Loss of surface immunoglobulin expression precedes B cell death by apoptosis in the bursa of Fabricius.

The vast majority of lymphocytes generated daily in the chicken bursa of Fabricius do not emigrate to the periphery but die in situ. Apoptotic cells in the bursa can be readily detected by the presence of fragmented DNA and by the large numbers of condensed cellular nuclei observed by electron microscopy. Consequently, most newly generated lymphocytes die by programmed cell death. We show that bursal cells divide rapidly and apoptotic cells are derived from rapidly dividing precursors. Analysis of the phenotype of bursal cells undergoing apoptosis demonstrated that cell death does not occur in the most mature bursal cell population and is therefore not random. High levels of surface Ig are expressed on bursal cells entering S phase of the cell cycle. In contrast, bursal cells in the early stages of apoptosis in vivo express very low to undetectable levels of surface Ig but were unequivocally confirmed as being of the B lineage by polymerase chain reaction (PCR) detection of rearranged Ig genes. Bursal cells induced to undergo apoptosis in vitro express high levels of surface Ig demonstrating that induction of apoptosis does not in itself induce a loss of surface Ig expression. Consequently, loss of surface Ig expression precedes bursal cell death by apoptosis in vivo, suggesting that maintenance of a threshold level of surface Ig may be a requirement for the continued progression of chicken B lymphocyte development in the bursa.

Survivors of bursal B cell production and emigration.

The bursa of Fabricius is the site of primary B cell generation in the chicken. Analysis of the rate of bursal cell emigration demonstrated that about 1% of the blood B cell pool was replaced per hour by bursal emigrants. Surgical bursectomy distinguished three populations of blood B cells. About 60% of blood B cells in 3-wk-old chickens were short lived, with a lifespan of 2 to 3 d, and included most bursal emigrants. These cells migrate directly from the bursal follicular cortex to the periphery, express the LT2 antigen, and are proposed to represent a diversified repertoire of B cell specificities that have emigrated from the bursa in the absence of interaction with environmentally derived antigens in the follicular medulla. About 35% of blood B cells were much longer lived cells, having a lifespan exceeding 2 wk. These cells represent about 10% of bursal emigrants, which do not express the LT2 antigen and do not divide in the peripheral blood following emigration from the bursa. This population may represent cells that have undergone positive selection in the bursa by interacting with environmentally derived antigens and should, therefore, contain a restricted repertoire of B cell specificities. The third population, about 5% of blood B cells, were short-lived cells that represent the progeny of postbursal B cell production. The frequency of these cells progressively increases with time, taking over from the short-lived bursal emigrants as the bursa involutes, likely as a diversified repertoire of B cell specificities.

Lymphoid signal transduction mechanisms linked to cellular prion protein.

The normal cellular isoform of the prion protein (PrPC) is a glycosylphosphatidylinositol-anchored cell surface protein that is expressed widely, including in lymphoid cells. We compared lectin-induced mitogenesis and selected cell signaling pathways in splenocytes from wild-type BALB/c mice and Zrch Prnp0/0 (PrP0/0) mice bred on a BALB/c background for more than 10 generations. 3H-thymidine incorporation induced by concanavalin A (Con A) or phytohemagglutinin (PHA) was significantly reduced in PrP0/0 splenocytes, most prominently early in activation (24 and 48 h). Con A activation in PrP0/0 splenocytes was associated with differences in the phosphorylation (P) patterns of protein kinase C (PKC alpha/beta, but not delta) and the PKC downstream effectors p44/42MAPK (mitogen-activated protein kinase). P-PKC and P-MAPK profiles were similar in wild-type and PrP0/0 splenocytes following PMA treatment, indicating that the ability of these 2 enzymes to be phosphorylated is not impaired in the absence of PrPC. Con A-induced calcium fluxes, monitored by indo-1 fluorescence, were equivalent in PrP0/0 and PrP+/+ splenocytes, suggesting that calcium-dependent mechanisms are not directly implicated in the differential phosphorylation patterns or mitotic responses. Our data indicate that PrP0/0 splenocytes display defects in upstream or downstream mechanism(s) that modulate PKCalpha/beta phosphorylation, which in turn affects its capacity to regulate splenocyte mitosis, consistent with a role for PrPC in immune function.

