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Early in embryonic development of mice, from day 12.5 after conception, myeloid-lymphoid bipotent progenitors, expressing receptors both for IL7 and CSF-1, migrate from embryonic blood into developing fetal liver. These progenitors also express multiple chemokine receptors, i.e., CCR7, CXCR3, CXCR4, and CXCR5, all on one cell. Their migration through LYVE-1+ vascular endothelium is guided by CCR7, recognizing the chemokine CCL19, and by CXCR3, recognizing CXCL10/11, chemokines which are both produced by the endothelium. Once inside fetal liver, the progenitors are attracted by the chemokine CXCL12 to ALCAM+ liver mesenchyme, which produces not only this chemokine, but also the myeloid differentiation-inducing cytokine CSF-1 and the lymphoid differentiation-inducing cytokine IL7. In this mesenchymal environment B-lymphocyte lineage progenitors are then induced by IL7 to enter differentiation and Ig gene rearrangements. Within 3-4 days surface IgM+ immature B-cells develop, which are destined to enter the B1-cell compartments in the peripheral lymphoid organs.

We have previously shown that long-term in vitro proliferating fetal liver pre-B cell lines derived from autoimmune-prone (NZB x NZW)F1 (BW) mice, but not normal (B6 x DBA2)F1 mice, can differentiate in severe combined immunodeficient (SCID) mice to produce elevated levels of serum immunoglobulin (Ig) M and IgG, and high titers of antinuclear antibodies The contribution of parental NZB and NZW strains to B cell abnormalities of BW hybrid mice was investigated here by preparing pre-B cells and transferring them into immunodeficient SCID- and RAG-2-targeted mice. We show that transfer of NZB pre-B cells led to a marked IgM hypergammaglobulinemia and to the production of limited amounts of IgG2a. On the other hand, the transfer of NZW pre-B cell lines led to moderately elevated IgM levels and marked hypergammaglobulinemia of IgG2a. High IgM and low IgG anti-DNA titers are found in the recipients of NZB pre-B cells, whereas those receiving NZW pre-B cells contained lower levels of IgM and high titers of IgG anti-DNA. In marked contrast, essentially identical titers of antibodies directed against a non-self-antigen, DNP, are found in all group of pre-B cell recipients. Thus, B-lineage cells of both NZB and NZW parental strains manifest abnormalities associated with the development of this lupus-like disease. Therefore, the present study strongly suggests a complex inheritance of B cell abnormalities in autoimmune-prone (NZB x NZW)F1 mice and emphasizes the critical importance of intrinsic B cell defects in the development of murine systemic lupus erythematosus.

We describe a novel regulatory myosin light chain gene (termed precursor lymphocyte-specific regulatory light chain or PLRLC) that is expressed specifically in precursor B and T lymphocytes. PLRLC is the first example of a regulatory myosin light chain gene which displays specific expression in non-muscle cells. PLRLC is expressed in adult bone marrow derived normal and transformed pre-B cells; in the former, PLRLC expression levels are induced by the pre-B cell specific growth factor interleukin-7 (IL-7). PLRLC is not expressed in either transformed pre-B cells derived from fetal liver or in normal fetal liver pre-B clones grown in the presence of IL-7. Therefore this gene provides the first marker that clearly distinguishes these two pre-B subsets. Finally, several of the different PLRLC transcripts potentially encode regulatory myosin light chains with unique structural features. The unique distribution, regulation and structural features of the PLRLC gene products suggest an important role for PLRLC during lymphocyte development.

A series of checkpoints for antigen receptor fitness and specificity during B cell development ensures the elimination or anergy of primary, high-avidity-autoantigen-reactive B cells. Defects in genes encoding molecules with which this purging of the original B cell repertoires is achieved may break this B cell tolerance, allowing the development of B cell- and autoantibody-mediated immune diseases. Furthermore, whenever tolerance of helper T cells to a part of an autoantigen is broken, a T cell-dependent germinal center-type response of the remaining low--or no--autoreactive B cells is activated. It induces longevity of these B cells, and expression of AiD, which effects Ig class switching and IgV-region hypermutation. The development of V-region-mutant B cells and the selections of high-avidity-autoantigen-reactive antibodies producing B cells by autoantigens from them, again, can lead to the development and propagation of autoimmune diseases such as lupus erythematosus or chronic inflammatory rheumatoid arthritis by the autoantibody BcR-expressing B cells and their secreted autoantibodies.

