Ontogeny of B cells and pathogenesis of humoral immunodeficiencies

CLINICAL

LMMUNOLOGY

AND

40. 5-12

IMMUNOPATHOLOGY

(1%~)

Ontogeny of B Cells and Pathogenesis Humoral Immunodeficiencies’ R. LAWTON

ALEXANDER

Studies

of

B-cell

ontogeny

have

played

an

important

standing of the pathogenesis of immunodeficiencies. both T and B cells begins during the first trimester Fetal and neonatal B cells have a limited capacity responses. and B-cell

although interactions

precursors in the

present and functional, This paper will review functions of several normal

associated humoral progression

expressing neonate are

but their the major

with each immunodeficiency of B-ceil

capacity steps

role

in furthering

our

under-

Development of clonal diversity for and is far advanced hy midgestation. to express IgG and IgA antibody

these immunoglobulin classes are present. dominated by suppression. T helper cells to drive in ontogenetic

differentiation diseases

differentiation.

of

IgG

and IgA development

Tare

responses is impaired. of 6 cells and the

stage. PosGble pathogenetic mechanisms are reviewed from the perspective of the c I%+ .Ac:Kl~,“,z Pre,,. l,lC

INTRODUCTION

Ontogenetic studies were crucial in tracing the steps of primary B-cell development in mammals. Birds have a specialized lymphoepithelial organ, the bursa of Fabricius. for which the only known function is the genesis of B cells. Once extirpation studies had demonstrated this function, establishing the sequential order of B-cell differentiation (from cortical to medullary bursal lymphocytes, to surface-immunoglobulin-positive [slg+] B cells in circulation and germinal centers in spleen, and finally to plasma cells). was a relatively straightforward process. An exact homolog of the bursa could not be found in mammals. The embryonic liver and the bone marrow were known to contain stem cells capable of reconstituting the hemopoietic and lymphoid systems of lethally irradiated mice, but whether these sites contained the specific inductive microenvironments for primary B-cell development was not clear. The crucial experiments on this issue were done by Owen et al. in 1974 (1). They demonstrated that cultured fragments of liver from 12- to 13-day-old mouse embryos could generate slg+ B lymphocytes de novo with kinetics similar to those observed in ~il*o. They then made the serendipitous observation that cells with lymphoid morphology containing cytoplasmic p. chains appeared in fetal liver several days before slgM+ B lymphocytes. Cells with this phenotype (slg- ct.~+) were provisionally called pre-B cells (2). Subset Presented Prevention. ton. D.C’.

as part and

of a symposium entitled “Childhood February 25-26. 1985.

Management.”

lmmunodeficiency National Academy

Disorders: of Sciences.

