Repopulation of SCID mice with fetal-derived B-lineage cells.
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Animals↗
Biomedical subjects
R R Hardy
Publications and source records attributed to R R Hardy.
Variable gene usage, physiology and development of Ly-1+ (CD5+) B cells.
Two related features of Ly-1+ B cells (CD5+ B cells in man) are their biased specificities and variable gene usage, and their relationship to the bulk of Ly-1- ('conventional') B cells. An emerging model views Ly-1+ B cells as a memory population in the adult animal, whose generation is largely limited to fetal and neonatal development. Selection based on germline-encoded self-reactivity then shapes the adult repertoire.
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Developmental origins, specificities and immunoglobulin gene biases of murine Ly-1 B cells.
Ly-1 B cells in mouse show numerous phenotypic and functional features that distinguish them from the bulk of IgDhigh/Ly-1- B cells. Their association with autoantibody production and the presence of Ly-1 on a group of murine B lymphomas that also exhibit certain specificities enriched in the normal population has stimulated continuing interest in this population. We have taken two approaches in our investigations of these cells: 1) defining the origins of Ly-1 B cells (the "lineage question"); and 2) studying the expression of particular specificities and associated immunoglobulin V genes enriched in this population. In this review we present the experimental background that supports our current understanding of Ly-1 B cells as the remnant of a fetal B cell differentiation pathway and suggest that the selection of cells from this fetal/neonatal population into the adult long-lived pool results in the over-expression of certain germline-encoded autoreactivities, such as antibody to bromelain-treated mouse red blood cells and intact thymocytes.
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Murine CD4+ T-cell subsets generated by antigen-independent and dependent mechanisms.
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Animals↗
A developmental switch in B lymphopoiesis.
B and T lymphocytes are generated from hematopoietic stem cells during both fetal and adult life. A critical unresolved issue is whether the differentiation pathways in lymphopoiesis are the same in fetal and adult animals or whether they differ, similar to the hemoglobin switch in erythropoiesis. We report here that a developmental switch occurs in B lymphopoiesis. We isolated "pro-B" cells (i.e., cells that have initiated, but not completed, heavy-chain gene rearrangement) from fetal and adult sources and investigated their B-cell progeny generated both in vitro and in vivo. Most of the cells from fetal liver, but few from adult bone marrow, expressed CD5. Further, fetal pro-B cells failed to generate cells expressing high levels of IgD in severe combined immunodeficiency mice, whereas adult pro-B cells gave rise to CD5-B cells bearing IgD at levels comparable to the bulk of cells in the spleen of adult mice. Thus, all committed B progenitors in fetal liver of day 16 gestation mice give rise to phenotypically distinct progeny when compared to cells at a comparable differentiation stage in the bone marrow of adult animals. We conclude that the cohort of B-lineage progenitors in early fetal development is committed to a differentiation pathway distinct from that seen in the adult.
Aging↗
Structural basis of a conserved idiotope expressed by an autoreactive human B cell lymphoma. Evidence that a VH CDR3 mutation alters idiotypy and specificity.
Our laboratory has previously investigated the relationship of autoimmune disease and B cell neoplasia in a patient with a diffuse, well differentiated splenic B cell lymphoma and associated autoimmune hemolysis due to an anti-Pr2 antibody. EBV-immortalized B cell clones, established from this lymphoma, were shown to secrete the same pathologic anti-Pr2 antibody. The antiidiotypic mAb, RI.1, defined a private Id (IdRI.1) of the anti-Pr2 antibody that was related to the Ag-binding site and was expressed by both the lymphoma and derived cell lines. This unique Id was expressed by the majority of splenic tumor B cells and also was conserved over a period of 4 yr. In this report, the structural basis of IdRI.1 expression was investigated by analysis of Id- variants isolated by flow microfluorimetry using RI.1. Six Id- cell lines that secrete IgM kappa but lack Pr2 specificity were generated from an Id+ cell line, LS2. These lines were shown to be related to LS2 and the lymphoma by karyotype and by restriction fragment analysis of Ig gene rearrangements. Shared and unshared nucleotide substitutions in the VH and VL regions of the six independent clones were used to construct a genealogic tree relating the Id- clonal members to a common Id+ precursor. The tree illustrates that the base changes occurred sequentially, suggesting that they were introduced by somatic point mutation. Only one VH CDR3 bp difference from the LS2 nucleic acid sequence is common to all Id- sequences, resulting in an amino acid substitution of cysteine 108 to tyrosine. Taken together, these findings suggest that both the expression of IdRI.1 and Ag binding are affected by a single mutation localized to the D region of the anti-Pr2 antibody.