Preface.- Strategies of NK cell receptor recognition and signaling.- Signal Transduction in Natural Killer Cells Alexander.- Transcriptional Regulation of NK Cell Receptors.- Extending Missing-Self? Functional Interactions Between Lectin-Like Nkrp1 Receptors on NK Cells with Lectin-Like Ligands.-Immunobiology of Human NKG2D and its Ligands.- NKG2 receptor-mediated regulation of effector CTL functions in the human tissue microenvironment.- The Dendritic Cell/NK Cell Cross -Talk: Regulation and Physiopathology.- NK cell activating receptors and tumor recognition in human.- NK cell recognition of mouse cytomegalovirus-infected cells.- NK cell receptors involved in the response to human cytomegalovirus infection.- The Impact of Variation at the KIR Gene Cluster on Human Disease.- NK cells in autoimmune disease.- Subject index

This chapter will review the scenario of normal B cell development--from the decision of a lymphoid progenitor to enter the B-lineage, over the stages of the generation of the repertoires of antigen-receptor (immunoglobulin)-expressing cells, to the response of mature B cells to develop memory and plasma cells--highlighting some of the cellular stages and the molecular mechanisms that can generate and select transformed states of cells. The scenarios for pre-B lymphoma (lymphocytic leukaemia) development are discussed in more detail.

Drugs and drug resistance.- Quinolines and Artemisinins: Chemistry, Biology and History.- Antimalarial multidrug resistance in Asia: mechanisms and assessment.- Antimalarial drug resistance in Africa: strategies for monitoring and deterrence.- Malaria the disease.- Uncomplicated malaria.- Metabolic complications of severe malaria.- The clinical and pathophysiological features of malarial anaemia.- Malaria in the pregnant woman.- Biology.- Host receptors in malaria merozoite invasion.- A post-genomic view of the mitochondrion in malarial parasites.- The plastid of Plasmodium spp. a target for inhibitors.- Hemoglobin degredation.- Bioavailable iron and heme metabolism in Plasmodium falciparum.- Plasmodium permiomics: membrane transport proteins in the malaria parasite.- Plasmodium ookinete invasion of the mosquito midgut.- Molecular genetics of the mosquito resistance to malaria parasites.- Functional proteome and expression analysis of sporozoites and hepatic stages of malaria development.- Subject index

Preface.- Selection of CD4+CD25+ Regulatory T Cells by Self-Peptides.- The Role of TCR Specificity in Naturally Arising CD25+ CD4+ Regulatory T Cell Biology.- Thymic Commitment of Regulatory T Cells Is a Pathway of TCR-Dependent Selection That 'Isolates' Repertoires Undergoing Positive or Negative Selection.- Selection and Behaviour of CD4+CD25+ T Cells in vivo: Lessons from T Cell Receptor Transgenic Models.- Migration Matters: Functional Properties of Naive- and Effector/Memory-Like Regulatory T Cell Subsets.- Peripheral Generation and Function of CD4+CD25+ Regulatory T Cells.- Dendritic Cells, Key Cells for the Induction of Regulatory T Cells?.- Autoimmune Gastritis Is a Well-Defined Autoimmune Disease Model for the Study or CD4+CD25+ T Cell-Mediated Suppresion.- Regulatory T Cells in Experimental Colitis.- Autoimmune Ovarian Disease in Day 3-Thymectomized Mice: the Neonatal Time Window, Antigen Specificity of Disease Suppression, and Genetic Control.- Regulatory T Cells in Transplantation Tolerance. Infectious Tolerance.- CD4+CD25+ Regulatory T Cells in Hematopoietic Stem Cell Transplantation.- ...- Phenotypic and Functional Differences Between Human CD4+CD25+ and Type 1 Regulatory T Cells.- Subject Index.