Diagnosis. Washing-

h

ALEXANDER

K. 1 AW-I‘ON

quent studies in man and rabbit established that cells having this phenotype u’ctc the first immunoglohulin-containing cells to appear during ontogeny and that the\ were found nor-mally only in embryonic liver and fetal or adult bone marI.OM ii. 4). Once the role of these cells as precursor\ of B lymphocytes wa\ cstablishcd. this restricted tissue distribution helped to establish fetal liver and marrow ;I\ the sites of primary B-cell development in man. Somewhat earlier it was shown that agammaglobulinemic patients could be subdivided into groups based on the presence of slg + B lymphocytes in thcircirculation (5. 6). Boys with X-linked agammaglobulinemia (XL,A), with rare eyceptions. had very few B lymphocytes. while other agammaglobulinemic patients, particularly those with hyperplastic lymphoid tissues. had normal numbers of circulating slg + cells (7. 8). Subsequent analysis of bone marrow samples from XLA patient5 revealed normal numbers of pre-B cells. establishing this condition as an arrest of B-cell differentiation at the pre-B-cell stage (9). PRE-B CELLS The biologic function of pre-B cells is the generation of clonal diversity. The cytoplasmic expression of p chains signals successful rearrangement of three gene segments, V,, D, and J,. which are joined to make a functional p-chain gene. The details of this process are discussed by Waldmann in this symposium (IO). Suffice to say that the creation of immunoglobulin variable region genes by splicing together several smaller gene segments tremendously amplifies the specificity information which is carried in the germ line. First, the potential number of heavy-chain V regions which can be generated is the product of the numbers of V, (hundreds). D, (210). and J, (6) germ-line genes. In addition, the splicing mechanism is tlexible with regard to the precise sites of joints, permitting additions or deletions of one or more bases and codon frame shifts. This flexibility greatly amplifies potential diversity but at the cost of a high frequency of abortive rearrangements. Pre-B cells and their immediate precursors which are in process ol V,-D,-J, rearrangement divide rapidly, with a cell cycle time of 8- 10 hr. The large rapidly dividing cells give rise to smaller cells with the same phenotype which apparently do not divide (I I ). Rearrangement of. light-chain V genes to their respective J genes then follows in an ordered manner. Kappa gene rearrangements are attempted first. If neither kappa locus yields a functional V,-J,-C, gene, then the lambda light-chain loci undergo rearrangement ( 13). In a manner yet to be defined, a functional rearrangement at one of the two heavy- or four light-chain loci inhibits further attempts at rearrangement of the homologous loci. accounting for the phenomenon of allelic exclusion. Expression of a light chain is followed by assembly of monomeric IgM molecules which arc then expressed on the cell membrane. This event marks the transition from pre-B cell to a B lymphocyte capable of interacting with some epitope. How might a regulatory gene on the X chromosome interfere with the pre-B cell to B lymphocyte transition’? Schwaber (‘1 r/l. (13) have reported that pre-B cells from marrow of three patients with XLA did not react with antibodies spe-

B-CELL

ONTOGENY

AND PATHOGENESIS

7

cific for Vu determinants. Approximately 5% of normal marrow pre-B cells, and a larger proportion of fetal liver pre-B cells, were reported to have a similar phenotype. Hybridomas constructed from XLA marrow or fetal liver produced a shorter than normal p-chain mRNA which was shown by primer extension analysis to have a 5’ deletion of a size consistent with Vu (13). This intriguing result has not yet been confirmed in other laboratories. Such a defect might originate from an X-linked “recombinase” recognizing the highly conserved heptamernonamer sequences which flank V, D, and J genes (14). A problem with this notion is that T-cell-receptor chains are assembled in an identical way from V. D. and J genes flanked by the same recognition sequences (IS). Light-chain expression is a potential site for the regulatory defect in XLA. The ordered sequence of immunoglobulin gene rearrangements (p. -+ K ---f A) and the phenomenon of allelic exclusion imply the existence of regulatory elements acting between different chromosomes to trigger or suppress immunoglobulin gene rearrangement. Direct evidence for active suppression of light-chain rearrangement mediated by formation of a functional immunoglobulin molecule was recently gained by ingenious experiments employing kappa chain transgenic mice (16). In short, hybridomas expressing the exogenous kappa gene as a part of an immunoglobulin molecule had not rearranged endogenous kappa genes. Indirect evidence for a positive signal came from an experiment in which a cloned, virus-induced pre-B cell line was fused with a myeloma variant lacking light-chain expression. Several hybridomas from this fusion produced kappa light chains encoded by rearrangements of different VK genes of the pre-B cell parent ( 17). In this experiment the myeloma fusion partner apparently provided a regulatory signal which activated VK-CK rearrangement in the pre-B cell. Failure to produce such a regulatory signal could explain the differentiation block in XLA. Conley (18) has recently reported a detailed observation on the phenotypic characteristics of the few circulating B cells in XLA patients. Most of the cells had an immature phenotype, with a high density of sIgM. low density of HLADR, and absence of the C3d receptor. The low numbers and immature phenotype of these cells suggested a generalized abnormality of proliferation, affecting all stages of B-cell development (18). An alternative explanation comes from consideration of the role of idiotypic networks in B-cell maturation. Given the immense diversity of antibody V regions which may be produced by gene shuffling and somatic mutations, Jerne’s seminal idea that the universe of internal idiotypes may mirror that of external epitopes has become extremely compelling (19). A logical consequence of the existence of idiotypic network interactions is that they might play a dominant role in functional maturation of the immune system: idiotypes, rather than external antigenic determinants. might drive some of the early steps in B-cell differentiation. It might be predicted that panhypogammaglobulinemia of any type, or severe restriction of antibody, diversity without panhypogammaglobulinemia, would be associated with immature B lymphocyte phenotype and function. It has been demonstrated that experimentally induced B-cell deficiency profoundly alters the repertoire and function of helper and suppressor T cells (20. 21).

