Project
1: Function of the Enzyme Terminal Deoxynucleotidyl Transferase
Why are antibodies less diverse in the fetus and neonatal life compared to those in adults and is this functionally important in the immune response?
The
immune system develops in a series of programmed developmental stages.
The limited diversity of the early fetal repertoire of B cells is
guided
by genetic mechanisms involved in the production of the B cell receptor
(BCR) that are active during fetal development. These characteristics
may
affect the establishment of the adult B cell repertoire. Indeed,
interference
with the developmental program in early life results in long-lasting
alterations
to the expressed B cell repertoire. Of particular relevance is that
during
this period of development (in mice up to 3 wks of age, in humans up to
1.5 to 2 yrs) neonates respond very poorly if at all to bacterial
capsular
polysaccharides. This has the important consequence that in addition to
Gram negative enteric infections, Gram positive organisms including Streptococcus
pneumoniae and Group B streptococci are the leading cause of
serious
bacterial infections in neonates in both developed and developing
countries.
Terminal Deoxynucleotidyl Transferase (Tdt) and B cell Development
Of
these mechanisms, the lack
of expression and activity of the enzyme mouse terminal
deoxynucleotidyl
transferase (Tdt) clearly distinguishes B cell development in the fetus
from that in adult bone marrow. Tdt adds non-templated nucleotides at
the
junctions of the VDJ genes during splicing to form the T and B cell
receptor
genes. The release of transcriptional and post transcriptional control
mechanisms involved in Tdt expression beginning at birth and continuing
into adult life results in N region addition and subsequent
diversification
of the B cell repertoire. In mice there are two forms of Tdt: one with
a 20 amino acid deletion resulting from mRNA splicing which gives rise
to a short form (TdtS) which has been shown clearly to produce N region
additions during Ig gene rearrangements. Experimental evidence for the
function (or lack of) of Tdt (TdtL) is contradictory. We propose that
the
two forms of Tdt have distinct but complementary functions in
lymphocyte
development and that TdtL has a dominant negative effect on TdtS
function.
Approaches
Generation of Tdt Tg mice
To
increase diversity in the
fetal B cell repertoire, we generated transgenic mice in which
junctional
diversity was introduced into Ig gene rearrangements via the activity
of
Tdt, which is normally not expressed in fetal mice. We generated lines
expressing the short splice variant (TdtS) and others containing the
long
splice variant (TdtL) to determine whether one or both variants were
capable
of adding N regions. We could show in vitro that
both forms are
expressed in the nuclei of LPS-activated B cells (Figs. 1 and 2). By
sequencing
heavy and light chain immunoglobulin genes from fetal liver we could
show
also that N regions were present when they normally are not in short
form
but not in long form mice.
Consequences of Premature N Region Addition
Does
this premature N region addition affect the adult antibody repertoire?
We studied the antibody response to phosphorylcholine (PC) a component
of the cell wall in Streptococcus pneumoniae which
can induce protective
antibody-mediated immunity to this organism. In mice the response to PC
is dominated by a very restricted population of antibodies with
identical
heavy and light chains. For this reason these chains are said to be
canonical
and the genes that code for them are fetal-like in that they do not
contain
N regions. When these Tdts transgenic mice were challenged as adults
with
S. pneumoniae they could no longer make these
canonical antibodies
because of N region addition. As a result their serum no longer
contained
protective antibodies.
Our
results show that during
fetal development this window, when Tdt is not expressed, is necessary
for the development of these protective clones of B cells which then
last
for the life of the mouse and are generated only poorly in the adult.
These
antibodies are important in protection against S. pneumoniae
infections.
References
Benedict, C. L. and Kearney, J. F. Increased junctional diversity in fetal B cells results in a loss of protective anti-phosphorylcholine antibodies in adult mice. Immunity 10:607-617, 1999.
Benedict, C.L., Gilfillan, S., Thai, T-H., and Kearney, J.F. Terminal deoxynucleotidyl transferase and lymphocyte repertoire development. Immunol. Rev. 175:150-157, 2000.
Benedict,
C.L., Gilfillan, S., and
Kearney, J.F. The long isoform of terminal deoxynucleotidyl
transferase
enters the nucleus and, rather than catalyzing nontemplated nucleotide
addition, modulates the catalytic activity of the short isoform. J.
Exp.
Med. 193:89-99, 2001.
In the Future
Our
plans are to now determine
the structure/function relationship of both forms of Tdt, and to
determine
their role in B- and T-cell development. This will involve the
production
and characterizion of mouse monoclonal and rabbit polyclonal antibodies
to TdtS and TdtL, and using these and other molecular approaches to
determine
the expression of TdtS and TdtL during B cell development, ontogeny,
and
cell cycle.This will include intracellular localization using confocal
microscopy and biochemical methods. We will express and purify both
forms
to determine the in vitro functions of TdtS and
TdtL. By using the
lines of transgenic mice we will determine in vivo
functions of
the two forms and whether they regulate each other.
Significance of Our Studies
As well as the fuctions of Tdt outlined above, historically, Tdt is used as a marker for the diagnosis of lymphomas, however, it has not been determined whether the high level expression of Tdt in these tumors is the cause or consequence of the cancer or whether one form is preferentially expressed. The new reagents that we plan to make will provide a unique tool to re-evaluate these tumors for Tdt expression. Recently, a BRCT domain (BRCA1 terminus) has been identified in the N terminal region of Tdt. The BRCT domain is also present in many proteins known to be involved in DNA double strand break repair and cancer susceptibility such as BRCA1 (the breast cancer susceptibility protein), the 53BP1 (p53-binding protein), the yeast RAD9 protein involved in DNA repair, the mammalian DNA ligases also known to be involved in DNA repair and V(D)J recombination. Moreover, RAG1 and RAG2 have been shown to act as transposases capable of mediating translocation of a piece of rearranged DNA to a non-related plasmid suggesting their roles in the generation of lymphomas which resulted mainly from DNA translocation. Since Tdt is a member of this constellation of DNA modifying proteins these studies may also have particular relevance to mechanisms of tumor induction.