Nineteen different mu heavy-chains, seven of them not capable of forming a pre B cell receptor were expressed in Drosophila melanogaster Schneider cells together with either VpreB1, VpreB2, lambda5, or the complete surrogate light-chain to study their interactions in the formation of the pre B cell receptor. The lambda5 protein alone was unable to bind properly to any of the mu heavy-chains, while the VpreB proteins alone formed complexes with five of the mu heavy-chains. All mu heavy-chains incapable of forming a pre B cell receptor with surrogate light-chain were also incapable of complex formation with VpreB. The possible role of the VpreB/mu heavy-chain in allelic exclusion of the heavy-chain locus during B cell development is discussed.

During B-lymphocyte development in mouse fetal liver and bone marrow, a pre-B I cell stage is reached in which the cells express B-lineage-specific genes, such as CD19, Ig alpha and Igbeta and VpreB and lambda5, which encode the surrogate light (SL) chain. In these pre-B I cells both alleles of the immunoglobulin heavy (IgH) chain locus are D(H)J(H) rearranged. Transplantation of pre-B I cells from wild-type (e.g. C57Bl/6) mice in histocompatible RAG-deficient hosts leads to long-term reconstitution of some of the mature B-cell compartments and to the establishment of normal IgM levels, a third of the normal serum IgA levels, and IgG levels below the detection limit. Neither T-lineage nor myeloid cells of donor origin can be detected in the transplanted hosts, indicating that the pre-B I cells are committed to B-lineage differentiation. Consequently, the B-cell-reconstituted hosts respond to T-cell-independent antigens but not to T-cell-dependent antigens. Responses to T-cell-dependent antigens can be restored in the pre-B I-cell-transplanted, RAG-deficient hosts by the concomitant transplantation of mature CD4+ T cells. The transplanted wild-type pre-B I cells do not home back to the bone marrow and become undetectable shortly after transplantation. B-lymphocyte development in Pax-5-deficient mice becomes arrested at the transition of pre-B I to pre-B II cells i.e. at the stage when V(H) to D(H)J(H) rearrangements occur and when the pre-B-cell receptor, complete with muH chains and SL chains, is normally formed. T-lineage and myeloid cell development in these mice is normal. Pre-B I cells of Pax-5-deficient mice have a wild-type pre-B I-cell-like phenotype: while they do not express Pax-5-controlled CD19 gene, and express Ig alpha to a lesser extent, they express Igbeta, VpreB and lambda5, and proliferate normally in vitro on stromal cells in the presence of interleukin (IL)-7. Clones of these pre-B I cells carry characteristic D(H)J(H) rearrangements on both IgH chain alleles. However, removal of IL-7 from the tissue cultures, unlike wild-type pre-B I cells, does not induce B-cell differentiation to surface IgM-expressing B cells, but induces macrophage differentiation. This differentiation into macrophages requires either the presence of stromal cells or addition of macrophage colony-stimulating factor (M-CSF). Addition of M-CSF followed by granulocyte-macrophage colony-stimulating factor induces the differentiation to MHC class II-expressing, antigen-presenting dendritic cells. In vitro differentiation to granulocytes and osteoclasts can also be observed in the presence of the appropriate cytokines. Moreover, transplantation of Pax-5-deficient pre-B I clones into RAG-deficient hosts, while not allowing B-cell differentiation, leads to the full reconstitution of the thymus with all stages of CD4-CD8- and CD4+CD8+ thymocytes, to normal positive and negative selection of thymocytes in the thymus, and to the development of normal, reactive mature CD4+ and CD8+ T-cell compartments in the peripheral lymphoid tissues, all carrying the clone-specific D(H)J(H) rearrangements. On the other hand, Ig alpha, Igbeta, VpreB and lambda5 are turned off in the thymocytes, demonstrating that the expression of these genes does not commit cells irreversibly to the B lineage. Further more, Pax-5-deficient pre-B I cells are long-term reconstituting cells. They home back to the bone marrow of the RAG-deficient host, can be reisolated and regrown in tissue culture, and can be retransplanted into a secondary RAG-deficient host. This again develops thymocytes and mature T cells and allows the transplanted clonal pre-B I cells to home to the bone marrow.

