D-J joining and D-J proteins in B cells and T cells

D-J joining and D-J proteins in B cells and T cells

ImmunologyToday,voL6, No. 4, 1985 128 ~ ( E ' ~ l ~ ) Newdirectionsinresearch D-J joining and D-J proteins in B cells and T cells The phylogeny a...

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ImmunologyToday,voL6, No. 4, 1985


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D-J joining and D-J proteins in B cells and T cells The phylogeny and ontogeny of lymphocytes of T and B lineages are so parallel that it would have been profoundly disturbing if B cells and T cells achieved specific antigen recognition by using genetic elements and mechanisms which showed no evidence of a common evolutionary ancestry. Fortunately this is not the case. Rather, those T-cell receptor (Tcr) genes so far described show almost as close a familial relationship with immunoglobulin (Ig) genes as that between Ig light (L) and heavy (H) chain genes themselves 1 4. One may guess that the divergence of primordial Ig and Tcr genes fairly closely preceded that of IgL and IgH genes, perhaps roughly coinciding with the origin of the chordate line. The nonamer and heptamer recombination signals have been rigorously preserved in IgL, IgH and the genes encoding the Tcr[1chain (TO) , as has the 12 or 23 nucleotide spacing between nonamer and heptamer and the organization of recombination signals within loci (the 'one turn-two turn rule'5,6). The structural homology oflg and Tfl loci encourages one to anticipate that the dynamics of Tcr genes may be described using similar principles to those developed over the last few years to describe the dynamics of Ig loci 7-1°. Thus we might expect that recombination of Tcr loci will be errorprone, implying some sort of proof-reading mechanism which in turn may act to establish stable phenotypic expression of only one V(D)J combination from only one of an allelic pair oflocP °. The similarity of recombination signals in Tfl and Ig loci is such that it would be surprising if the apparatus which fuses IgV(D) andJ elements in B lineage cells were incapable of recombining their T o homologs. Moreover we know that IgH D a n d J elements are frequently fused in T lineage cells 11, so that at the moment, arguing from parsimony, it must be considered likely, though of course not proven, that a single apparatus is responsible for V(D)J joining at TO, IgH and IgL loci. This attractively simple idea poses no new problems of genetic control and is not inconsistent with the observation of T-cell-specific To rearrangement (or ofpre-B-cell rearrangement of IgH and not IgL loci). It is known that there is only limited, if any, heterogeneity in R N A polymerase II, yet extremely fine regulation can be exerted over patterns of gene transcription by controlling the structure of the template so as to allow or deny productive access of polymerase to promoters; an analogous process of regulation could apply to recombinase. Comparison of rearrangement and transcription may, however, be more than a useful analogy: the capacities ofloci for transcription and rearrangement seem to be closely coupled. Thus, in some Abelson virus transformed pre-B cell lines, IgH genes are both transcribed and rearranged while IgK genes are neither12; J n loci can be both transcribed and rearranged in T cells and B cells whereas To loci are transcribed and rearranged in T cells but are apparently inactive in both processes in B cells 1,~. The ability of a locus to undergo recombination then may depend on two conditions: that the recombinase be present and that the locus be accessibl~ to it (the © 1985, Elsevier Science Publishers B.V., Amsterdam 0167 - 4919/85/$02.00

