Protein evolution by hypermutation and selection in the B cell line DT40

Genome-wide mutations and selection within a population are the basis of natural evolution. A similar process occurs during antibody affinity maturation when immunoglobulin genes are hypermutated and only those B cells which express antibodies of improved antigen-binding specificity are expanded. Protein evolution might be simulated in cell culture, if transgene-specific hypermutation can be combined with the selection of cells carrying beneficial mutations. Here, we describe the optimization of a GFP transgene in the B cell line DT40 by hypermutation and iterative fluorescence activated cell sorting. Artificial evolution in DT40 offers unique advantages and may be easily adapted to other transgenes, if the selection for desirable mutations is feasible.     

Quantitative changes in sperm head morphology during passage through the male excurrent duct system of the rabbit

A fine adjustment of sperm head size and shape occurs during maturation and storage within the male excurrent duct of the rabbit. This remodelling, as judged by morphometric values of area, perimeter, length, width, and shape factors, takes place mostly in passage from the seminiferous tubules of the testis to the distal caput of the epididymis. The dimensions of sperm heads from the distal corpus of the epididymis break the general tendency toward a reduction in size and more elliptical shapes. A period of transport and storage within the epididymal cauda and vas deferens follows in which there are no further changes in sperm head morphometry. It can be concluded that the period immediately following sperm release from the testis is crucial to the final morphological maturation of spermatozoa. Moreover, the fact that changes are detected in the appearance of sperm heads at successive stages of sperm maturation suggests that the dimensions of a particular epididymal spermatozoon may be taken as an approximate indication of its relative maturity. Mol. Reprod. Dev. 51:203–209, 1998. © 1998 Wiley-Liss, Inc.     

A mathematical model for germinal centre kinetics and affinity maturation

We present a mathematical model which reproduces experimental data on the germinal centre (GC) kinetics of the primed primary immune response and on affinity maturation observed during the reaction. We show that antigen masking by antibodies which are produced by emerging plasma cells can drive affinity maturation and provide a feedback mechanism by which the reaction is stable against variations in the initial antigen amount over several orders of magnitude. This provides a possible answer to the long-standing question of the role of antigen reduction in driving affinity maturation. By comparing model predictions with experimental results, we propose that the selection probability of centrocytes and the recycling probability of selected centrocytes are not constant but vary during the GC reaction with respect to time. It is shown that the efficiency of affinity maturation is highest if clones with an affinity for the antigen well above the average affinity in the GC leave the GC for either the memory or plasma cell pool. It is further shown that termination of somatic hypermutation several days before the end of the germinal centre reaction is beneficial for affinity maturation. The impact on affinity maturation of simultaneous initiation of memory cell formation and somatic hypermutation vs. delayed initiation of memory cell formation is discussed.     

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.     

Reconciling repertoire shift with affinity maturation: the role of deleterious mutations

The shift in Ab repertoire, from Abs dominating certain primary B cell responses to genetically unrelated Abs dominating subsequent "memory" responses, challenges the accepted paradigm of affinity maturation. We used mathematical modeling and computer simulations of the dynamics of B cell responses, hypermutation, selection, and memory cell formation to test hypotheses attempting to explain repertoire shift. We show that repertoire shift can be explained within the framework of the affinity maturation paradigm, only when we recognize the destructive nature of hypermutation: B cells with a high initial affinity for the Ag are less likely to improve through random mutations.     

Codon insertion and deletion functions as a somatic diversification mechanism in human antibody repertoires

   

It has been suggested that codon insertion and/or deletion may represent a mechanism that, along with hypermutation, contributes to the affinity maturation of antibodies. We used repertoire cloning to examine human antibodies directed against 3 carbohydrate antigens and 1 protein antigen for the presence of such modifications. We find that both the insertion and deletion of codons occur frequently in antigen-specific responses following vaccination. Codon insertions and deletions were observed most often in the complementarity determining regions, and less frequently in the framework regions, of VH, Vκ, and Vλ gene segments, and involved motifs known to be preferred targets of somatic hypermutation. Clonal lineage analysis shows that these events occur through out the course of the somatic maturation of individual antibody clones. We also determined that these alterations of paratope structure have varying effects on the relative affinity of the binding site for its cognate antigen.     