Amino Acid Sequence↗
The role of clonal selection in the pathogenesis of an autoreactive human B cell lymphoma.
To study the association of autoimmunity and human B cell neoplasia, we have established a model of a B cell lymphoma which expresses a pathogenic autoantibody of defined specificity. The Ig VH gene expressed in this neoplasm was analyzed longitudinally using clinical specimens taken from the splenic lymphoma (S) at diagnosis and from lymph node relapses 3 and 4 yr later (N3 and N4). Southern analysis and oligonucleotide hybridization experiments demonstrated that clonally related predominant and minor tumor cell populations were present in S at diagnosis, and that the minor population became the predominant population in the relapse specimens, N3 and N4. Although the Ig specificity and idiotype were the same at diagnosis and at both relapses, analysis of the expressed VH gene sequences showed 14 base changes between S and N3, and 2 further changes at N4. Little sequence heterogeneity was observed at each sampling time, indicating that the ongoing mutation frequency was low. The relevant germline precursor VH gene was determined from autologous germline DNA and compared to the expressed genes. Based on the pattern of shared and unshared mutations, we were able to establish the genealogic relationship of the germline VH gene and the expressed clonotypes of S, N3 and N4. Taken together, the findings from Southern blotting, oligonucleotide hybridization, and sequence analysis permit us to describe a molecular aspect of tumor progression, "clonotypic shift", wherein subpopulations of the malignant clone, marked by different V gene clonotypes, emerge and predominate at different time points in the evolution of the lymphoma. Furthermore, the sequential and nonrandom pattern of the VH mutations, correlated with the observed conservation of autospecificity and idiotype, implies that clonal selection may have influenced the pathogenesis of the lymphoma.
Amino Acid Sequence↗
Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow.
We have resolved B220+ IgM- B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin, CD43), BP-1, and 30F1 (heat stable antigen). Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction, lacking S7, consists of pre-B cells whereas the others, expressing S7, include B lineage cells before pre-B. These S7+ fractions, provisionally termed Fr. A, Fr. B, and Fr. C, can differentiate in a stromal layer culture system. Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7- pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1, DFL16.1, and Jk1, we find that the Ig genes of Fr. A are in germline configuration, whereas Fr. B and C are pro-B cell stages with increasing D-J rearrangement, but no V-D-J. Finally, functional analysis demonstrates that the proliferative response to IL-7, an early B lineage growth factor, is restricted to S7+ stages and, furthermore, that an additional, cell contact-mediated signal is essential for survival of Fr. A.
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Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice.
Self-reactive B cells can be regulated by either deletion or inactivation. These manifestations of self-tolerance have been dramatically shown in transgenic mice in which the number of self-reactive cells has been artificially expanded. We have now extended these models to ask if B-cell tolerance as described for non-disease-associated antigens also operates for the targets of autoimmunity. The target we have chosen is DNA. Anti-DNA antibodies are diagnostic of certain autoimmune syndromes in humans and are a characteristic of the murine model of systemic autoimmunity, the MRl/lpr mouse. Antibodies to both single-stranded and double-stranded DNA have been implicated in disease. By generating anti-DNA transgenic mice, we have addressed the question of whether DNA-specific B cells are regulated in normal (non-autoimmune) mice. We indeed found that most transgenic B cells bind DNA, yet we failed to detect secreted anti-DNA. We suggest that as a consequence of their self-reactivity these B cells are developmentally arrested.
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CD5 B cells: a separate lineage at last?
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Journal Article↗
Natural autoantibodies to thymocytes: origin, VH genes, fine specificities, and the role of Thy-1 glycoprotein.