frequency with which rearrangement will occur may depend on additional determinants'4). Identical factors may control access both to R N A polymerase and to recombinase; such factors could operate by direct steric effects, but might also determine D N A topology, by definition crucial for the recognition of elements containing precisely spaced sequence motifs, as do recombination signals. The process of assembly of T o genes in T cell development seems to mirror that oflgHgenes in B cells so that in //loci, an early phase of D - J fusion may precede V - D fusion 15, as seems to be the case for IgHloci in developing B cells. The structural homology between T o and IgHloci extends to some fine details whose significance is not yet clear. Most mouse IgH D segments and both of the characterized mouse To D segments, D~1_1 and Dtn_l, have open reading frames in their immediate 5' flanking sequences, as does the human homolog of D¢1_116'17.As there are three termination codons, one expects to encounter one of them roughly once every 20 codons in any reading frame in a random sequence. Long sequences without a terminator, or open reading frames (ORFs), are therefore deemed highly significant as putative protein coding sequences. mRNA-like transcripts of/a loci lacking V genes have been extensively described 1a-21 and it is now clear that some of those which originate from D - J recombinant loci encode truncated ' D - J ' proteins which resemble normal /~chains but lack V sequences 22. The multiplicity of/~ gene transcripts from loci lacking V genes complicates assessment of their protein coding potential. UnrearrangedJH-C~ loci are transcriptionally active, and in unrearranged or D-J recombinant loci, there are multiple transcriptional initiation sites 21; most transcripts lacking V sequences are not translatable 23. Schwaber et al. 24 provided initial evidence of/a proteins without V segments, observing that while bone marrow from patients with X-linked agammaglobutinaemia (XLA) entirely lacks cells with surface/a chain, around 20% of fixed cells can be stained with anti-C/a, but not with antiserum directed against V n framework determinants. About 5 % of ~ positive cells from normal bone marrow show the same phenotype. Hybridomas with this phenotype could be derived from X L A bone marrow and normal foetal liver, and analysis revealed the synthesis of truncated/a chains programmed by m R N A lacking V sequences, which might derive from D - J recombinant loci. More recently Reth and Alt 22 have analysed many Abelson virus transformed mouse pre-B cell lines and have found several that can produce a/~ protein lacking V sequences. The ability to produce such a protein depends on the existence ofrecombinant~IgHloci containing an inframe fusion of a Jn segment with an O R F of a D segment. The predicted D - J proteins contain N-terminal sequences which somewhat resemble known signal peptides, though the similarity is rather weak for some D segments (see Ref. 25 for a recent compilation of signal

Immunology Today, vol. 6, No. 4, 1985

peptide sequences). Signal peptides vary so m u c h in sequence, however, that, lacking identity with a k n o w n signal sequence, m e m b r a n e insertion o f a polypeptide has to be demonstrated by direct experiment and cannot be confidently inferred from sequence alone. A t least one D segment, DQ~2, the one closest to the JH cluster, lacks a flanking O R F 27. m R N A - l i k e transcripts arising from T o loci containing Jt~ segments fused with D O elements, lacking VO, are c o m m o n in T cells and in T-cell lines~6'~7; indeed two of the first four mouse T o e D N A clones to be described 2 turned out to be copied from such transcripts. T h e transcriptional activity of u n r e a r r a n g e d T o loci has yet to be closely examined. T h e 5' flanking sequence of D0I_ 1 has one O R F ~6including a methionine codon 46 codons from the D segment, followed by what, it has been suggested, might be a signal sequence, and including the D segment itself, in the frame in which it appears in 86 T1, a c D N A clone derived from thymocyte m R N A z. Dr32_ 1 has an O R F at least 56 codons long, which includes the D segm e n t as it appears in the m R N A sequence expressed in the helper T cell line C5, and, the authors report, an A U G codon followed by a putative signal sequence TM. T r a n scripts f r o m 773 loci containing in-frame fusions of D01_ 1 o r Dt~2_ 1 w i t h J might therefore be translated, though no D - J protein has yet been demonstrated. Clark et al. ~7h a v e pointed out extensive homologies in sequences flanking mouse Dt~I _ ~ and D/32_ i, and h u m a n D0I _ 1. H o w e v e r , as frameshifts must be introduced to maintain homology, these similar D N A sequences would encode quite dissimilar proteins. T h e evolutionary conservation of these sequences m a y therefore reflect a D N A or R N A role, rather than a protein function. Do D - J proteins have any function? An important role would be established if it could be demonstrated that the formation of a D - J recombinant locus with an O R F spanning the D and J elements was an obligate step in functional lymphocyte development. T h e data from X L A patients suggests that D - J joining and V - D j o i n i n g m a y occur in separable phases orB-cell development, and that pre-B cells in these patients are prevented from m o v i n g from the D - J joining phase into that of V - D j o i n i n g 24. T cell precursors also are capable of DH-JH j o i n i n g but a p p a r e n d y not of V H - D H joining. M i g h t synthesis of a DH-JH protein be a necessary prerequisite of a pre-B cell m o v i n g from one phase to the next? X L A might then be a lesion in the m e c h a n i s m of sensing the presence of D - J proteins. R e t h and Alt 22 show that D - J recombinant loci which produce D - J proteins can undergo V - D j o i n i n g which m a y or m a y not result in the expression of complete /~ chains, as can allelic D - J loci which do not produce a D - J protein. Yaoita et aL 28 describe an Abelson line, P, which contains allelic D - J recombinant i g H loci, neither of which could be expressed as a D - J protein; a subclone, S, has u n d e r g o n e V - D joining on one of the allelic loci in the absence of any further r e a r r a n g e m e n t of the other. It would appear that in Abelson virus transformed cells, the ability to produce a D - J protein is not an obligate prerequisite of the formation of complete V - D - J loci. A scan of published sequences of expressed V - D - J I g H loci in lines derived from m a t u r e B cells - hybridomas and plasmacytomas - reveals that, almost all of the time, D