IMMATURE

B CELLS

Normal expression of functional light chain\ i\ rapidly followed by cxprccGon of intact slgM molecules on the cell surfLice. since the frequency of prc-B cell\ expressing p and light chains is quite low. Thc\e immature \IgM + lymphoc],tc\ are uniquely susceptible to tolerance induction if euposed to the appropriate mutivalent antigen (reviewed in (12)). This may well be an important mechanism [or induction of B-cell tolerance to widely distributed self-antigens. ‘4s the divcrsitb of the immune system is both very large and largely generated by stochastic combinations of genetic elements. the generation of an abundance of beIf-rcactivc clones is inevitable. The absence of expression of cell-surface antigen receptor> by pre-B cell protects the integrity of the diversity-generating system. while the tolerance-susceptibility of the most immature precursor5 ensures purging of the repertoire of clones with high-affinity receptors for abundant self-antigens. ISOTYPE

DIVERSITY

The next step in the differentiative process is generation of isotype diversity. By a process called isotype switching. each clonal precursor expands to form a family of B lymphocytes retaining the original clonal specificity but secreting antibodies of each of the different heavy-chain classc~. The ultimate mechanism of switching involves a second rearrangement of the expressed immunoglobulin gene by which the VDJ coding segment for the variable region is detached from the 5’ side of the C~.Lgene and spliced to LI new location 5’ to another CH gene. The CIJ, and other C, genes located 5’ to the new splice site are deleted. Allelic deletion is not the only mechanism for expression of isotype diversity. however. While plasma cell tumors or hybridomas expressing isotypes other than IgM have undergone this DNA rearrangement, B lymphocytes may simultaneously express multiple isotypes of cell surface immunoglobulin without disturbance of the germ-line C, gene configuration. The most frequent class of B lymphocytes express both IgM and 1gD. Yuan c,t trl. have carefully dissected the mcchanismh regulating coexpression of sIgM and sIgD on mouse B cells as a function ofdevclopment (73). Transcriptional. post-transcriptional, and post-translational controls are all involved. Mature mRNA for 6 chain is formed by processing of a large (29 kb) polycistronic nuclear RNA encoding VDJ-Cb-Cfi. Mt.-Chain mRNA may bc formed similarly. but is also derived from a shorter nuclear message which dots not include a Cb. In addition to transcriptional controls and RNA processing. differences in the half-lives of k and 6 mRNAs and of the corresponding cell-aurface proteins regulate the relative quantities of IgM and IgD on the cell surface. While cells bearing sIgM and slgD constitute the most frequent subclass, other combinations of isotypes are found on B lymphocytes. During ontogeny B cells expressing k. y. and (Ydeterminants all appear at about I I - 12 weeks ofgcstation. a few weeks after the first TV+ B cells are detectable (3). Invariably. cells bearing y or (Y determinants are also p ’ . but do not express 6 (34). In neonatal blood. nearly all cy+ or y + cells express both k and ii as well. while coexpression of u and y is rare. Most LY+ and y’ cells from adult blood express only the single isotype.