Memory B lymphocytes migrate to bone marrow in humans.

IgM-bearing B lymphocytes with mature phenotype (CD10- CD24(lo) IgD+) are acquired after birth in the bone marrow of humans. These B cells are defined here as relatively large, nondividing lymphocytes, variable proportions of which express cell surface molecules indicative of relatively recent activation. Analysis of V(H)5(2) (heavy chain variable region) gene transcripts indicated point mutations throughout the Ig variable region from the mature IgM+ B population but not from the immature B cells in the bone marrow. The mutations were concentrated in the complementarity determining regions, and amino acid substitutions were favored over silent mutations, findings indicative of antigen selection within germinal centers in peripheral lymphoid tissues. The V(H) sequence analysis also revealed the existence of clonal relatives in individual bone marrow samples. These antigen-experienced lymphocytes did not secrete Ig spontaneously but could be induced to do so in vitro. The data suggest that a subpopulation of memory B lymphocytes generated during antigen responses recirculates to the bone marrow in humans.

The end can justify the means.

The generation of a pre-immune repertoire of B lymphocyte specificities is a prerequisite for subsequent antigen-dependent antibody responses. In avian species the bursa of Fabricius plays a central role in the development of B lineage cells and in the generation of antibody diversity. Apart from its segregation into the bursa, the pathway of avian B cell development differs from that seen in mammalian species by a number of other criteria. In particular the chicken has evolved a remarkable mechanism for the diversification of immunoglobulin V region genes. Here we review some of the key features by which avian and mammalian B cell development differ.

Bursa-dependent subpopulations of peripheral B lymphocytes in chicken blood.

By selective labeling of juvenile chicken bursal cells with colloidal fluorescein isothiocyanate in situ, the emigration rate of bursal lymphocytes to the periphery was estimated at approximately 0.84% and 0.96% of the peripheral blood lymphocyte (PBL) and splenic B cell pool per hour, respectively. Emigrant bursal cells were found primarily in blood and spleen, with very small numbers migrating to thymus, bone marrow, and gut-associated lymphoid tissues. Emigrant bursal cells expressed high levels of both major histocompatibility complex class II antigen and the Ov alloantigen, a phenotype found on a population comprising approximately 4% of bursal cells from which the bursal emigrants may be derived. Surgical bursectomy at 3 weeks of age revealed that peripheral blood B cells could be divided into three distinct populations. Specifically, 60% of the peripheral blood B cells were short lived with a half-life of about 30 h in the blood. These cells accounted for the great majority of emigrants from the bursa to the peripheral blood. Approximately 35% of PBL B cells had a half-life of 12 days following bursectomy and comprised cells which did not divide in the periphery. Consequently, we propose that physiological differences between this population and the majority of bursal emigrants are established intrabursally. The remaining PBL B cells, whose relative proportion increases with age from about 5% of PBL B cells at 2-3 weeks of age, are short lived and are being continually produced from (a) post-bursal site(s) of B cell production.

B cell emigration directly from the cortex of lymphoid follicles in the bursa of Fabricius.

In the peripheral blood (PBL) of juvenile chickens three populations of B cells have previously been distinguished based on life-span and origin of cells within each population. In this report we show that the largest PBL B cell subset, population 1 B cells, which are short-lived cells corresponding to about 60% of PBL B cells and the vast majority of bursal emigrants, exit from the bursa directly from the follicular cortex. This conclusion is based on the specific labeling of rapidly dividing cortical lymphocytes with bromodeoxyuridine, followed by their detection in the periphery prior to the appearance of bromodeoxyuridine labeled cells in the bursa medulla. Furthermore, the rate of emigration of cortical lymphocytes, 1.00 +/- 0.1% of PBL B cells per hour, is indistinguishable from the emigration rate of B cells from the bursa as a whole. The anatomical organization of the bursa has evolved to focus gut-derived antigens from the bursal lumen into the lymphoid follicles. The emigration of cortical bursal cells is discussed in relation to the exposure of bursal lymphocytes to extrinsic antigen.

