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R R Hardy

Publications and source records attributed to R R Hardy.

At least 55 records · Page 3Linked to original sources
PubMed1995

Differential expression of the blk and ret tyrosine kinases during B lineage development is dependent on Ig rearrangement.

Expression of Ig transgenes in recombination-deficient mutant scid and Rag-1-mice results in the generation of pre-B and B cells, which are normally absent from these animals. In screening for protein tyrosine kinases (PTKs) that may play a role in this progression beyond the pro-B stage, we have identified five differentially regulated PTKs and compared their gene expression in defined stages of early B-lineage cells from normal, mutant, and Ig-transgenic mutant mice. Three PTKs (fgr, flk2/flt3, and tsk) show a comparable decrease at an early stage in all mice. In contrast, the decreasing expression of ret and the increasing expression of blk seen in differentiating B cells from normal mice are not observed in the mutant mice, unless they carry Ig transgenes. Therefore, our results show that the expression of certain PTKs is dependent on productive Ig rearrangement and suggest important roles for both Ret and Blk at distinct stages in the Ig-dependent progression of B cell differentiation.

Animals↗
PubMed1995

The site and stage of anti-DNA B-cell deletion.

Antibodies to DNA and nucleoproteins are found in sera of individuals with systemic autoimmune disease. In the population (and in the autoimmune mouse strain MRL/lpr) there is a great variety of such antinuclear antibodies, but individuals with systemic lupus erythematosus or single MRL mice express a subset only of the antinuclear specificities found in the population. These observations have been interpreted to mean that these antibodies arise by immunization. The oligoclonal nature of the autoantibody response and the evidence of selection acting on somatically mutated autoantibodies favour this interpretation. Specific activation of autoantibodies in disease implies either that autoantibodies are regulated in non-diseased individuals or that autoantigen availability is variable. The former has been demonstrated in anti-DNA transgenic mice. In normal mice, transgene-encoded antibodies against double-stranded (ds) DNA are not expressed in serum or on B cells. Here we describe modified anti-dsDNA transgenic mice which allow us to study the site and developmental stage at which such B-cell regulation occurs. This model shows that in normal mice B cells expressing anti-DNA specificity are deleted in the bone marrow at a pre-B to immature B transitional stage.

Animals↗
PubMed1994

Altered major histocompatibility complex restriction in the NK1.1+Ly-6Chi autoreactive CD4+ T cell subset from class II-deficient mice.

We previously demonstrated selective enrichment of major histocompatibility complex (MHC) class II-specific autoreactive T cells in a subset of mouse CD4+ thymocytes. Here we show that a significant fraction of these autoreactive cells in the normal adult thymus expresses NK1.1 and high levels of Ly-6C and also exhibits flexibility in MHC restriction. In normal mice, this NK1.1+Ly-6Chi subfraction accounts for 10-50% of the CD4+ autoreactive subset and is enriched for MHC class II-restricted autoreactive cells as determined by mixed leukocyte reaction frequency analysis, similar to NK1.1-Ly-6C-CD4+ autoreactive cells. In contrast, in the thymus of class II-deficient littermate mice, NK1.1+Ly-6Chi cells account for most of the mature heat stable antigen (HSA)-CD4+ fraction and exhibit MHC-restricted non-class II autoreactivity. Thus, NK1.1+Ly-6ChiCD4+ T cells show flexibility in MHC class restriction, but their autoreactivity remains MHC dependent.

Aging↗
PubMed1994

Absence of MHC class II expression distinguishes fetal from adult B lymphopoiesis in mice.

Early B-lineage progenitor cells (Pro-B) isolated from murine fetal liver and adult bone marrow can differentiate to the immature B cell stage in a stromal cell-dependent culture system and to mature B cells upon transfer into immunodeficient SCID mice. By using immunofluorescence analysis, we found that progenitor cells from day 16 fetus differentiating in culture lacked MHC class II expression during the Pre-B and immature B cell stages, whereas such expression was readily apparent on the surface of corresponding adult-derived populations. RT-PCR analysis of RNA message levels for the four class II genes (A alpha, A beta, E alpha, E beta) yielded completely concordant results. B cells of fetal progeny did eventually express class II upon further maturation in vivo. Thus, the onset of class II expression is uniquely delayed during fetal B cell differentiation. This result explains an apparent paradox, i.e., that class II expression is absent from B cells in neonatal spleen but present as early as the Pre-B cell stage in adult bone marrow. Furthermore, we suggest that such distinct programs of class II expression during fetal and adult lymphopoiesis could result in differences in susceptibility to tolerance.

Animals↗
PubMed1994

Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice.

