Immunology Today M a y 1984


I m m u n e dysfunction is one of many defects from Richard Gatti Ataxia-telangiectasia (AT), a progressive and uniformly fatal inherited neuroimmunological disorder, affects approximately I in 40 000 children. Patients are born to normal parents and appear to develop normally for about two years. They t h e n b e g i n to stagger (ataxia) a n d show signs of degenerating cerebellar function; by five years of age, they have dilated blood vessels (telangiectases) over the exposed bulbar conjunctiva and skin of the ear. By 10 years of age, they are usually confined to a wheel-chair. Most AT patients have IgA and IgG2 deficiency as well as various T cell-associated i m m u n e dysfunctions. Because one of every five patients develops cancer, usually lymphoid, d u r i n g their shortened life-span, it is believed that u n r a v e l l i n g the pathogenesis of A T will also shed light upon the genetics of cancer susceptibility and upon the relationship of immunodeficiency to oncogenesis. A recent conference* focused on four areas of this complex disorder: DNA repair/replication, genetics, neuropathology and immunopathology. An early clinical observation that A T patients are unusually sensitive to X-rays led to the demonstration that fibroblast strains from these patients are about three times more sensitive to killing by ionizing radiation than are the corresponding cells from normal individuals. This unique sensitivity to X-rays and )'-rays, but not to short wavelength uv light, is now a hallmark of the disease. Thus far, there has been no unequivocal demonstration of a defect in any particular step in a DNA repair pathway. DNA strand breaks produced by X-rays are rejoined normally in A T cells and the cells are not sensitive to some other agents, such as methylmethane sulfonate, that produce strand breaks. R. Painter (Univ. of California, San Francisco) has found that chain elongation of DNA during synthesis in A T cells is unaffected by as much as 5 000 rads of X-rays. The deficiency could be in a regulatory process which normally delays synthesis on the damaged D N A templates until they have been repaired.

*A Kroc Foundation conference held on 16-20 January 1984 at Solvang, California. Ray A. Kroc, the founder of the foundation and of McDonald's food chain, died on 14January 1984.

Y. Shiloh (Children's Hospital, Boston) described a sensitivity of A T cells to bleomycin and other agents, such as neocarzinostatin and hydrogen peroxide, which have in common with

X-rays the property of generating free radicals that attack the deoxyribose sugar moiety. Shiloh suggested that AT cells may be deficient in repair of only specific types of D N A lesions, such as those that result in a gap with a 3' phosphate end which cannot be ligated unless the phosphate group is first removed. C. Arlett (Univ. of Sussex, Brighton, UK) reported that untransformed AT fibroblasts are hypomutable for the production of 6-thioguanine resistance to ),-rays so that the putative translesion DNA synthesis is evidently not error prone; it is likely that the lesion simply results in lethality. Curiously, Arlett also finds that when an A T fibroblast strain is transformed with SV40 virus, it becomes mutable and less sensitive to y-rays. M. Paterson (Chalk River Nuclear Labs, Ontario, Canada) suggested, by analogy with properties of recA mutants in bacteria and rad52 mutants in yeast, that the primary defect in A T could be an enzyme involved in genetic recombination. Continued on p. 122

The biochemistry and genetics of complement

Agreement n o w the norm from Michael Hobart Many immunologists hold that complement is baffling or irrelevant or, most conveniently, both but a recent meeting* emphasized that complement is interesting and that it may be important, even if only as an elegant model system. Moreover agreement is now the norm. The biochemistry has now advanced to the point where the outlines of the reaction mechanisms are agreed and work is under way with detailed analysis and model building. The latter approach (M. Colomb, Grenoble; J. Fothergill, Aberdeen; and J. Gagnon, Oxford) provides stimulating questions for the sequencers (themselves included), though unfortunately not yet for crystallographers. It is now agreed that the early components of the complement pathways are serine proteases with peculiar properties of structure and * Held at the Royal Society, London, on 25 and 26 January 1984.

specificity. They fall into fanfilies: C l r with Cls, perhaps distantly related to factor D, and C2 with factor B. Their substrates are other complement components and the control of their actions is subtle. We still need a fully satisfying account of how C 1r is able to effectively cleave C 1s into its fully active form only when C l q interacts with a suitable molecule (normally fixed antibodies). C 1s can cleave the dissimilar molecules C4 and C2 but not their close relatives C3 and ~~i~.~

