Immunology: B cells break the rules

properties of the newly prepared dendralenes depend on the number of C=C bonds in the molecule: the properties of the even-numbered members of the series are distinctly different from those of its odd-numbered members. A good example is the thermal stability of the compounds. Dendralenes that have an even number of C=C bonds can be kept at room temperature for weeks without any significant decomposition, whereas their odd analogues have much shorter half-lives. A similar dichot-omy occurs for the electronic spectra of these compounds, and in their chemical behaviour.

Perhaps the most likely initial use of dend-ralenes will be in organic synthesis, acting as sources of dienes in ‘cycloaddition’ reactions. The most widely used cycloaddition reaction is the Diels–Alder addition, because this is the best method for preparing rings of six carbon atoms. When dendralenes are used in Diels–Alder additions, the reaction product will contain a new diene fragment, which can in principle undergo another Diels–Alder addi-tion, and so on, until no more diene units can be generated (Fig. 2b). Such ‘diene-transmis-sive Diels–Alder processes’8 allow the rapid generation of molecular complexity from relatively simple starting materials in a one-pot operation.

Payne et al.1 found that the reactivity of dendralenes in Diels–Alder additions again depends on the number of C=C bonds in the molecule: odd-numbered dendralenes react faster than their even-numbered counterparts. Furthermore, only the endmost dienes of odd-numbered dendralenes take part in reactions,

MgCl

ClClCl

Br

a

b

Diels–Alder

reaction

Diels–Alder

reaction

Nickel catalyst

Figure 2 | Preparation and reactions of selected dendralenes. Payne et al.1 have prepared dendralenes by stitching together unsaturated hydrocarbon fragments from other compounds. a, In these examples, the diene fragment (red) of a magnesium-containing compound is coupled in nickel-catalysed reactions to hydrocarbon fragments (various colours) of halogen-containing compounds, to make the first three members of the dendralene family. b, The authors also investigated the reactivities of dendralenes in Diels–Alder additions. In these reactions, a diene fragment (red) reacts to form a six-membered ring. Another diene is formed in the product, which can, in principle, take part in another Diels–Alder reaction. The cycle continues until no more dienes are formed.

whereas diene subunits throughout the even-numbered dendralenes react. The authors rationalized this surprising chemical effect using quantum mechanical calculations, which suggest that the geometries of the bonds in the

dendralenes are at least partly responsible. In the odd-numbered dendralenes, the endmost diene subunits adopt a conformation that has long been known to be optimal for Diels–Alder reactions. These subunits therefore react quickly, and preferentially to the other diene subunits. But all of the diene subunits in the even–numbered dendralenes adopt an unfa-vourable conformation for Diels–Alder addi-tions; their reactions are therefore slower than in the odd-numbered dendralenes, and no particular diene subunit reacts preferentially to the others.

With the dendralenes now available in sufficient amounts for further study, we can expect the discovery of many new reactions. The resulting products should show interesting chemical and structural properties, and would not have been available using conventional methods of synthesis. ■

Henning Hopf is at the Institute of Organic

Chemistry, Technical University Braunschweig,

Hagenring 30, D-38106 Braunschweig, Germany.

e-mail: [email protected]

1. Payne, A. D., Bojase, G., Paddon-Row, M. N. & Sherburn,

M. S. Angew. Chem. Int. Edn 48, 4836–4839 (2009).

2. Hopf, H. Classics in Hydrocarbon Chemistry (Wiley-VCH,

2000).

3. Maas, G. & Hopf, H. Chemistry of Dienes and Polyenes Vol. 1

(Ed. Rappoport, Z.) 927–977 (Wiley, 1997).

4. Hopf, H. Angew. Chem. Int. Edn 23, 948–960 (1984).

5. Krause, N. & Hashmi, A. S. K. (Eds) Modern Allene

Chemistry Vol. 1 & 2 (Wiley-VCH, 2004).

6. Corriu, R. J. P. & Masse, J. P. J. Chem. Soc. Chem. Commun.

144a (1972).

7. Tamao, K., Sumitani, K. & Kumada, M. J. Am. Chem. Soc.

94, 4374–4376 (1972).

8. Tsuge, O., Wada, E. & Kanemasa, S. Chem. Lett. 12, 1525–1528 (1983).

IMMUNOLOGY

B cells break the rules Marilyn Diaz and Janssen Daly

A study of lymphocytes that lack a DNA-repair enzyme challenges long-standing dogma about the spatial separation of processes that rearrange antibody genes, and provides clues about the origins of B-cell cancers.