We have examined the regulatory role of the individual components of the immunoglobulin antigen receptor in B-cell development by transgenic complementation of Rag-1 deficient (Rag-1-) mice. Complementation with a membrane mu heavy chain (mu HC) gene allows progression of developmentally arrested Rag-1- pro-B-cells to the small pre-B cell stage, whereas the introduction of independently integrated mu HC and kappa light chain (kappa LC) transgenes promotes the appearance of peripheral lymphocytes which, however, remain unresponsive to external stimuli. Complete reconstitution of the B-cell lineage and the emergence of functionally nature Rag-1- peripheral B cells is achieved by the introduction of cointegrated heavy and light chain transgenes encoding an anti-H-2k antibody. This experimental system demonstrates the competence of the mu HC and kappa LC to direct and regulate the sequential stages of B-cell differentiation, defines the time at which negative selection of self-reactive B cells occurs, and shows that elimination of these cells occurs equally well in the absence of Rag-1 as in its presence. These data also support the hypothesis that Rag-1 directly participates in the V(D)J recombination process.

Animals↗
PubMed1994

Deletion and editing of B cells that express antibodies to DNA.

We previously demonstrated that in mice transgenic for genes coding for an anti-ssDNA autoantibody B cells were functionally inactivated but not physically deleted. We have now extended this model by introducing an arginine into the CDR2 of the heavy chain transgene. This change alters the specificity of the Ab from anti-ssDNA to anti-dsDNA and increases the affinity for ssDNA. Mice carrying this transgene displayed a significant reduction of peripheral B cells and anti-dsDNA B cells were not recovered from the spleens. The remaining B cells escape deletion by revising their Ag receptors in several ways: 1) elimination of the transgenic heavy chain gene via intrachromosomal recombination, followed by rearrangement and expression of endogenous VH genes; 2) ongoing rearrangement of endogenous kappa light chain genes to generate a non-dsDNA-binding Ab; and 3) expression of a rare V lambda gene, V lambda x, to generate a non-DNA-binding Ab.

Amino Acid Sequence↗
PubMed1994

Two distinct non-T helper type 2 interleukin-4+ cell subsets in mice as revealed by single-cell cytokine analysis.

We have previously defined four murine CD4+ peripheral T cell subsets, fractions (Fr.) I-IV, based on expression of the 6C10 and 3G11 determinants (Hayakawa, K. and Hardy, R. R., J. Exp. Med. 1988. 168: 1825). These subsets also show distinctive levels of other cell surface markers: the two minor subsets, Fr. III and Fr. IV, are both CD45RBlow/-, L-selectin (Mel-14)- and CD44hi, characteristic of secondary T cells. The patterns and levels of cytokine production by individual cells in each subset were determined by bioassay for interleukin (IL)-2/IL-4 or IL-4/interferon (IFN)-gamma production after anti-CD3 stimulation. Our data revealed that these four phenotypically defined subsets largely coincide with clusters of cells showing uniform distinctive cytokine profiles, i.e. IL-2+/IFN-gamma-/IL-4- (Fr. I and Fr. II, L-selectin+), IL2+/IFN-gamma +/IL-4+ (Fr. III, L-selectin-), and IL-2-/IFN-gamma low/-/IL-4+ (Fr. IV, L-selectin-). Besides these subsets, an L-selectin-negative cell subfraction within Fr. II appears to represent a transitional population between the IL-2+/IFN-gamma-/IL-4- stage and the IL-2+/IFN-gamma +/IL-4+ stage. Taken together, these results demonstrate the presence of two IL-4+ secondary T cell subsets with distinct cytokine production patterns, and show that the majority of IL-4+ cells found in healthy adult laboratory mice co-produce IFN-gamma, and thus are not typical T helper type 2 cells.

Animals↗
PubMed1994

T cell receptor V beta 8.2 gene germ-line transcription: an early event of lymphocyte differentiation.

Rearrangement of the T cell antigen receptor (TCR) beta chain genes is highly regulated in both a developmental and a tissue-specific manner. T cell precursors originate from the yolk sac or fetal liver during gestation and from the bone marrow during adulthood. They initiate the recombination of TCR genes primarily during differentiation in the thymus. It has previously been suggested that transcription of immunoglobulin genes in germ-line configuration is linked to recombination events within these loci. Here, we examine whether germ-line transcription of TCR variable genes coincides with their rearrangement or whether it marks even earlier stages of T lymphocyte development. During gestation, we found V beta 8.2 germ-line transcripts in the fetal liver and the fetal thymus, but not in the yolk sac. This transcription precedes V beta 8.2 to D beta J beta rearrangement. In adult animals, we found these transcripts in the thymus, the spleen, the liver and the bone marrow. However, in the liver, this transcription is dependent on the presence of mature lymphocytes. This transcription does not happen in non-lymphoid cells. In the B lymphocyte lineage, V beta 8.2 germ-line transcripts are detected only in the earliest stages of differentiation (pre-pro- and pro-B cells), but not in pre-B cells and mature B lymphocytes. Altogether, our results show that transcription of the unrearranged V beta 8.2 gene is an early event of lymphocyte development, taking place in lymphocyte precursors, long before V beta 8.2 rearrangement.

