDlowCD11blowB220low cells. Most of the peritoneal B-1 cells also express low levels of CD5 (B-la cells) whereas a minority lack CD5 completely (B-1b cells). So far, we have not been able to unequivocally detect the presence of B-1 lineage cells (or their homologues) in rats.2–4 In adult animals, CD5-expressing B cells are absent, whereas in neonates all (splenic) B cells seem to express low amounts of CD5, which become gradually lower with age. Possible detection of B-1 cell (homologues) in rats might be hampered by the notion that in this species there are virtually no B cells in the PerC, in marked contrast to mice.2,3 Here we studied whether the relative absence of B cells from the rat PerC is an inherent property of the leukocytes themselves or due to non-leukocyte factors, such as a difference in microscopical anatomic organization or properties of mesothelial cells. To this end, we constructed xenogeneic rat-to-mouse SCID chimeras by injecting rat fetal liver (FL) cells into lightly irradiated C.B17 SCID mice. As shown by Surh and Sprent rat B cells develop readily in the mouse SCID environment.5 Here we demonstrate that the PerC of these xenogenic rat-to-mouse SCID chimeras harbor high numbers of B cells. In addition, we show that a relative high proportion of the PerC B cells have a unique, previously undetected phenotype (IgMhighIgDlowHIS24/B220lowThy-1high), to some extent resembling splenic marginal zone B cells. We speculate that this novel B cell subset might represent the rat homologue of the B-1 cells in the mouse.