A/T-targeted somatic hypermutation: critique of the mainstream model

The "affinity maturation" of the humoral immune response is driven by antigen-activated somatic hypermutation (SHM) of the genes that encode antibody variable regions and the subsequent antigenic selection of mutant clones. The molecular mechanism of SHM is yet to be completely elucidated. SHM affects cytosine-guanine (C/G) and adenine-thymine (A/T) pairs with approximately equal frequency in vivo. The proposition that error-prone DNA-dependent DNA synthesis explains A/T-targeted hypermutagenesis seems to have mainstream support within the hypermutation research community at present. A major feature of SHM in vivo is that C/G hypermutation is strand unbiased, whereas A/T hypermutation is strand biased. We show that the "DNA-based polymerase error" model of A/T-targeted hypermutagenesis does not explain this important aspect of SHM.     

Antigen-driven selection in germinal centers as reflected by the shape characteristics of immunoglobulin gene lineage trees: a large-scale simulation study

During the immune response, the generation of memory B lymphocytes in germinal centers involves affinity maturation of the cells’ antigen receptors, based on somatic hypermutation of receptor genes and antigen-driven selection of the resulting mutants. Affinity maturation is vital for immune protection, and is the basis of humoral immune learning and memory. Lineage trees of somatically hypermutated immunoglobulin genes often serve to qualitatively illustrate claims concerning the dynamics of affinity maturation in germinal centers. Here, we derive the quantitative relationships between parameters characterizing affinity maturation dynamics (proliferation, differentiation and mutation rates, initial affinity of the Ig to the antigen, and selection thresholds) and the mathematical properties of lineage trees, using a computer simulation which combines mathematical models for all mature B cell populations, stochastic models of hypermutation and selection, lineage tree generation and measurement of graphical tree characteristics. We identified seven key lineage tree properties, and found correlations of these with initial clone affinity and with the selection threshold. These two parameters were found to be the main factors affecting lineage tree shapes in both primary and secondary response trees. The results also confirm that recycling from centrocytes back to centroblasts is highly likely.     

Glossina morsitans morsitansandGlossina palpalis palpalis:Dosage Compensation Raises Questions about the Milligan Model for Control of Trypanosome Development

Gooding, R. H., and McIntyre, G. S. 1998.Glossina morsitans morsitansandGlossina palpalis palpalis: Dosage compensation raises questions about the Milligan model for control of trypanosome development.Experimental Parasitology90, 244–249. Evidence that dosage compensation occurs in tsetse flies was obtained by comparing the activities of X chromosome-linked enzymes, arginine phosphokinase and glucose-6-phosphate dehydrogenase inGlossina m. morsitansand hexokinase and phosphoglucomutase inGlossina p. palpalis, with the activity of an autosome-linked enzyme, malate dehydrogenase, in each species. The shortcomings of the X chromosome model for the control ofTrypanozoonmaturation in tsetse are discussed in light of these findings and previously published reports on the lack of fitness effects of matureTrypanozooninfections in tsetse and on published results on antitrypanosomal factors in male and female tsetse flies.     

Gene conversion and hypermutation during diversification of VH sequences in developing splenic germinal centers of immunized rabbits

The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine gamma-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.     

Mutation pattern of immunoglobulin transgenes is compatible with a model of somatic hypermutation in which targeting of the mutator is linked to the direction of DNA replication.

We have previously demonstrated that B lymphocyte specific somatic mutations are introduced into the variable regions of immunoglobulin kappa transgenes in two independent transgenic mouse lines. The frequency, distribution and nature of these mutations strongly suggest that they arose as a result of the process of somatic hypermutation, which is responsible, in part, for affinity maturation during an immune response. Unexpectedly, in these multiple copy transgenic lines, many of the transgene copies showed no evidence of somatic mutation. This paradox was addressed by determining the sequence of each transgene copy in several B cell hybridomas derived from a mouse line carrying three copies of the kappa transgene. It was found that the somatic hypermutation process in different B cells from the same mouse preferentially targets one, but not the same, transgene copy. We present a model, based on the pattern of this targeting, which links somatic hypermutation to the orientation of the Ig gene relative to the direction of DNA replication.     

Maturation of the immune response in germinal centers.

Germinal centers develop in peripheral lymphatic tissue during the primary immune response and may play a crucial role in affinity maturation. We have compared the diversification of the antigen-specific repertoire of B cells, both from within and from outside the germinal centers, during the murine response to 2-phenyloxazolone (phOx). By sequencing V kappa Ox1 L-chains characteristic of phOx-specific antibodies, we show that somatic mutations accumulate in germinal center B cells and that a mutation conferring high affinity binding is found with increasing frequency. An analysis of V/D/J rearrangements suggests that this mutation occurred independently in many B cells, which were then preferentially expanded. We conclude that, although the hypermutation mechanism may be activated before germinal centers develop, affinity maturation by hypermutation and selection takes place in the germinal centers.     