Animals↗
PubMed1994

Monoclonal anti-lambda 5 antibody FS1 identifies a 130 kDa protein associated with lambda 5 and Vpre-B on the surface of early pre-B cell lines.

mAbs specific for mouse lambda 5 protein were prepared by fusion of spleen cells from a hamster immunized with recombinant lambda 5 protein synthesized in bacteria and the mouse myeloma cell line SP2/0-Ag14. Here we report the characteristics of the antibodies produced by the FS1 hybridoma. FS1 antibody stains a variety of mouse pre-B cell lines but not B cell lines or T cell lines. The staining of the pre-B cell lines A-1 and C-7 by phycoerythrin (PE)-conjugated FS1 (FS1-PE) can be blocked by preincubation of these cells with unconjugated FS1 antibody or with affinity purified polyclonal lambda 5 specific Ig but not with normal hamster or mouse IgG or with affinity purified polyclonal anti-Mb-1 Ig. From these experiments we concluded that FS1 specifically recognizes lambda 5 protein. We used FS1-PE to probe for surface (s) lambda 5+ cells in normal BALB/c mouse bone marrow. Such cells were undetectable when total bone marrow or FACS sorted subpopulations were analyzed. However, when B220+, CD43+, s lambda 5-bone marrow cells were cultured for 4 days on the stromal cell line FLST2 in the presence of IL-7, s lambda 5 expression became apparent. Further expansion of these cells in IL7 alone augmented the s lambda 5 expression to readily detectable levels. This modulation may indicate that s lambda 5 expression on normal bone marrow cells in vivo is transient and that at any given moment only a small fraction of bone marrow cells expresses low levels of lambda 5 protein on the surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Distinctive developmental origins and specificities of murine CD5+ B cells.

CD5+ B cells constitute a small fraction of cells in the spleen of adult mice that exhibit numerous features serving to distinguish them from the bulk of IgD++CD5- "conventional" B cells. In this review we focus on two major questions relating to this population: 1) the relationship of CD5+ B cells to other B cells; and 2) the distinctive enrichment of particular autoreactive specificities in this subset. The nature of their origins is clarified by a thorough analysis of intermediate stages of early B-cell development in both fetal and adult tissues. The reactivity to bromelain-treated mouse red blood cells serves as a prototype system for the investigation of biased specificities in CD5+ B cells. These lines of investigation lead us to propose that CD5+ B cells in the adult are the remnant of a distinct fetal B-cell differentiation pathway wherein selection of cells from this fetal/neonatal population into the adult long-lived pool results in the over-expression of certain germline-encoded autoreactivities.

Amino Acid Sequence↗
PubMed1993

The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver.

The expression of B lineage associated genes during early B cell differentiation stages is not firmly established. Using cell surface markers and multiparameter flow cytometry, bone marrow (BM) cells can be resolved into six fractions, representing sequential stages of development; i.e., pre-Pro-B, early Pro-B, late Pro-B/large Pre-B, small Pre-B, immature B, and mature B cells. Here we quantitate the levels of several B lineage associated genes in each of these fractions by RT-PCR, demonstrating different patterns of expression. We find that expression of terminal deoxynucleotidyl transferase (TdT), lambda 5, and VpreB is predominantly restricted to the Pro-B stages. Rag-1 and Rag-2 expression is also tightly regulated, and is found largely in the Pro-B through small Pre-B stages. Mb-1 is present from Pro-B throughout the pathway at high levels. Finally, Bcl-2 is expressed at high levels only at the pre-Pro-B and mature B stages, whereas it is low during all the intermediate stages. We also correlate this expression data with an analysis of the onset of Ig gene rearrangement as assessed by amplifying D-JH, VH-DJH, and VK-JK. Finally, we report differences in gene expression during B lymphopoiesis at two distinct ontogenic timings, in fetal liver and adult BM: both TdT and the precursor lymphocyte regulated myosin-like light chain are expressed at high levels in the Pro-B cell stage in bone marrow, but are absent from the corresponding fraction in fetal liver. In contrast, lambda 5, VpreB, Rag-1, and Rag-2 are expressed at comparable levels.

Animals↗
PubMed1993

Selective enrichment of major histocompatibility complex class II-specific autoreactive T cells in the thymic Thy0 subset.