Single cell PCR assays have been further developed that detect over 80% of all VkappaJkappa, VkappaRS, and VlambdaJlambda rearrangements at efficiencies between 70% and 90%. These IgL chain gene rearrangement assays were used with small pre-BII cells that develop in comparably high numbers in the bone marrow of wild-type, Ckappa-deficient, and JCkappa-deficient homozygous and heterozygous mice. In all of these mice, only 15%-25% of all small pre-BII cells carry VlambdaJlambda rearrangements. These results confirm that lambdaL chain gene rearrangements occur independently of kappaL chain gene rearrangement and expression. They also show that a large part of the small pre-BII cells that express the rearrangement machinery can develop without IgL chain gene rearrangements.

Ordered rearrangements of immunoglobulin (Ig) gene loci, first as DH to JH, then as VH to DHJH, and finally as VL to JL segment-specific recombinations occur 'in-frame' and 'out-of-frame'. 'In-frame' rearrangements lead to the expression of truncated DHJH-microC proteins and to microH chains. These H chain proteins have two major effects on precursor B cells. They suppress (as DJC mu proteins) or enhance (as full microH chain) the proliferation of precursor cells at the point where these precursors express these proteins. At the same time, they signal allelic exclusion of the microH chain alleles, so that VH to DHJH rearrangement at the second allele is suppressed. Regulation of precursor B cell proliferation and H chain allelic exclusion is mediated by a pre-B cell receptor that is composed of the microH chains and a surrogate L chain. This surrogate L chain is made up of two proteins encoded by the Vpre-B and lambda 5 genes that are expressed only at the early precursor cell stages just before and when H chain genes are first expressed. They are not found in later B cell development, when L chains are expressed, nor in any other cell of the body tested so far. The physiological roles of surrogate L chain and of the pre-B receptor have been clarified by generating mutant mice in which the lambda 5 gene has been inactivated by targeted disruption. Molecular mechanisms and cellular developments, by which the pre-B receptor controls proliferation and allelic exclusion, are discussed.

A chromosomal translocation (Tx) that interrupts the transcription of either c-Myc or Pvt 1 is the principal lesion in many B cell malignancies including Burkitt's Lymphoma (BL), AIDs-NHL, mouse plasmacytoma (Pct) and possibly multiple myeloma (MM). There is a restriction associated with this Tx such that only the immunoglobulin (Ig) heavy chain gene is found juxtaposed to c-Myc and only the Ig light chain gene is found juxtaposed to Pvt 1. Over the past several years, our laboratory has been instrumental in the elucidation of the structure of the mouse Pvt 1 locus as a means of understanding the relationship between these two divergent Txs which, nevertheless, produce indistinguishable disease phenotypes. In the mouse, we have identified a uniform Pvt1/Ig Ck fusion product which is consistently found in all tumors harboring Pvt 1 associated Txs. We have recently constructed transgenic mice harboring a translocated Pvt 1/Ck segment in order to determine whether 1). these mice produce the Pvt 1/Ck fusion product 2). these mice are immunocompromised and 3). these mice develop tumors of a B cell origin.

We have investigated the capacity of precursor B cells from normal (BDF1) and V(D)J recombinase-deficient (RAG-27) or defective (SCID) mice to be induced by a CD40-specific monoclonal antibody and IL-4 to epsilon H chain gene transcription and to S mu-S epsilon switch recombination. In differentiating precursor B cells from all three strains of mice, the development of similar numbers of CD19+, CD23+, CD40+, and MHC class II+ expressing B lineage cells and similar levels of epsilon H chain gene transcription were induced. Efficient S mu-S epsilon switching occurred in normal and RAG-2-deficient, but not in SCID, precursor B cells. Thus, the transcription of the epsilon H chain is independent of the RAG-2 and the SCID gene product, while the S mu-S epsilon switch recombination requires the SCID gene-encoded DNA-dependent protein kinase, but not the RAG-2 protein.