High levels of CD45 are coordinately expressed with CD4 and CD8 on avian thymocytes.

In this paper we describe the avian homolog of mammalian CD45. We show that this Ag is expressed on all leukocytes but not on erythroid cells or their immediate precursors. Immunoprecipitations demonstrated that B lineage cells from the bursa of Fabricius expressed a higher molecular mass variant (215 kDa) than did T lineage cells from the thymus (190 kDa), and crucially, these high molecular mass molecules had intrinsic phosphotyrosine phosphatase activity characteristic of mammalian CD45. We show that levels of CD45 expression as detected by mAb LT40 in the avian thymus are heterogeneous and further that mAb LT40 can deplete all phosphotyrosine phosphatase activity from thymocyte membrane preparations. Therefore total levels of CD45 are heterogeneous among avian thymocytes. Specifically, 87 to 89% of thymocytes expressed fourfold higher levels of surface CD45 (CD45hi) than the remaining 11 to 13% (CD45lo). The CD45lo population contained exclusively thymocytes with the phenotype CD3-4-8lo, characteristic of the immediate precursors to the CD3-4+8+ thymic population which are CD45hi. The shift from low to high levels of surface CD45 expression therefore occurred at the same stage as the transition from CD4-8lo to CD4+8+ and before the expression of CD3. The protein tyrosine kinase activity associated with CD4 and CD8 (p56lck) and the phosphatase activity of CD45 have been implicated elsewhere in jointly regulating peripheral T cell signal transduction and subsequent cellular responses. The coordinated expression of high levels of CD45 with both CD4 and CD8 in the avian thymus supports the possibility that these molecules may function together in regulating thymocyte growth and/or differentiation.

B lymphocyte migration to the bone marrow of humans is not random.

This research is a result of analysing a total of 20 independently isolated immunoglobulin gene sequences from a defined population of mature B cells found in the bone marrow of five healthy adults. Each individual exhibited at least two identical gene sequences in our sample. Each variable immunoglobulin sequence is the product of the recombination of three gene segments, V, D and J genes. All sequences sampled contained a specific V gene, V5-51. Comparing the particular J and D genes expressed together with V5-51 as well as the junctional modifications between these genes established the relatedness of the sequences. The likelihood of finding at least two out of k identical gene sequences, if they occur randomly, is found and compared to the experimentally determined sequences. We conclude that it is unlikely that the B cells containing the related sequences found in the bone marrow is by chance. This suggests that this particular population of B cells migrate to the bone marrow in a co-ordinated fashion.

Identification and analysis of the expression of CD8 alpha beta and CD8 alpha alpha isoforms in chickens reveals a major TCR-gamma delta CD8 alpha beta subset of intestinal intraepithelial lymphocytes.

Expression screening has been used to clone cDNAs encoding the alpha- and beta-chains of chicken CD8. Amino acid sequence similarities with the mammalian sequences were about 30%. Many amino acid residues of structural or functional importance were more highly conserved, as were the overall structures of both chains. Like human CD8 alpha, the chicken alpha-chain lacked sites for N-linked glycosylation, but the beta-chain contained three such sites. In COS cells transfected with CD8 beta cDNA, surface expression of the beta-chain was dependent on co-transfection of the alpha-chain cDNA, indicating that, as in mammals, chicken CD8 can be expressed as a CD8 alpha alpha homodimer or as a CD8 alpha beta heterodimer. Immunofluorescence analysis with mAbs that were shown to identify the CD8 alpha- and CD8 beta-chains revealed that the vast majority of the CD8+ cells in the thymus, spleen, and blood of adult chickens express both CD8 alpha- and CD8 beta-chains. However, a relatively large proportion of the CD8+ TCR-gamma delta cells in the spleens of embryos and young chicks express only the alpha-chain of CD8. Among intestinal epithelial lymphocytes the major CD8+ T cell populations present in mice are conserved, but there is a population of TCR-gamma delta CD8 alpha beta cells that is not found in rodents. This observation is important in interpretation of experiments examining the pathways of development of intestinal intraepithelial lymphocytes in chickens.