Long-lived organisms are constantly being attacked by a myriad of pathogens that have evolved mechanisms to evade the host immune system. To counter this onslaught, vertebrate T and B lymphocytes have an extraordinar-ily diverse repertoire of surface receptors that recognizes an array of foreign antigens. The generation of this wide range of surface B-cell receptors (membrane-bound immunoglobu-lin) takes place in developing B lymphocytes in the bone marrow through a process that involves breakage and recombination of vari-able (V), diversity (D) and joining (J) segments of immunoglobulin genes. Mature B cells in peripheral tissues (the spleen and lymph nodes) also rearrange immunoglobulin genes by DNA breakage and repair, but through a different

mechanism — class-switch recombination. In an exciting study in this issue (page 231),

Wang et al.1 find that a special type of V(D)J recombination — receptor editing — can take place in the periphery in mature B cells that are simultaneously undergoing class-switch recombination. In the absence of a DNA-repair enzyme, these cells experienced frequent chromosome translocations at the sites of immuno globulin genes. These find-ings refute the long-standing belief that recep-tor editing and class-switch recombination are restricted to distinct anatomical locations and specific stages of B-cell development, and provide insight into the mechanism of gene translocations.

The immunoglobulin molecule (antibody)

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consists of two heavy-chain proteins that are noncovalently bound to two light chains (either two λ- or two κ-light chains). The genes encod-ing the heavy chain undergo V(D)J recombi-nation, and those encoding the light chains VJ recombination, to form the V(D)J exon, which encodes the region of the immunoglobulin molecule that determines its specificity. This reaction is initiated by the RAG enzyme com-plex, which induces double-stranded DNA breaks in the V, D and J regions (Fig. 1a). After recombination, the breaks are repaired through a pro cess known as non-homologous end-joining (NHEJ). In receptor editing, devel-oping B cells in the bone marrow undergo successive rounds of RAG-mediated V(D)J recombination to exchange the light chains of an autoreactive immunoglobulin molecule so that it is no longer autoreactive2,3.

Class-switch recombination, which changes the effector function of immuno globulins, is a process by which the exons for the constant domain of the heavy chain of IgM are swapped with downstream exons to generate different classes of antibody, such as IgG, IgE or IgA. Class-switch recombination is also initi-ated by DNA breaks, in this case induced by the AID enzyme (Fig. 1b). These breaks can also be repaired by NHEJ or by an alternative end-joining (A-EJ) pathway. Another AID-mediated mechanism of gene rearrangement in peripheral B cells is somatic hypermutation. Here, mutations accumulate in the rearranged immunoglobulin genes, potentially increasing antibody-binding specificity.

That immunoglobulin-gene rearrangements are associated with double-stranded DNA breaks underscores the enormous selective pressures driving the evolution of these proc-esses — when not repaired correctly, DNA breaks can lead to chromosome translocations, which predispose to cancer. Indeed, certain types of human B-cell tumour (lymphomas) frequently contain translocations that merge antigen-receptor genes with a proto-oncogene (a gene with the potential to promote cancer).

There are several biological mechanisms that reduce the tumour-causing potential of DNA breaks in B cells, including restricting these processes to distinct tissues, such as the bone marrow for V(D)J recombination and peripheral tissues for class-switch recombi-nation. However, as Wang et al.1 show, these apparent safeguards underestimate the plastic-ity of B cells.

The authors examined mice in which the NHEJ double-strand-break-repair protein XRCC4 is deleted in mature B cells. They report that a subset of activated peripheral B cells with defective NHEJ simultaneously harbour double-stranded DNA breaks associated with V(D)J recombination and class-switch recom-bination. Surprisingly, when these cells are activated by signals that lead to class-switch recombination in the DNA locus encoding the immunoglobulin heavy chain (Igh), they also re-initiate V(D)J recombination at the

immunoglobulin-λ light-chain locus (Igl). In Wang and colleagues’ study, the splenic

B cells that reactivate V(D)J recombination are not undergoing the conventional V(D)J recom-bination used by developing B cells to generate the initial immunoglobulin repertoire. Instead, the authors argue, these cells are undergoing receptor editing, which was thought to be con-fined to immature B cells in the bone marrow. It has previously been suggested4 that editing can occur in peripheral B cells during the gen-eration of memory B cells in specific regions of the spleen — the germinal centres — through a mechanism termed receptor revision. How-ever, the peripheral editing in the B cells in Wang et al.’s study1 seems to be distinct from the receptor-revision mechanism, because the B cells lack germinal-centre markers and are not activated by signals that normally lead to germinal-centre formation.

Incorrect repair of the breaks initiated by receptor editing and class switching frequently resulted in chromosome translocations1 involv-ing Igh and Igl (Fig. 2, overleaf). Although neither Igh nor Igl are chromosomal regions that promote cancer, translocations involv-ing Igh or Igl with a proto-oncogene, such as c-myc, can result in lymphomas. Indeed, dele-tion of both Xrcc4 and p53 (a gene encoding a tumour-suppressor protein) in mature B cells

in mice leads to lymphomas, known as CXP lymphomas, the cells of which reveal evidence of receptor editing and class switching5. Thus it is likely that the XRCC4-depleted cells in Wang and colleagues’ study are the progeni-tors of mouse CXP lymphoma cells, and that B cells with similar mutations may contribute to some human B-cell lymphomas.