Progenitor and precursor B lymphocytes with the capacity of long-term proliferation on stromal cells in the presence of interleukin-7 (IL-7) can be cloned ex vivo from fetal liver, neonatal blood, and spleen, and from adult bone marrow (BM) in frequencies that are similar in different strains of mice and that change with age. A wave of clonable cells appears before birth and disappears after birth in liver. Up to 2 weeks after birth, high frequencies of clonable cells are present in spleen but become undetectable at 6 to 8 weeks of age. In BM, high frequencies (1 in 50) of clonable cells are present early after birth, and then decrease continuously to 10- to 20-fold lower levels at 6 to 8 months of age. The earliest clonable cells have at least part of their IgH genes in germline configuration. Clones of pro/pre B cells apparently continue to rearrange DH to JH segments on both chromosomes. Rearrangements without insertion of N-sequences at the DHJH joints are found in fetal liver, while DHJH joints in pre B cells of spleen and BM throughout life have N-regions inserted. At least half of all primary pre B-cell clones develop mitogen-reactive B cells after differentiation to sIg+ B cells. Clonable pro and pre B cells are enriched in B220- c-kit(low) as well as in B220+ c-kit+ and B220+ CD43+ cell populations of BM. The frequencies of clonable cells in the B220- c-kit(low) BM cell population decrease 10- to 20-fold during 8 months of life, while those in the B220+ c-kit+ population remain constant, although their absolute numbers drop 5- to 10-fold during that time. All long-term proliferating clones express the surrogate L chain VpreB/lambda 5 as well as c-kit and CD43 on all cells. The number of total clonable pro and pre B cells is at best 10% of the number of cells required to produce the estimated daily output of 5 x 10(7) B-lineage cells in a mouse. This suggests that the production of a relatively constant number of B cells during adulthood may be effected by precursors, which are not clonable on stromal cells and IL-7 with long-term proliferative capacity. On the other hand, BM transplantation experiments indicate that a mouse retains B220- progenitors throughout life, from which pre B and B cells can be generated in old mice in frequencies characteristic of young mice.

BAFF, a member of the family of tumor necrosis factor ligands, is essential for the development of peripheral, mature, long-lived B lymphocytes. It binds to three different receptors (BCMA, TACI and BAFF-R), which are all members of the family of tumor necrosis factor receptors. Defects in the genes encoding either BAFF or BAFF-R abolish the generation of mature B cells. BAFF is made by myeloid cells while BAFF-R is expressed preferentially on B cells. BAFF induces polyclonal maturation of resting, short-lived immature cells to resting, long-lived mature B cells without proliferation. Lupus erythematodes-prone mice have elevated levels of BAFF in their blood, and treatment of these mice with BAFF decoy receptor (BCMA-Ig) prevents the onset of this autoimmune disease. Human lupus patients also show elevated levels of BAFF in their blood. Treatments with BAFF-neutralizing agents should prevent, delay or, at least, slow down the disease.