We show here a unique enrichment of autoreactive T cells in the CD4+ mouse thymic subset, Thy0. A single- and 10-cell AMLR (autologous mixed leukocyte reaction) assay demonstrates that more than 30% (one cell per well) and almost all (10 cells per well) Thy0 cultures from normal mice exhibit reactivity specific to autologous cells, resulting in induction of interleukin 3 secretion. In contrast, no other mature thymic or splenic CD4+ T cell subsets showed such a high frequency. The majority of this AMLR reactivity in the Thy0 subset is accounted for by reactivity with self-major histocompatibility complex class II. Furthermore, antigenic selection in generating Thy0 subset is suggested by studies with T cell hybrids from a T cell receptor (TCR) V beta transgenic mouse line, 2B4 beta EH. TCR V-gene analysis of T cell hybrids revealed that those from Thy0, half of which responded to self-class II, consisted predominantly of cells that expressed endogenous TCR V beta s alone (without the transgene), unlike hybrids generated from peripheral naive T cells. Thus, we suggest that the presence of Thy0 results from selective stimulation of cells expressing TCR with sufficient affinity for autoantigens in the thymic CD4+ T cell repertoire.

Animals↗
PubMed1993

The 3G11+ antigen, a marker for murine CD4+ TH1 lymphocytes, is a ganglioside.

Two monoclonal antibodies (mAbs), SM3G11 and SM6C10, can be used to discriminate between functionally distinct murine CD4+ T cell subsets. In this study we use high-performance thin-layer chromatography and immunostaining techniques to show that the 3G11 mAb reacts with two bands of a ganglioside fraction from murine spleen and thymus, and rat spleen. The 6C10 antibody shows no evidence of glycolipid reactivity. The 3G11+ bands have a mobility between those of the reference gangliosides GD1a and GD1b from human brain. The 3G11+ reactive bands were eluted in the disialyl fraction of rat spleen gangliosides using DEAE anion-exchange chromatography. Treatment of spleen gangliosides with endoglycoceramidase eliminates 3G11 antibody binding over time, indicating that the antigen contains a Glc beta 1-1'ceramide linkage, characteristic of a glycosphingolipid. Treatment of thymus or spleen gangliosides with sialidase eliminates binding of 3G11, thus indicating that the 3G11 epitope is dependent on the expression of one or more sialic acid residues. Immunostaining studies with a variety of reagents indicate that the 3G11+ gangliosides: (i) probably do not contain either the asialo-GM1 or the GM1 core structures; (ii) are not recognized by mAbs specific for the oligosaccharides of asialo-GM2, GM2, GD2 and GD3 gangliosides; and (iii) are also not recognized by antibodies or reagents that are specific for several structures representative of other major glycosphingolipid classes. Overall, these studies strongly suggest that the 3G11+ gangliosides have structures that have not been previously recognized in murine lymphoid tissue. Structures that could account for the known properties of the 3G11+ molecules are described. Finally, ways in which the selective expression of 3G11+ gangliosides might be linked to functionally distinct T-cell behaviours are discussed.

Animals↗
PubMed1993

B-lineage cells in mu-transgenic scid mice proliferate in response to IL-7 but fail to show evidence of immunoglobulin light chain gene rearrangement.

The severe combined immunodeficiency (scid) mouse mutation impairs the recombination of Ig and TCR genes. Mice homozygous for this mutation (scid mice) lack pre-B, B, and T lymphocytes. Earlier we introduced a functionally rearranged mu-heavy chain gene into the scid mouse genome and found that this resulted in the development of pre-B cells in the bone marrow of these mice; however, sIgM+ B cells were not detected. We have now investigated the growth properties and rearrangement status of Ig genes in early B-lineage cells arising in mu-transgenic scid mice. We find that the presence of a functional mu-transgene allows pro-B cells from these mice to proliferate in short-term culture with IL-7. Nevertheless, rearrangements of Ig light chain genes are not detected in the bone marrow of such mice. Furthermore, the frequency of rearrangement detected at the endogenous Ig heavy chain locus in scid pro-B and pre-B cells is reduced relative to that in wild-type cells.

Animals↗
PubMed1992

Murine thymic CD4+ T cell subsets: a subset (Thy0) that secretes diverse cytokines and overexpresses the V beta 8 T cell receptor gene family.

We demonstrate here the presence of a distinct mature CD4+8- T cell subset in mouse thymus. This subset, termed "Thy0," is delineated by the absence of 3G11 expression from about half of the 6C10-/HSAlow/- fraction of CD4+8- thymic cells. Thy0 is detectable from the neonatal period and largely contributes the Th0-type diverse cytokine production previously reported for the HSAlow/-CD4+ thymic population. Further, cells expressing the T cell receptor V beta 8 gene family are found at increasing frequency in Thy0 with age, comprising 40-60% of Thy0 in adult BALB/c mice. This alteration of V beta 8+ cell frequency is unique to Thy0, since no other CD4+ subset in thymus or spleen shows such V beta 8 overusage. All functional CD4+ T cell subsets, including Thy0, show appropriate V beta clonal deletion associated with endogenous superantigens. Thus, it appears that Thy0 is an intrathymically generated secondary cell subset produced after CD4+ T cell selection.

Age Factors↗