Antibody diversification by somatic mutation: from Burnet onwards

The clonal selection theory proposed by Burnet required a genetic process, for which there was then no precedent, which randomizes the region of the gene(s) responsible for the specification of gamma-globulin molecules. Work over the subsequent half-century substantiated Burnet's speculation, revealing two distinct novel genetic processes. During early development (when Burnet first thought the randomization took place) programmed gene segment rearrangement catalysed by the RAG1/RAG2 recombinase generates a substantial diversity of immunoglobulin molecules (the primary repertoire). Somatic hypermutation (triggered by the activation-induced deaminase (AID) DNA deaminase) then occurs following antigen encounter in man and mouse, yielding a secondary repertoire. This hypermutation allows both limitless diversification as well as maturation of the antibody response by a process of somatic evolution akin to that envisioned by Burnet in later formulations of the clonal selection theory. AID-triggered antigen receptor diversification probably arose earlier in evolution than RAG-mediated repertoire generation. Here I trace our insights into the molecular mechanism antibody somatic mutation from when it was first proposed through to our current understanding of how it is triggered by targeted deamination of deoxycytidine residues in immunoglobulin gene DNA.     

Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity

Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.     

AID is essential for immunoglobulin V gene conversion in a cultured B cell line

Following productive V gene rearrangement, the functional immunoglobulin genes in the B lymphocytes of man and mouse are subjected to two further types of genetic modification. Class-switch recombination, a region-specific but largely nonhomologous recombination process, leads to a change in constant region of the expressed antibody. Somatic hypermutation introduces multiple single nucleotide substitutions in and around the rearranged V gene segments and underpins affinity maturation. However, in chicken and rabbits (but not man or mouse), an additional mechanism, gene conversion, is a major contributor to V gene diversification. It has been demonstrated recently that both switch recombination and hypermutation are ablated in mice and humans lacking AID, a B cell-specific protein of unknown molecular activity. Here we show that disruption of AID in the DT40 chicken B cell lymphoma leads to a failure to perform immunoglobulin V gene conversion. Thus, AID is required for all three immunoglobulin gene modification programs (gene conversion, hypermutation, and switch recombination) and acts in the initiation or execution of these processes rather than in bringing the B cell to an appropriate stage of differentiation.     

The block in immunoglobulin class switch recombination caused by activation-induced cytidine deaminase deficiency occurs prior to the generation of DNA double strand breaks in switch mu region

Affinity maturation of the Ab repertoire in germinal centers leads to the selection of high affinity Abs with selected heavy chain constant regions. Ab maturation involves two modifications of the Ig genes, i.e., somatic hypermutation and class switch recombination. The mechanisms of these two processes are not fully understood. As shown by the somatic hypermutation and class switch recombination-deficient phenotype of activation-induced cytidine deaminase (AID)-deficient patients (hyperIgM type 2 syndrome) and mice, both processes require the AID molecule. Somatic DNA modifications require DNA breaks, which, at least for class switch recombination, lead to dsDNA breaks. By using a ligation-mediated PCR, it was found that class switch recombination-induced dsDNA breaks in S mu switch regions were less frequent in AID-deficient B cells than in AID-proficient B cells, thus indicating that AID acts upstream of DNA break induction.     

Checkpoint kinase 1 negatively regulates somatic hypermutation

Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.     

Precise Dosage of an Endogenous Mutagen in the Immune System

Four decades ago, it was hypothesized that lymphocytes mutate the immunoglobulin loci at a rate much greater than the spontaneous rate. Over time this Ig somatic hypermutation became the prime example of in vivo, site-directed mutagenesis. But recent studies have demonstrated that this is not necessarily the case, and that hypermutation occurs not only at the Ig locus but also at various locations throughout the genome. Here, we propose that hypermutation can be genome-wide with minimal injury, if a sufficient mutation rate is coupled with strong clonal selection.     

THE DENDRITIC CELL: ITS POTENT ROLE IN THE RESPIRATORY IMMUNE RESPONSE

The importance of the dendritic cell for the capture of antigens and initiating an immune response is now well recognized. Whereas much is known about their structure and function, their lineage is still not clear. Studiesin vitrohave demonstrated that the regulated maturation of function that occurs in culture explains many of thein vivoevents relating to antigen capture and presentation. The control over maturation and migration of these cells to the immune system is decisive as to whether an immune response is mounted or not. ‘Danger ’ signals provided by conserved bacterial products or by microenvironmental cytokines are important regulators. Dendritic cells have been clearly involved in the development of respiratory disease and our understanding of their involvement will have an impact on our future therapeutic strategies.