A gene, called m-mb-1, was isolated from a murine pre-B-minus T lymphocyte subtracted library. It was found expressed as mRNA at low to medium abundance in early progenitors of the B lineage, in pre-B and mature B lineage cell lines, in normal resting B lymphocytes and in polyclonally activated B cell blasts. The gene was not expressed in plasmacytomas, in cell lines of the monocyte/macrophage, the T lymphocyte or the fibroblast lineages, nor in thymus, liver, heart, kidney, lung or brain. The nucleotide sequence of the m-mb-1 gene encodes a putative membrane glycoprotein with 220 amino acids, which includes a leader sequence, a putative extracellular domain with two potential N-glycosylation sites, a transmembrane portion and a putative intracellular domain. The partial sequence of a human homologue, h-mb-1, shows nearly 90% homology in nucleotide as well as amino acid sequences to the murine form of a stretch of the putative intracytoplasmic domain. Antibodies raised against a fusion protein of m-mb-1 with protein A, affinity purified for their m-mb-1 specificity, stained pre-B and mature B cell lines on their surface, but did not stain T cell lines and fibroblasts. Antibodies raised against a stretch of 20 amino acids of the putative intracellular domain with 90% homology between the mouse and human protein did not stain the surface of any cell lines tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Peanut agglutinin (PNA) binding by mouse bone marrow cells and fractionation by the fluorescence-activated cell sorter have previously been shown to separate high concentrations of pre-B cells, as identified by cytoplasmic mu-chains (c mu). PNA+ and PNA- marrow cell fractions have now been assayed for the presence of functional pre-B cells able to generate mature B cells in culture, as defined by three criteria, the appearance of cell surface mu-chains (s mu), immunoglobulin secretion in response to bacterial lipopolysaccharides, and B cell colony formation. Small PNA+ cell fractions contained pre-B cells that developed into mature B lymphocytes in 1/2 to 1 day but did not sustain B cell production. Large PNA+ cells included pre-B cells that gave rise to mature B lymphocytes after an interval of 1 1/2 to 3 days and were able to sustain B cell genesis in vitro for at least 3 to 5 days thereafter. PNA- cell fractions contained mature B cells but lacked pre-B cell activity. The results demonstrate that PNA binding allows the separation of functional subsets of pre-B cells from bone marrow and that the three in vitro assays used in this study are closely comparable with one another as functional pre-B cell criteria. The findings suggest correlations between functional assays, c mu expression, PNA receptors, and cell size in characterizing stages of pre-B cell development.

Nine hybridomas producing monoclonal autoantibodies specific for kidney proximal tubular brush border were found in 600 hybridomas derived from (C57BL/6J X DBA/2)F1 mice injected with DBA/2 T cells. None of the 1100 hybridomas derived from nonautoimmune (C57BL/6J X DBA/2)F1 mice produced antibodies with a similar specificity. Four of these nine monoclonal antibodies were characterized further. They did not bind to cryosections of liver, lung, stomach, or intestine. Three bound to kidney proximal tubular brush border of mouse, cattle, sheep, pigs, rabbits, rats, and humans, whereas the fourth was specific only for murine brush border. All four precipitated from mouse kidney microvilli, a protein with an apparent molecular weight of 160,000 under reducing as well as nonreducing conditions. Removal of asparagine-linked carbohydrate with EndoF reduced the molecular weight of the 160,000 protein by about 20,000. One of the three multi-species-specific antibodies bound to pig kidney aminopeptidase, a glycosylated enzyme located on the microvilli of kidney proximal tubular brush border. Three antibodies have a heavy-chain variable region encoded by VH genes of the J558 family, whereas the heavy-chain variable region of the fourth is encoded by a VH gene of the 7183 family. Attempts to passively transfer immune complex glomerulonephritis to normal mice by injection of the purified monoclonal antibodies or by growth of the corresponding hybridoma cells in mice have so far been unsuccessful. However, antibodies recognizing the 160,000 molecule are present in the serum of mice with chronic graft-versus-host disease and can be eluted from kidneys with immune complex glomerulonephritis.

lambda 5 is an immunoglobulin lambda light chain-related gene which is selectively transcribed in murine pre-B lymphocytes to yield a 1.2 kb poly(A)+ mRNA. Comparison of the nucleotide sequence of a 1 kb cDNA clone with the sequence of a genomic clone isolated from 70Z/3 murine pre-B lymphoma cells shows lambda 5 is composed of three exons spanning a 3.75 kb DNA segment. Conserved splice signal sequences at all exon/intron boundaries and the presence of a long open reading frame indicate that a functional mRNA molecule can be made. Exon I contains a cap-site and a potential ATG start codon as well as sequences encoding a signal peptide. This gene could encode a lambda 5 protein of 209 amino acids which has, however, not yet been identified. The 3' portion of exon II and all of exon III shows strong sequence homologies to J lambda L and C lambda L exons. Homology to the lambda L chain genes is lost in the 5' portion of exon II and throughout exon I. In exon I short homologies to leader sequences and to VH framework 1 sequences are seen.