Continued on p. 122

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122 Ataxia-telangiectasia: continuedfrom p. 121 in the laboratories of Painter and Attempted replication Over a lesion that Jaspers. As W. Salser (UCLA) pointed constituted a strand break should result out, the lingering hypothesis whereby a topologically in a chromosomal break large deletion of adjacent genes might and this could account for the chromo- ~expl~'p the pleomorphism of AT pathsomal fragility that is characteristic of 01ogy co~a]d be dismissed on the basis of AT cells. Although the diminished inthis evidence. Although it has not yet hibition of DNA synthesis in irradiated been possible to correct the in-vitro A T AT cells suggests that the primary defect defect of radiation resistance by transinvolves an abnormality in DNA replicafecting the fibroblasts with DNA from tion, N. G. J. Jaspers (Erasmus Uniindividual normal, chromosomes, this versity, Rotterdam) pointed out that AT seemed to some a promising approach to cells also respond untowardly to X-rays the localization of the AT gene(s) in the in the absence of DNA synthesis in that human genome. D. Botstein (MIT, increased frequencies of chromosomal Cambridge) pointed out the potential aberrations are observed after radiation difficulties of genetic mapping by exposure to AT cells in the G2,phase. restriction fragment length polymorphIt was generally agreed that published isms (RFLPs) in this disorder, considerdata of in-vitro post-radiation survival ing its autosomal recessive nature, the curves showed significant: differences lack, thus far, of a reliable method for between controls and obligatory AT heterozygote identification and the probheterozygotes as groups; however, no ability that the AT phenotype may single assay appeared to discriminate reflect as many as a half-dozen different individual heterozygotes from nongenetic addresses. carriers sufficiently well to be used in Within the confines of the above genetic counselling. Paterson presented reservations, linkage data were predata showing good separation of 10 sented which suggest that the AT gene is heterozygotes from controls when he not closely linked to the Gm loci in the exposed fibroblasts from these patients to IgH complex on chromosome 14q32, an chronic, low-dose irradiation. Perhaps area where break points for tandem this assay, or combination of assays, will chromosomal translocations have been prove sufficiently reliable to distinguish d e s c r i b e d in A T patients. AlphaAT heterozygotes within families, if not fetoprotein (AFP) is raised in all but a few within populations. The time has come AT patients; those who have normal for 'blind' samples to be submitted to levels are suspected of belonging to a those laboratories confident that their separate subset of the disorder. Informaassay separates A T heterozygotes from tion regarding the cloning and mapping controls. of the AFP gene was presented by A. Dugaiczyk (Univ. of California, RiverGenetics side). M. Swift (Univ. of N. Carolina, The neuropathology session was Chapel Hill) presented a preliminary unique in being the first of its kind which analysis of an on-going epidemiological specifically addressed AT before a multistudy of American families with AT. disciplinary audience with expertise in Despite a very low incidence of conmolecular biology, genetics, immunsanguinity in these American families, ology and medicine. H. Vinters (Univ. the autosomal recessive pattern of of Western Ontario, London) presented inheritance was supported by an work performed at U C L A which showed incidence of affected members approxithat because basket cells represent a footmating 30%. Cancer incidence among print of Purkinje cells and were present 211 patients was 19%, although more in normal numbers in the cerebellum of than half these patients are still alive and AT patients, normal numbers of Purcancer incidence among those dead was kinje cells must have existed previously 36%. The data also suggest an increased in these patients and deteriorated cancer incidence among A T heteroprogressively with time. This picture zygotes and a carrier frequency as high as correlates well with the progressive 1:100; this would make AT the most nature of the ataxia. P. Rakic (Yale common cancer susceptibility gene in the Univ.) noted that Vinters' ectopic general population. Purkinje cells in the molecular layer of Before strategies for mapping and the cerebellum would have had to arrive eventual cloning of the AT gene could there by the thirteenth week of gestation, considered, the issue of complementasuggesting that the genetic disorder tion groups was once again raised. expresses itself much earlier than had Recent fusion experiments between 10 been previously appreciated. Since A T fibroblast strains identify at least four Purkinje cells do not divide after that complementation groups. These results time, the cause of their disappearance in derived from independent experiments Continuedon p. 123

Immunology Today, vol. 5, No. 5, 1984

Complement:continuedfiom p. 121 factor B. Both C2 and factor B require to be complexed with their partners, C4b and C3b before they can be efficiently cleaved by the pre-existing enzymes C 1~ and factor D. Now that several of the complement proteins have been cloned, as was reported at the meeting, we may expect some of the three-dimensional questions to be answered indirectly by site specific mutagenesis experiments. There is now good agreement on the outline of the mechanism by which the triggered enzymes of the complement system lead to cell lysis. Both S. Bakdi (Giessen) and H. M/.iller-Eberhard (La Jolla) produced beautiful electron micrographs of the membrane attack complex (p. 124). This is composed of one molecule each of C5, C6, C7 and C8 and a variable number (8-18) of C9 molecules. While the C5-8 molecules attach to the cell surface, C9 inserts itself into the membrane bilayer and polymerizes into a ring structure, leaving a central channel through which ions and other small molecules can pass. Poly-C9 can be produced by in-vitro manipulation of the purified component, and this will insert into pure lipid liposomes, though not into cells, probably because of electrostatic repulsion. It requires but a few of these unscalable punctures to lyse a nonnucleated cell. The formation of the membrane attack complex is a remarkable process: water-soluble proteins develop an affinity for surface structures and one of their number undergoes a polymerization process in the course of which it develops a strongly hydrophobic property. The proper study of complement genes has begun with the cloning of C3 (G. Fey, La Jolla), C4 (M. Carrol, Oxford), C2 and factor B (R. Campbell and D. Bently, Oxford) and, most recently, Clq (K. Reid, Oxford). The latter work is at a preliminary stage, with interesting results anticipated from the study of families in which normal C l q appears to be replaced by an abnormal protein. Though sequence analysis reveals that there is an abnormal structure with a kink in the collagenous part of the molecule, it remains possible that this abnormal molecule is present in normal subjects, albeit at a much lower level of expression than conventional C 1q. In contrast, investigation of the C3 gene is at an advanced stage in mouse and man. C3 is coded by a single gene in both species and the sequences show strong homologies. Significant homologies are also found with another protein containing a thioester bond, a2-macroglobulin. The scene is set here Continuedon p. 124


