Return to Table of Contents

DISTINCTIONS BETWEEN FETAL AND ADULT B LYMPHOCYTE DIFFERENTIATION

RICHARD R. HARDY, Ph.D., Member
YUE SHENG LI, Ph.D., Postdoctoral Associate
ROBERT WASSERMAN, M.D., Postdoctoral Fellow, University of Pennsylvania
SUSAN F. SAUDER, B.S., Graduate Student, University of Pennsylvania (from June 1994)
SUSAN A. SHINTON, B.S., Research Assistant

B lymphocytes are generated from hematopoietic stem cells by a complex program of cell differentiation in the liver before birth and in the bone marrow afterward. B lineage cells in the bone marrow can be recognized by the expression of the high molecular weight form of the common leukocyte antigen CD45, B220. This population is very heterogeneous, consisting of cells at all stages of differentiation, from newly committed progenitor to mature B cells. Initially, we used multiparameter four-color flow cytometry to resolve these stages into seven fractions, based on expression of several cell surface molecules (leukosialin/ CD43, heat stable antigen, BP-1, IgM, and IgD). We found that these phenotypic subsets showed meaningful molecular and functional distinctions. From these studies, we defined early fractions, showing only incomplete heavy chain rearrangements (D-JH); later fractions, possessing complete rearrangements (VH-D-JH); and still later ones, showing Vk-Jk light chain rearrangement (Figure 1). We employed this system to study the expression of a number of genes thought to be critical to B lymphopoiesis by using the polymerase chain reaction to amplify cDNA generated from sorted cells. This RT-PCR analysis yielded data that both validated the pathway and also revealed greater detail concerning the regulated expression of genes during B cell differentiation.

In the past year, we have focused on extending our analysis to earlier stages, investigating the initiation of germline transcription and the expression of the recombinase activating genes throughout the pathway. In other work, we have examined the effect of heavy chain expression on the regulation of protein tyrosine kinases, enzymes that are involved in signal transduction pathways controlling cell proliferation and differentiation.

Another area of interest in our laboratory is the origin and autoreactive propensity of a distinctive subset of peripheral B cells known as CD5+ B cells, so called because they bear the pan-T cell marker CD5 on their surface. An important issue for us is determining the relationship of these cells to the majority of B cells that lack CD5. Cell transfer experiments performed several years ago suggested that, whereas fetal lymphoid precursors could generate CD5+ B cells, those in the adult could not. Using the cell fractionation scheme described above, we have carried out a careful comparison of gene expression between corresponding B cell stages isolated from fetal and adult tissues. Our recent work suggests that a gene thought to be a positive mediator of programmed cell death, interleukin-beta convertase (ICE), is expressed at significantly lower levels in the fetus compared to the adult. We are currently testing whether this difference plays any role in permitting the autoreactive bias in fetal CD5+ B cells.

FIGURE 1. Diagram of markers used to define fractions (Fr.) A0 through F of B lineage cells in mouse bone marrow. The approximate level of expression is indicated by the line thickness. The predominant rearrangement stages, heavy chain D to JH, heavy chain VH to DJH and light chain Vk to Jk also are shown.

TIMING OF Ig GERMLINE TRANSCRIPTION AND RECOMBINASE ACTIVATING GENE EXPRESSION DURING EARLY B CELL DIFFERENTIATION. HARDY, LI

We previously identified seven fractions of B lineage cells in mouse bone marrow with a differentiation order from Fr. A to Fr. F. In this study, we further resolved the earliest fraction (Fr. A) into two subfractions, A1 and A2, based on the surface expression of CD45 (B220), HSA, AA4.1, and CD4, and defined a B220­ precursor fraction, termed A0 (Figure 1). All three fractions lack HSA. A0 is B220­AA4.1+ and shows low CD4 expression. A1 and A2 are B220+AA4.1+ and are discriminated as CD4+ and CD4­, respectively. Together with our previous classification, we now delineate nine B lineage subsets in bone marrow, from the earliest progenitor to mature B cells. We have employed this scheme in our investigation of expression of several B-lineage related genes using semi-quantitative RT-PCR. One of the earliest markers of B lineage commitment is mu germline (Mu0) transcription. We found a high level of Mu0 in A1 and A2, prior to extensive D-J rearrangement (Fr. B). Kappa germline transcript, associated with Ig kappa light chain rearrangement, is sharply up-regulated in Fr. C' after completion of productive heavy chain VDJ rearrangement.

We also determined expression of the recombinase activating genes Rag1 and Rag2 in these fractions. Significant expression of Rag1 and Rag2 appears after Fr. A1, following the onset of high levels of mu germline transcription. Very interestingly, we found that there are two peaks of Rag1 and Rag2 expression, one prior to C' and the other afterward, segregating Ig heavy and light chain rearrangement, respectively. The level in Fr. C', in which all cells contain productive mu rearrangements, is essentially undetectable. It appears likely that association of mu heavy chain with the surrogate light chain complex (lambda5 and VpreB) results in down-regulation of Rag1 and Rag2 (Figure 2). The disappearance of surrogate light chain correlates with the reinduction of Rag1 and Rag2 during the light chain rearrangement phase of B cell development. Final generation of a complete heavy-light chain immunoglobulin molecule results in final extinction of Rag1 and Rag2 expression.


FIGURE 2. Diagram of the Ig complex at intermediate stages of B cell development with distinct levels of Rag-1, Rag-2, blk, and ret indicated. Lambda5 and VpreB are the components of the surrogate light chain, a complex that associates with mu heavy chain prior to kappa light chain rearrangement. Pluses (+) indicate relative levels of expression, minus (-) indicates no expression detected.