129 segments are used in only one reading frame (e.g. Refs 29-31; see Ref. 32 for a possible exception), which seems not to be a frame capable of supporting D - J protein synthesis 22. T h e positional flexibility of formation of D - J and V - D joints denies any mechanistic constraint on the reading frame of D, so that the p r e d o m i n a n c e of one out of two or three translatable D reading frames in mature B cells, remarkable in an area where one anticipates maximized diversity, presumably results from a potent, but obscure and probably antigen-independent, selection pressure. In any event it appears as though most functioning I g H loci in B cells derive from precursor D - J loci which were not expressed as D - J proteins, although precursor cells could have expressed D - J proteins from their second alleles. O n balance, an obligate role for D - J proteins in B cell development seems unlikely. At the m o m e n t we m a y well consider the transcriptional activity of incompletely rearranged I g H and T o loci significant, as a possible concomitant of recombinational competence. Conservation of sequence elements flanking D segments m a y indicate a function and the prevalence of O R F s suggests an importance to translation products of these sequences, though this has yet to be established. T h a t such proteins exert their function, if any, by display on the cell surface should be regarded as tentative. [:[-1 CHRISTOPHER COLECLOUGH Roche Institute ofMolecular Biology, Roche Research Center, Nutley, NJ O7H O, USA.

References 1 Yanagi, Y., Yoshikai, Y., Legatt, K., Clark, S. P., Aleksander, I. and Mak, T. W. (1984)Nature(London) 308, 145-149 2 Hedrick, S. M., Nielsen, E. A., Kavaler,J., Cohen, D. I. and Davis, M. M. (1984) Nature (London) 308, 153-158 3 Chien, Y., Becker, D. M., Lindsten, T., Okamura, M., Cohen, D. I. and Davis, M. M. (1984) Nature (London) 312, 31 35 4 Salto, H., Kranz, D. M., Takagaki, Y., Hayday, A. C., Eisen, H. N~ and Tonegawa, S. (1984) Nature (London) 312, 36-40 5 Early, P., Huang, H., Davis, M., Calame, K. and Hood, L. (1980) Cell 19, 981-992 6 Sakano, H., Maki, R., Kurosawa, Y., Roeder, W. and Tonegawa, S. (1980) Nature (London) 286, 676-683 7 Alt, F. W., Enea, V., Bothwell, A. and Baltimore, D~ (1980) Cell 2t. 1-12 8 Hieter, P. A., Korsmeyer, S.J., Waldmann, T. A. and Leder, P. (19Sl) Nature (London) 290, 368-372 9 Coleclough,C., Perry, R. P., Karjalainen, K. and Weigert, M. (198!) Nature (London) 290, 372-378 10 Coleclough,C. (1983) Nature (London) 303, 23 26 11 Kurosawa, Y., yon Boehmer, H., Haas, W., Sakano, H., Trauneker, A. and Tonegawa, S. (1981) Nature (London) 290, 565-570 12 Alt, F. W., Rosenberg, N., Enea, V., Siden, E. and Bahimore, I) (1981) c~a 27,381-390 i3 Saito, I~I.,Kranz, D. M., Takagaki, Y., Hayday, A., Eisen, H. N. and Tonegawa, S. (1984)Nature(London) 309, 757-762 14 Wood, D. L. and Coleclough,C. (1984) Proc. Natl Acad Sci. USA 81, 4756-4760 15 Born, W., Yagiie,J., Palmer, E., Kappler, J. and Marrack, P. (1985) Proe. Natl Acad. Sci USA (in press) 16 Siu, G., Kronenberg, M., Strauss, E., Haars, R., Mak, T. W. and Hood, L. (1984)Nature (London)311, 344 350 17 Clark, S. P., Yoshikai, Y., Taylor, S., Siu, G, Hood, L. and Mak, T. W. (1984)Nature (London)311,387-389 18 Kemp, D. J., Harris, A. W., Cory, S. and Adams,J. (1980) Proc. Natl Acad. Sci. USA 77, 2876-2880 19 Kemp, D. J., Harris, A. W., Cory, S. and Adams,J. (1980) Proc. Natl Acad. Sci. USA 77, 7400-7404 20 "Att, F. W., Rosenberg, N. E., Enea, V., Siden, E. and Baltimore, D. (1982) Mol. Cell. Biol. 2, 386-400 21 Nelson,K.J., Haimovieh,J. and Perry, R. P. (1983)Mol. Cell. Biol. 3, 1317-1332 22 Reth, M. G. and Att, F. W. (1984) Nature (London) 312,418-423