Immunology Today, ool. 5, No. 5, 1984 Continuedfrom p. 122 later life is difficult to reconcile with the suggestion that A T derives from a defect in a DNA replicating enzyme. R. Herndon (Univ. of Rochester) spoke on Purkinje cell vulnerability to various insults, such as hypoxia, and alcoholism, and noted that Purkinje cell loss is not specific to the A T defect and could represent simply another feature of premature aging in these patients in whom gray hairs and basal cell carcinomas are seen at an early age. Immunological studies E. Gelfand (Hospital for Sick Children, Toronto) reviewed the clinical and immunologic spectrum in A T patients. More than a dozen immunological parameters have been reported to be perturbed, but no one test is uniformly abnormal. These may change from time to time in the same patient; however, there is no firm evidence that the various cellular and humoral disturbances grow worse with the unrelenting progress of the neurological picture. IgA and IgG2 deficiencies are found in most A T patients from all countries and ethnic backgrounds that have been studied. I. Berkel (Hacettepe University, Turkey) tested titers of over 60 patients to Epstein-Barr virus antigens (VCA, EA and EBNA) but could find no clear correlation with the development of malignancies. R. Gatti (UCLA) described lympho-

cyte subpopulation studies which showed decreased pan-T (Leu4 and OKT3) percentages and absolute numbers, slightly reduced T suppressors (Leu2 ÷ and O K T 8 +), normal T helpers (Leu3 ÷ and O K T 4 +) and slightly elevated L e u l l + cells) these include a natural killer cell subset). Most patients studied by D. Nelson (NCI, Bethesda) had a deficient ability to generate virusspecific cytotoxic T cells and failed to produce specific antiviral antibodies in vitro. This latter defect involves both T helper and B cells. In general, those T cell responses requiring recognition of major histocompatibility complex (MHC) gene products appeared the most deficient. D. Cohen (Stanford) presented evidence that mouse T-cell receptor genes had been cloned and, in preliminary experiments, mapped to chromosome 5. The xid gene has also been cloned and appears to be differentially regulated during late B-cell maturation. It was suggested that B-cell maturation in A T patients could be evaluated by employing such probes. The immunology session also considered the significance of the various cellular and humoral findings in AT patients. A T patients with susceptibility to sinopulmonary infection and pulmonary insufficiency do not seem especially susceptible to opportunistic infections, such as Pneumocystis carinii, Mycobacterium avium or cytomegalovirus, as would be expected of patients with significant

deficiencies of the immune system. Why is this, and why, as Gelfand asked, have so many attempts been made to treat these patients with immunostimulants? N. Stebbing (Amgen, Newbury Park, CA) reviewed the applications of modulators of the immune response, such as the interferons and cyclophosphamide. There was a consensus that culturing of fibroblasts would be advisable for all A T patients to be included in research analyses. It was re-emphasized that, without exception in 30 patients, each A T fibroblast strain tested has shown an abnormal response to )'-radiation. Fibroblast response to radiation and/or radiomimetic agents appears to hold the greatest promise for reliably identifying heterozygotes. Fibroblast strains also allow for complementation group assignment. While an assay which substitutes lymphocytes for fibroblasts would be welcomed in clinical studies, the abnormal response to radiation has not been as reliably demonstrated in lymphocytes or in lymphoblastoid cell lines as in fibroblasts. The proceedings of this conference will be published later this year by Alan R. Liss, inc. in a monograph entitled Ataxia-telangiectasia: Genetics, Neuropathology and Immunology of a Degenerative Disease of Childhood. RichardA. Gatti is in the Department of Pathology, University of California at Los Angeles School of Medicine, Los Angeles, CA 90024, USA.

Immune dysfunction is one of many defects.

Ataxia-telangiectasea (AT), a progressive and uniformly fatal inherited neuroimmunological disorder, affects approximately 1 in 40 000 children. Patie...
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