B cells that undergo both class-switch recom-bination and receptor editing harbour AID- and RAG-dependent DNA breaks, and Wang et al.1 capitalize on this characteristic to identify fac-tors that may enhance translocations between immunoglobulin loci. One such factor may be the A-EJ pathway of DNA double-strand-break repair. As the XRCC4-depleted cells cannot repair DNA breaks with NHEJ and are forced to use the A-EJ pathway, the increase in trans-locations may reflect a propensity of the A-EJ mechanism to generate such translocations.

The authors found a strong correlation between translocations and proximity of Igh and Igl in the nucleus of B cells at interphase, a cell-cycle stage during which much of the gene expression occurs. Similarly, co-localization of c-myc and Igh in the nucleus correlated with translocations between these loci, although the rate-limiting factor was the frequency of breaks at c-myc, which is strongly AID-dependent6.

That the B cells studied by Wang et al.1 are

a b

Looped out

DNA

Class-switch recombination

Pro-B cell

Immature

B cell

Mature

B cell

Plasma cell

Heavy-chain-gene rearrangement

Light-chain-gene rearrangement

Receptor editing of light-chain gene

Activation

V(D)J recombination(bone marrow)

Class-switch recombination(peripheral lymphoid tissues)

SHM

V D

J

V(D)J recombination

VDJ

RAG

VDJ

VDJ

VDJ

AID

Immunoglobulin

heavy-chain gene

Immunoglobulin

gene

Memory B cell

B-cell developmentImmunoglobulin

Figure 1 | Immunoglobulin gene rearrangements. a, Recombination of variable (V), diversity (D) and joining (J) segments of immunoglobulin genes generate B-cell receptors during development in the bone marrow. If the immunoglobulin (antibody) on the developing B cell reacts against ‘self ’ antigen, the cell undergoes further light-chain-gene recombination (receptor editing), to generate a non-autoreactive immunoglobulin. b, Class-switch recombination occurs after activation of mature B cells in peripheral lymphoid tissues (the spleen and lymph nodes). In class switching, the μ exons are swapped with downstream exons to generate a different antibody class. Activated B cells also undergo somatic hypermutation (SHM) as they develop into memory B cells. Whereas the RAG proteins initiate V(D)J recombination (a), class-switch recombination and somatic hypermutation are triggered by the AID enzyme (b). All processes are initiated by or involve DNA double-strand breaks. Wang et al.1 find that, contrary to long-held dogma, mature B cells can simultaneously undergo both class switching and receptor editing in the periphery.

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the progenitors of mouse CXP lymphomas suggests that peripheral editing occurs in vivo and may contribute to the development of such

cancers. Paradoxically, in the authors’ study, the signals activating V(D)J recombination in B cells are typically associated with activation of class-switch recombination rather than autore-activity — the trigger for receptor editing in the bone marrow. So, if not the revision of an auto-reactive receptor, what is achieved by replac-ing the light chain in this subset of peripheral B cells? To fully understand the significance of this phenomenon, it will be important to determine the frequency of receptor edit-ing in peripheral B cells and its physiological function. Finally, this novel B-cell population can be exploited to elucidate the mechanisms that promote translocations between antigen-receptor loci and proto-oncogenes. ■

Marilyn Diaz and Janssen Daly are at the

Laboratory of Molecular Genetics, National

Institute of Environmental Health Sciences of the

National Institutes of Health, Research Triangle

Park, North Carolina 27709, USA.

e-mails: [email protected];

[email protected]

1. Wang, J. H. et al. Nature 460, 231–236 (2009).

2. Gay, D., Saunders, T., Camper, S. & Weigert, M. J. Exp. Med.

177, 999–1008 (1993).

3. Tiegs, S. L., Russell, D. M. & Nemazee, D. J. Exp. Med. 177, 1009–1020 (1993).

4. Hertz, M. & Nemazee, D. Curr. Opin. Immunol. 10, 208–213

(1998).

5. Wang, J. H. et al. J. Exp. Med. 205, 3079–3090 (2008).

6. Robbiani, D. F. et al. Cell 135, 1028–1038 (2008).

DNA

breaks

Igh

Igl

AID

dependent

RAG

dependent

A-EJ ?