15 SM/J mouse hybridoma antibodies that show antithymocyte autoantibody (ATA) activity by immunofluorescence staining were studied. Half of these antibodies react with determinants whose expression is associated with Thy-1, as shown by blocking experiments with anti-Thy-1 and loss of reactivity with Thy-1- mutant cell lines. The Thy-1 dependence of three of these ATA is further confirmed by their reexpression on a Thy-1 gene transfectant. However, the remaining antibodies exhibited binding that showed little or no dependence on Thy-1. Furthermore, we find that most ATA derives from the Ly-1 B subpopulation, as demonstrated by lipopolysaccharide-induced ATA secretion in vitro and by comparison of ATA hybridoma frequencies. VH region gene sequence data of 14 monoclonal ATA from Ly-1 B cell-derived hybridomas reveal the utilization of nine VH genes belonging to four different VH families (J558, 3609, Q52, and Vgam3.8). While we find that two of these hybridomas arose from a clonal expansion, we also find four examples of a 3609 family VH gene utilized in clonally independent lines showing similar specificity. Yet another example of identical VH gene usage by clonally unrelated cells is found in two J558 ATA of a distinct fine specificity. These data suggest that the enrichment of ATA B cells in the Ly-1 B subset is primarily due to repeated independent recruitment of B cells by antigen resulting in the expression of a restricted set of VH genes.
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Differences in the expression profiles of CD45RB, Pgp-1, and 3G11 membrane antigens and in the patterns of lymphokine secretion by splenic CD4+ T cells from young and aged mice.
Previous studies indicate that the 3G11, CD45RB, and Pgp-1 determinants are differentially expressed on CD4+ T cell subsets in the mouse. We used multicolor immunofluorescence staining and flow cytofluorometric analysis to examine the expression of each of these determinants on splenic CD4+ cells from young (age 3 to 6 mo) and aged (age 24 to 26 mo) C57BL/6 mice. The CD4+ pool from aged mice contained significantly reduced numbers of 3G11+ and CD45RBhi cells, but increased numbers of Pgp-1hi cells, in comparison with the young group. Analysis of the simultaneous expression of all three subset determinants on CD4+ cells revealed that, in young mice, the major fraction (greater than 50%) was 3G11+CD45RBhiPgp-1lo. Among the less prevalent cell phenotypes, reductions in 3G11 expression correlated with decreases in CD45RB levels and increases in Pgp-1 levels. The phenotype that dominated the young group (3G11+CD45RBhiPgp-1lo) was approximately fivefold less represented in the aged group. The CD4+ pool from aged mice was characterized by increases in the 3G11-CD45RBvariablePgp-1hi and the 3G11+CD45RBloPgp-1hi phenotypes. To evaluate possible age-associated differences in cytokine secretion patterns by splenic CD4+ cells, purified CD4+ cells from each age group were stimulated in vitro with immobilized anti-CD3 epsilon mAb and accessory cells. At various times thereafter, supernatants from cultures were tested for IL-2 and IL-4 content by using the CTLL.6 and 11.6 bioassays, respectively, and the CD4+ cells were assayed for [3H]TdR uptake. Cell cultures from the aged group exhibited similar peak IL-2 accumulation and lower peak [3H]TdR uptake, but greatly increased peak IL-4 accumulation, as compared with cell cultures from the young group. The expression patterns of subset determinants, in conjunction with cytokine secretion profiles, indicate that, in aged mice, marked alterations occur in the subset composition of the splenic CD4+ cell pool. These findings are discussed in the context of previous findings on changes in T cell reactivity with advancing donor age.
Aging↗
Rearrangement and selection of VH11 in the Ly-1 B cell lineage.
One of the predominant VH genes utilized to encode the anti-BrMRBC specificity is a member of the small VH11 family rearranged to JH1. Using the polymerase chain reaction (PCR) we have determined that the frequency of B cells with a VH11 rearrangement is 10-20 times higher in Ly-1 B than in Ly-1- "conventional" B cells regardless of location (spleen or peritoneal cavity). Conventional B cells rearrange this gene at comparable levels in pre-B cells and in mature B cells utilizing all JH gene segments. In contrast, the increased levels of VH11 rearrangement in Ly-1 B are restricted to JH1 (and some JH2) and therefore appear to be the result of selection. Furthermore, most peritoneal Ly-1 B cells with VH11 rearrangements fall in a fraction stained by anti-BrMRBC antibody, likely bearing multivalent natural (likely self) antigen constitutively bound to their surface Ig receptors. Thus, we suggest that autoantigens are largely responsible for the accumulation of autoantibody specificities in the Ly-1 B cell lineage with time, whereas they do not exert this effect in the conventional B cells.
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B cells are required for induction of T cell abnormalities in a murine retrovirus-induced immunodeficiency syndrome.