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130 23 Walker, I. D. and Harris, A. W. (1980) Nature (London) 288, 290-293 24 Schwaber, J., Molgaard, H., Orkin, S. H., Gould, H.J. and Rosen, F. S. (1983) Nature (London) 304, 355-358 25 Watson, M. E. E. (1984)NucleicAcids Res. 12, 5145-5164 26 Patten, P., Yokata, T., Rothbard, J., Chien, Y., Arai, K. and Davis, M. M. (1984) Nature (London) 312, 40-46 27 Sakano, H., Kurosawa, Y., Weigert, M. and Tonegawa, S. (1981) Nature (London) 290, 562 565

28 Yaoita, Y., Matsunami, N., Choi, C. Y., Sugiyama, H., Kishimoto, T. and Honjo, T., (1983) NudeicAcids Res. 11, 7303-7316 29 Bothwell, A. L. M., Paskind, M., Reth, M., Imanishi-~ari, T., Rajewsky, K. and Baltimore, D. (1981) Cell 24, 625-634 30 Kurosawa, Y. and Tonegawa, S. (1982)J. Exp. Med. 155, 201-218 31 Manser, T., Huang, S-Y. and Gefter, M. (1984) Science 226, 1283-1288 32 Near, R. I. K., Juszczak, E. C., Huang, S-Y., Sicari, S. A., Margolies, M. N. and Gefter, M. L. (1984) Proc. NatlAcad. Sci. USA 81, 2t67-2171

Leukocyte activation and the asthmatic response H u m a n asthmatic responses have traditionally been considered the result of the immediate effects of one or more mediators on the bronchial wall, leading to smooth muscle spasm, hypersecretion of mucus, and resultant airway narrowing. However, in recent years it has been recognized that the pathogenesis of asthma is more complex. In patients a prominent accumulation of inflammatory cells in the bronchial wall is common and after environmental exposure or inhalation challenge with antigens to which a subject shows IgE sensitivity there is often not only an immediate bronchospastic response but a similar reaction occurring 6-12 h later. It is also now clear that pharmacotherapeutic agents which are potent inhibitors of the immediate response often exert little if any effect on the delayed reaction. The mechanism underlying the inflammatory cell responses in asthma is not well defined. However, a potential mechanism was suggested by our finding I that bronchospasm induced during antigen inhalation challenge is accompanied by an increased serum level of a high molecular weight neutrophil chemotactic activity ( H M W - N C A ) . This increase in serum H M W - N C A correlated with the degree ofbronchospasm induced and persisted over a period of hours only in those individuals exhibiting late-onset as well as immediate bronchoconstriction 1-3. There was also a correlation between the increase in H M W - N C A and an increased circulating neutrophil leveP. Recently, K a y ' s group at the Brompton Hospital in London reported 5 that increased C3b receptor activity on both neutrophils and monocytes was temporally associated with the increase in serum H M W N C A and bronchoconstriction after antigen inhalation challenge of sensitive subjects. The increase in C3b receptors was found during both the immediate and late phase reactions in subjects who showed dual responses. Neither increased serum H M W - N C A nor increased leukocyte C3b receptors occurred with bronchospasm induced in a nonimmune reaction by methacholine 1'4'5. Two questions are raised by these interesting findings. W h a t is the mechanism underlying the increase in C3b receptors? Although a definite answer is not yet to hand, the most likely explanation is that increased C3b receptors reflect a perturbation of the membrane of the cells following interaction with H M W - N C A . Similar enhancement of C3b receptors on neutrophils follows in-vitro interaction of these cells with other chemoattractants such as F-MetLeu-Phe or leukotriene B4 6. A similar effect of H M W N C A on neutrophils would not be surprising since H M W - N C A is chemotactic for these cells. H M W - N C A has not been shown to be chemotactic for monocytes 1.zbut primary activation of neutrophils could result in secondary elaboration of a chemoattractant'for monocytes 7. This interaction could possibly cause a membrane © 1985;ElsevierSciencePublishersB.V., Amsterdam 0167- 4919/85/$02.00

