Table Of ContentCurrent Topics in
Microbiology
135 and Immunology
Editors
A. Clarke, Parkville/Victoria . R W. Compans,
Birmingham/Alabama . M Cooper, Birmingham/Alabama
H. Eisen, Paris . W. Goebel, Wilrzburg . H. Koprowski,
Philadelphia . F. Melchers, Basel . M Oldstone,
La Jolla/California· P.K Vogt, Los Angeles
H. Wagner, U1m . I. Wilson, La Jolla/California
Differentiation of
B Lymphocytes
Edited by
C.J. Paige and RH. Gisler
With 25 Figures
Springer-Verlag
Berlin Heidelberg NewY ork
London Paris Tokyo
Dr. CHRISTOPHER J. PAIGE
Dr. ROLAND H. GISLER
Basel Institute for Immunology
Grenzacherstrasse 487
CH-4058 Basel
ISBN-13: 978-3-642-71853-3 e-ISBN-13: 978-3-642-71851-9
DOl: 10.107/978-3-642-71851-9
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© Springer-Verlag Berlin Heidelberg 1987
Softcover reprint of the hardcover 1st edition 1987
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Preface
Rapid progress continues to be made in understanding the
molecular and cellular events that comprise B-Iymphocyte
differentiation. This is due in part to the high level of inter
est shown by many investigators from diverse disciplines,
who find this subject suitable for addressing some of the
fundamental issues of immunobiology. B-cell developmen
tal models are being extensively used to investigate cell-cell
interactions, molecular mediators of differentiation and
proliferation, differential onset of gene programs, and gene
rearrangement and expression, as well as the generation
of the immune response itself. Not surprisingly, increased
understanding of B-cell differentiation sometimes results
from the application of new techniques that permit greater
insight into the cells comprising the system and the genetic
mechanisms by which these cells express their differentiative
potential. However, experimental strategies based upon the
novel application of established technologies have also led
to the clarification of many issues, as well as to the discov
ery of previously unrecognized problems.
One problem, well recognized by those active in the
field, is how to keep up with significant developments as
they appear. The purpose of this book, part of a series
devoted to analysing current issues in biology, is to help
overcome this problem. No attempt at comprehensive cov
erage of all of the issues has been made. Rather, a more
thorough analysis of a few topics is presented. The first
section deals with cellular models of differentiation and
covers attempts, both in vivo and in vitro, to understand
B-cell development. As it has become increasingly apparent
that different species may well use different strategies to
achieve the requisite degree of antibody diversity needed
for the generation of an adequate repertoire, we have in
cluded chapters which present the avian and ovine systems
in contrast to the standard murine models. The second sec
tion deals with molecular analyses of immunoglobulin gene
formation, with particular emphasis on variable-region
gene utilization. Also discussed are the immunoglobulin
VI Preface
class switch during B-cell differentiation and attempts to
define genes expressed uniquely during B-cell development.
We thank Leslie Nicklin of the Basel Institute for Im
munology for editorial assistance in the preparation of this
volume.
Basel,1987 CHRISTOPHER J. PAIGE
ROLAND H. GISLER
Table of Contents
P.W. KINCADE, P.L. WITTE, and K.S. LANDRETH:
Stromal Cell and Factor-Dependent
B Lymphopoiesis in Culture. With 4 Figures . 1
K. DORSHKIND and O.N. WITTE: Long-Term Murine
Hemopoietic Cultures as Model Systems for
Analysis of B-Lymphocyte Differentiation. With
5 Figures . . . . . . . . . . . . . .. . 23
J.D. REYNOLDS: Peyer's Patches and the Early
Development of B Lymphocytes. With 3 Figures 43
J.R.L. PINK and O. LASSILA: B-Cell Commitment
and Diversification in the Bursa of Fabricius. With
1 Figure .................. 57
H. SAUTER and C.J. PAIGE: Differentiation of Murine
B-Cell Progenitors in Agar Culture: Determination
of the Developmental Potential of Clonable
Pre-B Cells ... . . . . . . . . . . . . 65
B.A. MALYNN, J.E. BERMAN, G.D. YANCOPOULOS,
C.A. BONA, and F.W. ALT: Expression of the
Immunoglobulin Heavy-Chain Variable Gene
Repertoire ............... 75
R.M. PERLMUTTER: Programmed Development of the
Antibody Repertoire. With 2 Figures . . . .. 95
J.-C. WEILL, M. LEIBOWITCH, and C.-A. REYNAUD:
Questioning the Role of the Embryonic Bursa in
the Molecular Differentiation of B Lymphocytes.