Cloned murine helper T cells, restricted to the Iab antigens of the major histocompatibility locus and specific for horse erythrocytes as a foreign antigen, produce, in cooperation with antigen and histocompatible adherent cells, soluble factors that replace the helper T cells in their action on B cells. Three types of factors can be distinguished on the basis of molecular weight: proteins having apparent Mr 30,000 (p30) that act antigen- and Ia-nonspecifically as replication- and maturation-inducing factors and proteins having apparent Mr 55,000 (p55) and 125,000 (p125) that act on resting B cells in an Ia-specific, restricted fashion. Neither horse erythrocytes (a T-cell specific antigen) nor p55 and p125, alone or together, stimulate resting B cells to proliferation and maturation. Double occupancy by antigen and p55 or p125, however, renders Ia-compatible, but not Ia-incompatible, resting B cells susceptible to stimulation. The subsequent addition of p30 to these "excited" B cells then results in the proliferation and maturation of clones of horse erythrocyte-specific resting B cells, which then replicate under the stimulatory action of p30. p30 do not bind antigen, nor do they bind anti-Ia or anti-immunoglobulin antibodies. p55 are bound by anti-heavy chain variable region antibodies. p55 are bound by anti-heavy chain variable region antibodies, but not by anti-heavy or anti-light chain constant region antibodies or anti-Ia antibodies. p125 molecules bind horse but not sheep erythrocytes and are bound by anti-heavy chain variable region, but not by anti-heavy or light chain constant region or anti-Ia antibodies. p55 and p125 are likely to be soluble analogues of the antigen-specific, Ia-restricted T-cell receptors of the cloned helper T cells.

A T cell line was established in long-term tissue culture from spleen cells of BALB/c mice immunized with influenza virus PR8 (A/PR/8/34(H1N1)) by continuous restimulation with PR8 virus and syngeneic x-irradiated spleen cells. This T cell line and clones derived from it have now been propagated for over 1 yr "in vitro". The T cell line and the clones, upon stimulation with Con A in the absence of antigen and irradiated spleen cells, as upon stimulation in the presence of antigen and adherent cells, produce T cell growth factors, B cell replication and maturation factors, and colony-stimulating factors, as tested by restimulation of proliferation of a clone of cytolytic murine T cells, of lipopolysaccharide-activated B cell blasts, and by macrophage/granulocyte colony formation of murine bone marrow cells, respectively. The T cell line and the clones help syngeneic B cells in the presence of syngeneic irradiated spleen cells and PR8 virus to proliferate and mature into clones of cells secreting virus-specific antibodies. In the presence of PR8 virus and of a bystander antigen, such as sheep red cells, B cells specific for this bystander antigen are also induced. Adoptive transfer of the T cells i.v. into syngeneic nu/nu BALB/c mice enable the recipient mice to produce, upon challenge with PR8, virus-specific antibodies and to clear virus infection of the lung. The virus-specific T cell line and the clones are restricted by H-2 antigens of the d-haplotype, probably by Iad, and recognize a determinant on the hemagglutinin (HA) molecule of PR8 virus. Fifty to 100 fmol of virus-associated or free HA suffice for stimulation as measured by proliferation assays. The stimulating determinant is present on the HA of all natural virus isolates of the H1 subtype, is absent from virus isolates of the H2 and H3 subtypes, and is abolished if glutamic acid at position 115 of the HA1 polypeptide of PR8 is replaced by lysine.