perturbation leading to increased expression of C3b receptors. It should be noted that there is no decrease in serum complement levels at the time the C3b receptor activity on these cells would be increased 1. The second question concerns the pathophysiologic implications of these findings. These should be viewed in the light of several recent studies. After in-vitro incubation with a variety of chemoattractants, neutrophils may exhibit decreased mobility, increased release oflysosornal enzymes and other granular contents. There is increased production of several proinflammatory mediators such as superoxide radicals, prostaglandins and leukotrienes 8. Under similar conditions, activated monocytes can release interleukin 1, elastase myeloperoxidase and lysozyme 9. Histologic studies have demonstrated the prominent and prolonged accumulation of granulocytes and mononuclear cells in allergic reactions 10.11. Furthermore, there is in-vitro and in-vivo evidence in animal studies that activation of these cell types leads to tissue damage and increased bronchial liability 12. Thus, the concept of the pathophysiology of asthma must be broadened to include not only ,the smooth muscle spasm and increased mucus production mentioned earlier, but also the recruitment and activation of inflammatory cells. Do these cellular inflammatory responses serve only a proinflammatory role? Do they explain in part the beneficial effects of corticosteroids? These and other questions will need to be answered but the important first step has been taken - the demonstration that the induction of asthma is accompanied by in-vivo activation of circulating leukocytes. ~[] References 1 Atkins, P. C., Norman, M., Weiner, H. and Zweiman, B. (1977) Ann. Intern. Med. 86, 415-418 2 Atkins, P. C., Norman, M. E. and Zweiman, B. (1978)J. AUergy Clin. Immunol. 62, 149-155 3 Nagy, L., Lee, T. H. and Kay, A. B. (1982) N. Engl. J. Med. 306, 497-501 4 Atkins, P. C., Norman, M., Zweiman, B. and Rosenblum, F. (1979)O( Allergy'Clin. Immunol. 64, 251-528 5 Durham, S. R., Carrol, M., Walsh, G. M. and Kay, A. B. (1984) N~ Engl. J. Med. 311, 1398-1402 6 Fearon, D. T. and Collins, L. A. (1983) Clin. Exp. Immunol. 38,294-299 7 Ward, P. A. (1968),]2 Exp. Med. 128, 1201 8 Gallin, J. I. (1984) Clin. Res. 32, 320-328 9 Cohen, A. B., Chenoweth, D. E. and Hugli, T. E. (1982) Am. Rev. Respir. Dis. 126, 241 10 Atkins, P. C., Green, G. R. and Zweiman, B. (1973)J. Allergy Clin. Immunol. 51, 763 11 Soney, G. O., Gleich, G. S., Jordan, R. E. and Schroeter, A. L. (1976) J. Clin. Invest. 58, 408 12 Fabberi, L. M., Aizawa, H., Alpert, S. E., Walters, E. H., Byrne, P. M., Gold, B. D., Nadel, J. A. and Haltzman, M. J. (1984) Am. Rev. Respir. Dis. 129, 288 PAUL. C. ATKINS BURTON ZWEIMAN

Department of Medicine, Allergy and Immunology Section, University of Pennsylvania, Philadelphia, PA 19104, USA