With 8 Figures .............. 111
P.D. BURROWS and H. KUBAGAWA: Immunoglobulin
Gene Rearrangements in Pre-B Cells. With
1 Figure .................,. 125
VIII Table of Contents
N. SAKAGUCHI, C.N. BERGER, and F. MELCHERS:
Cloning of Murine B-Lymphocyte Differentiation
Stage-Specific Genes. With 1 Figure 139
Subject Index 149
Indexed in Current Contents
List of Contributors
You will find the addresses at the beginning of the
respective contribution
ALT, F.W. 75 MELCHERS, F. 139
BERGER, C.N. 139 PAIGE, C.J. 65
BERMAN, J.E. 75 PERLMUTTER, R.M. 95
BONA, C.A. 75 PINK, J .R.L. 57
BURROWS, P.D. 125 REYNAUD, C.-A. 111
DORSHKIND, K. 23 REYNOLDS, J.D. 43
KINCADE, P.W. 1 SAKAGUCHI, N. 139
KUBAGAWA, H. 125 SAUTER, H. 65
LANDRETH, K.S. 1 WEILL, J.-c. 111
LASSILA, O. 57 WITTE,O.N. 23
LEffiOWITCH, M. 111 WITTE, P.L. 1
MALYNN, B.A. 75 YANCOPOULOS, G.D. 75
Stromal Cell and Factor-Dependent B Lymphopoiesis
in Culture
P.W. KINCADE, P.L. WITTE, and K.S. LANDRETH
1 Introduction 1
2 A Theoretical Model for B-Lymphocyte Formation
3 General Approaches Involving Lymphoid Cell Cultures 4
4 Long-Term Bone Marrow Cultures 5
5 Interculture Variability and the Nature of Cultured Lymphocytes 5
6 Nonlymphoid Adherent Cells 10
7 Cells with Potential for Growth in Long-Term Lymphocyte Cultures 11
8 A Cloned Tumor Cell Model 12
9 Soluble Factors Influencing B-Cell Precursors 14
10 Synthesis and Concluding Remarks 16
References 17
1 Introduction
Only a fraction of the cells responsible for humoral immunity (B lymphocytes)
are long lived. While some B cells are made by division of other B lymphocytes
during the propagation of immune responses, large numbers are continuously
produced from immature precursors within hemopoietic tissues such as bone
marrow. Recent technological advances have allowed substantial progress to
be made in understanding the processes involved in primary B-cell formation
and how they are regulated. This is particularly true for the final events in
the series because they can be observed in cell culture. On the other hand,
findings made with cell cultures provide exceptions to current hypotheses and
it is now appropriate to reevaluate those models. Our objectives here are to
present recent unpublished findings from in vitro studies, relate these to a general
model which has been derived largely from transplantation and whole-animal
work, and highlight new avenues of investigation which seem to hold particular
promise.
2 A Theoretical Model for B-Lymphocyte Formation
Panels of monoclonal antibodies can be used to identify committed progenitors
ofB cells, manipulate them experimentally, and construct a probable differentia-
Oklahoma Medical Research Foundation, 825 NE, 13th Street, Oklahoma City, OK 73104, USA
Current Topics in Microbiology and Immunology, Vol. 135
© Springer-Verlag Berlin' Heidelberg 1987
2 P.W. Kincade et al.
Large Small Newly
Hemopoietic ? B Lineage Pre-B Pre-B Formed
Stem Cell Precursor Cell Cell B Cell
Fig. 1. Possible relationships between precursors of murine B lymphocytes. The model emphasizes
the normal expression of an antigen on lymphocytes in adult bone marrow. Normal and leukemic
cells in culture provide exceptions to this apparent sequence of events, as detailed in the text
tion sequence for the final steps in de novo lymphopoiesis (KINCADE 1981;
KINCADE and PHILLIPS 1985; Fig. 1). Our model was constructed from several
experimental findings. All of the precursor cells in adult murine bone marrow
which could quickly become functional B cells in culture, and 70% of those
which did so in 10-day transfer experiments, could be depleted with our mono
clonal 14.8 antibody (KINCADE et al. 1981 b; LANDRETH et al. 1983). Most of
the lymphocytes in marrow could be highly enriched with this antibody and
the suspensions included large and small cells. Approximately one-half of the
selected 14.8-bearing (14.8+) lymphocytes which lacked surface immunoglobulin
contained the ~ heavy chains of IgM. These "pre-B" cells had earlier been
postulated to be the immediate precursors of B cells (RAFF et al. 1976; COOPER
1981). Other studies suggested that large pre-B cells normally divided just once
before giving rise to small pre-B cells, which then matured without further divi
sion by acquiring surface IgM (LANDRETH et al. 1981). Large cells sorted with
a similar monoclonal antibody had extensively rearranged heavy-chain immuno
globulin genes, whereas only the small cells had undergone light-chain gene
rearrangement (COFFMAN and WEISSMAN 1983). Not shown in our diagram is
the order of acquisition of various other surface markers (Lyb-2, Fc receptors,
Th-B, la, etc.) which characterize mature, functional B lymphocytes in mice.
Variations in assay techniques and the use of different inbred mouse strains
make it difficult to integrate the available information in this way.
While such theoretical models have been, and continue to be, most useful
for experimental design, some caution is advised about their literal use, especially
in interpreting culture results. We have stressed that the initial emergence of
B-Iymphocyte lineage cells in ontogeny might differ substantially from the pro
cess which occurs in adult marrow and have cited examples where initial surface
antigen expression was dependent on chronological age, rather than on position
in a differentiation sequence (KINCADE 1981, 1984; KINCADE et al. 1982). It
was found that most pre-B cells in early embryos lack detectable 14.8 (VELARDI
and COOPER, 1984; MEDLOCK et al. 1984), whereas the model indicates that
cytoplasmic ~-chains would only be seen in 14.8 + lymphocytes. After approxi
mately 16 days of gestation, all pre-B cells are 14.8+ and, while the marker
is expressed on a subpopulation of peripheral T cells, there is no evidence that
it is associated with myeloid cells (KINCADE et al. 1981 b; LANDRETH et al. 1983;
SCHEID et al. 1982).
