Project 2: 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.

Conversely, we have recently shown that in other dominant anti-polysaccharide responses exemplified by the anti 1-3 dextran response, Tdt activity is essential to generate the dominant B cell clones.  In Tdt KO mice this response is reduced or absent.

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.

Mahmoud, T.I., Kearney, J.F. Terminal deoxynucleotidyl transferase is required for an optimal response to the polysaccharide alpha-1,3 dextran.  J Immunol: 2010 Jan 15;184(2):851-8. Epub 2009 Dec 16.


 
 

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