EXPRESSION OF TYROSINE KINASE GENES REGULATED BY Ig REARRANGEMENT DURING B CELL DEVELOPMENT. HARDY, WASSERMAN

Early B cell differentiation proceeds in response to cytokines and stromal cell interactions, whereas differentiation beyond the Pro-B stage is dependent upon the successive rearrangement and expression of Ig heavy and light chain genes. It is likely that these stimuli, which drive B cell differentiation, activate protein tyrosine kinases (PTKs). We have used semi-quantitative RT-PCR to identify five candidate PTKs by showing that their gene expression changes during B cell differentiation in the bone marrow. Four genes are down-regulated (fgr, tsk, flk2/flt3, and ret), and one gene is up-regulated (blk). To establish a correlation between Ig gene rearrangement and expression of these five kinases, we carried out similar analyses with recombination defective mice (scid and Rag-1­) with or without rearranged Ig transgenes. Three PTKs (fgr, flk2/flt3,and tsk) showed the same sharp down-regulation between mutant and normal mice, confirming that some aspects of early B cell development can proceed independently of Ig rearrangement. However, blk showed an attenuated rise in expression in the recombination defective mice and could be increased to normal levels by expression of Ig transgenes. Finally, ret expression showed a slight increase in the mutant mice, but was strikingly down-regulated by the Ig transgenes. Thus, the induction of blk and down-regulation of ret correlate with the appearance of products of Ig rearrangement, mu heavy chain and kappa light chain (Figure 2). These results suggest that blk and ret have roles at distinct stages of B cell differentiation in the bone marrow.

DIFFERENTIAL EXPRESSION OF INTERLEUKIN-1beta CONVERTING ENZYME DURING FETAL AND ADULT B LYMPHOPOIESIS. HARDY, SAUDER

The pathway of B cell differentiation in the fetus differs in several key ways from that in adult bone marrow. It has been suggested previously that this represents a developmental switch in B lymphopoiesis between fetal and adult life. An important event during early B cell development likely involves selection (positive or negative) of B cells with particular specificities, and such selection may be dependent on expression of genes that regulate apoptosis, programmed cell death. Here we report that the expression of interleukin-1beta converting enzyme (ICE), thought to be a positive mediator of apoptosis, differs in distinct stages of B cell development. We isolated B cell progenitor populations from fetal liver and adult bone marrow and studied the expression of ICE mRNA using a semi-quantitative RT-PCR. We found that ICE is expressed in adult bone marrow B lineage fractions at consistently high levels. Interestingly, the fetal progenitors differed from their adult counterparts. The Pro-B fraction of fetal liver showed significantly lower expression of ICE compared to Pro-B cells derived from adult bone marrow. Whether this difference could be responsible for distinctions in the Ig-repertoires or phenotypes of B cell populations generated in the fetus remains to be determined.

PUBLICATIONS

CANDEIAS, S., R.R. HARDY, Y.S. LI, and U.D. STAERZ. T cell receptor Vbeta8.2 gene germline transcription: and early event of lymphocyte differentiation. Eur. J. Immunol. (in press).

CHEN, C., M.Z. RADIC, J. ERIKSON, S.A. CAMPER, S. LITWIN, R.R. HARDY, and M. WEIGERT. Deletion and editing of B cells that express antibodies to DNA. J. Immunol. 152: 1970­1982, 1994.

CHEN, C., M.Z. RADIC, R.R. HARDY, D. HUSZAR, S.A. CAMPER, and M. WEIGERT. The site and stage of anti-DNA B-cell deletion. Nature 373: 252­255, 1995.

HAYAKAWA, K., D. TARLINTON, AND R.R. HARDY. Absence of MHC Class II expression distinguishes fetal from adult B lymphopoiesis in mice. J. Immunol. 152: 4801­4807, 1994.

KARIV, I., R.R. HARDY, and K. HAYAKAWA. Altered major histocompatibility complex restriction in the NK1.1+Ly-6C hi autoreactive CD4+ T cell subset from class II-deficient mice. J. Exp. Med. 180: 2419­2424, 1994.

Papers in press at time of previous report:

HARDY, R.R., C.E. CARMACK, Y.-S. LI, and K. HAYAKAWA. Distinctive developmental origins and specificities of murine CD5+ B cells. Immunol. Rev. 137: 91­118, 1994.

KARIV, I., R.R. HARDY, and K. HAYAKAWA. Two distinct non-T helper type 2 interleukin-4+ cell subsets in mice as revealed by single-cell cytokine analysis. Eur. J. Immunol. 24: 549­557, 1994.

SHINJO, F., R.R. HARDY, and J. JONGSTRA. Monoclonal anti-lambda5 antibody FS1 identifies a 130 Kd protein associated with lambda5 and VpreB on the surfaceof early pre-B cell lines. Int. Immunol. 6: 393­399, 1994.

SPANOPOULOU, E., C.A.J. ROMAN, L. CORCORAN, M. SCHLISSEL, D.P. SILVER, U. STORB, D. NEMAZEE, M. NUSSENZWEIG, S.A. SHINTON, R.R. HARDY, and D. BALTIMORE. Expression of functional immunoglobulin transgenes allows ordered B-cell differentiation to progress in Rag-1 deficient mice. Genes & Dev. 8: 1030­1042, 1994.

Return to Table of Contents