Chromosome

Figure 2 | Chromosome translocations in B cells. Wang et al.1 show that mature B cells that lack an essential DNA-repair enzyme undergo both AID-induced class-switch recombination at the immunoglobulin heavy chain locus (Igh) and RAG-induced receptor editing at the immunoglobulin-λ light-chain locus (Igl). These breaks can lead to chromosome translocations involving Igh and Igl. Use of the alternative end-joining (A-EJ) pathway may contribute to the formation of such translocations.

MATERIALS SCIENCE

Nanotubes sorted using DNAMark C. Hersam

A vast number of DNA sequences are possible, and so finding the few that bind to a particular non-DNA entity is a daunting task. A systematic search algorithm has found sequences that target specific carbon nanotubes.

For nearly two decades, the carbon nanotube has been the poster child of nanotechnology. Researchers have used its exemplary physical and chemical properties in a diverse range of prototype devices, spanning such technolo-gies as alternative energy, biotechnology and computing. Underlying this success is the exquisite sensitivity of the nanotubes’ proper-ties to their physical size and atomic structure. However, this sensitivity also creates a funda-mental problem: because current syntheses of carbon nanotubes lack atomic-level control, samples produced are mixtures of nanotubes of different sizes and atomic geometries, and thus possess non-uniform properties. This non-uniformity has confounded their use in large-scale commercial applications, which invariably require materials that have consist-ent, reproducible performance.

Many researchers have therefore devised schemes for sorting carbon nanotubes accord-ing to their physical and electronic structures1.

Inspiration has often come from bioseparation methods, leading to the use of electrophore-sis2, ultracentrifugation3 and chromatography4 techniques. DNA has had a recurring support-ing role in these studies because of its ability to disperse carbon nanotubes in biologically compatible aqueous solutions5–7. But despite its ability to bind to specific molecules depending on its base sequence, DNA has not been system-atically explored as a means of isolating different types of carbon nanotube — until now. On page 250 of this issue, Tu et al.8 describe the heroic efforts that resulted in their identifying more than 20 DNA sequences that each selectively bind a specific carbon-nanotube structure. Their careful study uncovers distinct patterns of DNA sequences that will inform future efforts in nanotube separation, and provides fundamental insight into the chemical interactions between arguably the most important biomolecule and one of the most-studied nanomaterials.

To appreciate the magnitude of the authors’

task, consider that custom-made DNA sequences containing 100 nucleotides are read-ily available commercially. Because there are four DNA bases — adenine (A), thymine (T), guanine (G) and cytosine (C) — this amounts to 4100 (1060) sequences that could be screened for their nanotube-binding properties. This number is almost unfathomably large, and so the authors had to devise a systematic method to focus their search before they could attack this problem experimentally.

Initially, Tu et al. limited their search to DNA molecules containing 28 or 30 bases, thus restricting the number of possibilities to 430 (1018). Although this is a huge improvement over 1060, further refinement was clearly neces-sary. The authors therefore used a sequence-pattern-expansion scheme to come up with a manageable set of DNA sequences, starting with simple patterns and then adding complex-ity in a confined, progressive way. The scheme started with molecules that contained only one kind of base, thus yielding four sequences. Complexity was added in the next phase of the scheme — the second-order expansion — when all 16 variants of two-base repeats were added (for example, (AT)15). By following this proce-dure to a third and fourth order of complexity, Tu et al. constructed a search set containing approximately 350 different DNA sequences.

The authors used each of these sequences to disperse a randomly produced mixture of carbon nanotubes in water. They then used chromatography to separate the resulting 350 solutions into fractions based on the ionic charge of the solutes, and characterized each fraction spectroscopically to see if any of the DNA sequences had formed complexes specifi-cally with a single kind of nanotube. Although most of the sequences had not, a series of DNA molecules that contained alternating patterns of one or more purines (A or G) and pyrimidines (T or C) — such as (GT)15, (TCG)10 and (ATTT)7 — showed a differential affinity for nanotubes as a function of nanotube structure.

Recognizing the successful purine–pyrimi-dine motifs, Tu et al. performed more experi-ments in which they varied the length of their DNA sequences, and found that shorter DNA molecules (as short as eight bases) bind to nano-tubes with exceptional selectivity. In all, more than 20 distinct DNA sequences selected one kind of carbon nanotube from an as-prepared mixture. The purity of semiconducting nano-tubes isolated in this way approached 99%, equalling or exceeding those obtained by all pre-vious carbon-nanotube sorting techniques1.

Although the molecular-recognition mech-anism involved in this DNA–nanotube bind-ing8 is not fully understood, highly suggestive trends can be identified from the successful DNA sequences. For example, DNA molecules that contain alternating purine–pyrimidine patterns form stable, well-ordered, two-dimen-sional sheets through hydrogen bonding (see Fig. 2a on page 252) — structures that resemble the ubiquitous β-sheet motif in proteins. Fur-

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Immunology: B cells break the rules