During ontogeny of the mouse, cells bearing surface Ig first appear at approximately 16 days of gestation. To characterize B cell differentiation during embryogenesis further, we have measured several functional activities of B cells and their precursors. B cell function was measured in two assays, response to sheep red blood cells in a transplantation assay, and response in vitro to a B cell mitogen. By both assays, B function is first detectable at 16 days, the same time that Ig-positive cells appear. The immediate precursors of B cells, PB cells,3 can also be measured by a transplantation assay. This activity is first detectable in fetal liver at 13 days. The yolk sac at 13 days does not contain significant PB activity even though both fetal liver and yolk sac contain pluripotent stem cells, spleen colony-forming units, at that time. These data are consistent with the hypothesis that the development of B lymphocytes occurrs early in ontogeny and that the fetal liver may provide the inductive stimulus for B cell maturation.

Chromatography of seryl RNA and cysteinyl RNA on columns of methyiated albumin on kieselgur Two seryl t-RNA's (I and II) were partially separated by chromatography of [ 14 C]seryl s-RNA from yeast on 2.2 × 50-cm columns of methylated serum albumin on kieselgur. [ 35 S]Cysteinyl s-RNA from E. coli showed three peaks of radioactivity on these columns. The relative amounts of seryl t-RNA I and II were quite different between RNA samples from two yeasts of different origins, but were not influenced by the origin of the aminoacyl s-RNA synthetases (EC 6.1.1.) (yeast or rat liver) used in charging the s-RNA with serine. The slower moving serine t-RNA in counter-current distributions was identified with seryl t-RNA I from MAK-columns, the faster moving with seryl t-RNA II. The presence of a third serine t-RNA in yeast s-RNA is discussed. A partial loss of acceptor activity for serine was observed during chromatography on 2.2 × 50-cm MAK-columns; this loss was more pronounced in the preparative separations on 3.2 × 110-cm columns. Higher oligonucleotides containing [ 14 C]serine were found as one class of decomposition products of the [ 14 C]seryl t-RNA's.

Enzymatic splitting of serine-specific transfer -RNA Serine-specific t-RNA I and II fractions, which had been largely separated from each other and from other t-RNA's by countercurrent distribution, were investigated. The splitting products obtained with pancreatic and T 1 -ribonucleases (EC 2.7.7.16 and EC 3.1.4.8 respectively) were separated by column chromatography (Figs. 1 and 2) and further purified. Some oligonucleotides were further degraded enzymically and the products separated by column chromatography (Tables I–III). One each of N 2 -dimethyl-Gp, 5-methyl-Cp, rTp, Ip, 2′- O -methyl-Gp, 2′- O -methyl-Up; three ψ-Up's and two nucleotides with the properties of 4,5-dihydro-Up were found in defined sequences of the serine t-RNA's. Degradation of a serine t-RNA fraction with snake venom phosphodiesterase (EC 3.1.4.1) proceeded quickly at first and then slowed down (Figs. 3 and 4). The 5′-mononucleotides split off in the fast initial phase were analyzed.

Nucleotide sequences in serine-specific transfer ribonucleic acids The results of the preceding papers on the isolation of several serine t-RNA fractions and their splitting with pancreatic and T 1 -ribonucleases (EC 2.7.7.16 and EC 3.1.4.8 respectively) are discussed. By countercurrent distribution two serine t-RNA's (I and II) were largely separated from each other; they also differed in behaviour during methylated albumin-kieselgur column chromatography. The oligonucleotide patterns of serine t-RNA's I and II, on the other hand, were very similar. The nucleotide sequences found in the serine t-RNA's are summarized in Table I. Several overlapping sequences were found, with the odd nucleotides and the (Ap) n G-sequences as links. Some of the 11 odd nucleotides occur clustered in the RNA chains. Various possible “anticodons” appear in the RNA. There is very little similarity between the sequence model of serine t-RNA, proposed by Cantoni et al. , and the nucleotide sequences of the serine t-RNA's determined here. Possible base pairing between the nucleotide sequences of the serine t-RNA's (Table I) are discussed.