The role of B cells in induction of phenotypic and functional abnormalities of T cells in a murine retrovirus-induced immunodeficiency syndrome, MAIDS, was evaluated in mice depleted of mature B cells from birth with anti-IgM antibodies (mu-suppressed) and infected at 4 wk of age. Multicolor FACS analyses of CD4+ T cell subsets showed that development of phenotypic abnormalities of these cells at 9 wk after infection was completely inhibited by mu-suppression. Furthermore, induction of impaired proliferative responses to Con A and alloantigens and CTL responses to alloantigens was fully blocked in antibody-treated animals. The extent of virus replication was comparable in spleens of untreated and mu-suppressed mice. Retroviral induction of T cell dysfunction in MAIDS is thus dependent on the presence of B cells, and high level virus expression in mice without B cells has little or no effect on T cell function.
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A unique subset of normal murine CD4+ T cells lacking Thy-1 is expanded in a murine retrovirus-induced immunodeficiency syndrome, MAIDS.
Some strains of mice inoculated with LP-BM5 murine leukemia virus (MuLV) develop a syndrome, termed mouse acquired immunodeficiency syndrome (MAIDS), characterized by progressive lymphoproliferation and profound immunodeficiency. LP-BM5 MuLV is a virus mixture that contains ecotropic (eco) and mink cell focus-induced MuLV and a defective genome that is the proximal cause of disease. Flow cytometry analyses of spleen and lymph nodes from susceptible C57BL/6 mice infected with this virus mixture revealed the presence in spleen and peripheral lymph nodes of a previously unrecognized subset of CD4+CD3+ T cells that are Thy-1-. The frequency of these cells increased with progression of disease, eventually comprising between 30% and 50% of all CD4+ cells. Infection of A/J mice, a strain which is genetically resistant to development of MAIDS, did not induce an increase of this T cell population, indicating that infection with the virus mixture was insufficient to induce its proliferation. A central role for the defective virus in this process was suggested by the finding that C57BL/6 mice infected with LP-BM5 eco alone did not have increased frequencies of Thy-1-CD4+ cells in spleen. Studies of spleen and peripheral lymph node cells from normal mice demonstrated the presence of Thy-1-CD4+ cells at frequencies of 1%-2%. Studies using two anti-T cell monoclonal antibodies, SM6C10 and SM3G11, that define four CD4+ subsets showed that Thy-1-CD4+ T cells from normal and infected mice were present only in the 6C10- subsets.
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Development of B-lineage cells in the bone marrow of scid/scid mice following the introduction of functionally rearranged immunoglobulin transgenes.
Mice homozygous for the mutation scid (scid mice) are severely immunodeficient and generally lack detectable numbers of pre-B, B, and T cells. This condition is believed to result from a defect in the mechanism responsible for rearrangement of immunoglobulin and T-cell receptor genes in developing B and T lymphocytes. To test this hypothesis and evaluate whether scid affects only the process of gene recombination, we introduced functionally rearranged immunoglobulin genes into the scid mouse genome. As scid mice appear to contain early lymphoid cells committed to the B lineage (pro-B cells), we asked whether the introduction of an IgM heavy-chain gene alone (mu-transgenic scid mice) or both IgM heavy- and kappa light-chain genes (mu kappa-transgenic scid mice) would allow further differentiation of scid pro-B cells into pre-B and B cells. We found that normal numbers of pre-B cells appeared in the bone marrow of mu-transgenic scid mice and that both pre-B and B cells appeared in the bone marrow of mu kappa-transgenic scid mice. However, in the latter case, the number of pre-B and B cells was 2- to 3-fold less than in the controls (mu kappa-transgenic scid heterozygotes) and few, if any, B cells were detectable in the peripheral lymphoid tissues. The implications of these results for the above hypothesis are discussed.
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Development of murine B cell subpopulations.
The generation of diverse B cell subpopulations in the mouse is the subject of considerable current interest. In this review, I first describe a general framework for antigen-independent B cell differentiation in the adult bone marrow including results from in vitro culture systems. In following sections, effects on B cell development in several immunodeficient strains and transgenic lines are presented. Next, the CD5+ B cell subpopulation is covered in some depth, including data on development, physiology and repertoire selection. Finally, another selected population, memory B cells, together with their likely origins in the germinal center are discussed.
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