B-CELL

ONTOGENY

AND

PATHOGENESIS

9

Separate mature mRNAs for these isotypes are presumably produced by processing of a large polycistronic nuclear message (2%. as is the case for coexpression of sIgM and slgD (23). At some later point, B lymphocytes bearing these minor surface isotypes undergo switch recombination. Cells bearing sIgA or sigG as a single isotype constitute the majority of these subclasses in adult lymphoid tissues. Multiple isotype expression by B lymphocytes is therefore a useful marker for immaturity. For example, sIgA+ cells from nearly all patients with isolated IgA deficiency express sIgM as well (26). This immature phenotype might mean that the B cells from these patients are unable to undergo the final step of switch recombination to produce a complete a-chain gene (VDJ-Ca) or that the cells fail to receive the appropriate signals. Does expression of IgG. IgA, or one of their subclasses on the membrane of a B lymphocyte signal commitment of that cell and its progeny to secrete antibodies of the same isotype? In view of the distinctly different pathways through which mRNAs encoding heavy chains may be constructed. it is no longer surprising that this simple question has yielded such conflicting data and opinions (23-25. 27). Observations on patients having X-linked immunodeficiency with normal or elevated IgM are pertinent to this question. These boys are very deficient in both B lymphocytes expressing sIgG or sIgA and in plasma cells expressing these isotypes (28). Their B cells cannot be stimulated to secrete these isotypes irt i~itro, but their T cells can provide “help” for IgG and IgA responses (28, 29). These findings imply a regulatory factor encoded by the X chromosome which is involved in both initial expression of cell surface IgG or IgA by RNA processing and also in terminal switch recombination of DNA. Perlmutter and Gilbert (25) suggest a mechanism by which this linkage might occur. They used the fluorescence-activated cell sorter to obtain populations of mouse splenic B lymphocytes with the following phenotypes: sp+y, . SF+Y,+, SF-y,‘. sp.+a-. and SP+~+. Analysis of DNA demonstrated that the Cy, and Ca genes were not rearranged, and the Ck was not deleted in any of these populations. Nuclear RNA was examined by a hybridization sandwich technique; RNA bound to a nitrocellulose disk via a downstream probe (Cy, or Ca) was then hybridized to a radiolabeled Cp probe. A positive signal indicates a single RNA molecule with segments complementary to both probes. Cells expressing y, or o! chains had nuclear transcripts of upstream C genes (Ck*, or Ck + Crl. respectively) whether or not the products were expressed. Downstream transcripts were not observed; neither ~+y,- or IJ,+‘Y- cells had Crl or Ca transcripts and Y1+ cells lacked Ca transcripts. These observations indicated that transcription was terminated with the most downstream C gene being expressed. Upstream C genes were transcribed but not necessarily translated. The authors suggest that the switch regions on the 5’ side of C, genes may become available to DNA recombinase enzymes only when that chromosomal region is “open” to transcription. Thus the downstream RNA termination site would determine the isotype expressed and at least the limit for switch recombination. Premature termination of transcription might be a unitary lesion resulting in failure of membrane IgG and IgA expression and of switch recombination in the hyper-IgM syndrome.

IO

ALEXANDER

FUNCTIONAL

K.

LAWI‘ON

ONTOGENY

Initial studies on the ontogeny of immunoglobulin synthesis and antibody production in man antedated the identification of T and B lymphocytes and their role in immune responses (reviewed in (30)). Fetuses with intrauterine infections are capable of generating plasma cells and producing antibodies by about 20 weeks of gestation. The capacity to synthesize and secrete immunoglobulins develops in the sequence IgM - IgG --$ IgA. This pattern is recapitulated postnatally in the development of serum immunoglobulin isotype concentration. IgM reaches adult values by about I year, IgG by 5-6 years. and IgA in late childhood. These kinetics could not be explained by the appearance of B lymphocytes bearing the respective isotypes. since B lymphocyte isotype diversity was fully expressed by midgestation. These observations were among the first to raise the issue of T-cell regulation of isotype diversity. a topic still vigorously debated (3 I ). In early functional studies, stimulation of unfractionated cord blood lymphocytes with pokeweek mitogen (PWM) generated IgM responses which were IO-X% of those in adult controls. while IgG and IgA responses were virtually absent (32). Subscquently it was demonstrated that the B-cell response to PWM was T cell dependent and not restricted by histocompatibility differences. This made possible comparative analysis of T- and B-cell functions in newborns and adults. as well as patients with immunodeficiency. Three conclusions were drawn from initial experiments of this type: neonatal B cells had greater responses when cocultured with adult than neonatal T cells. but were still deficient with respect to synthesis of IgG and IgA isotypes. T cells from most newborns had dominant suppressor activity, which. however, was not selective for any immunoglobulin class. Nconatal T cells cocultured with B cells were less efficient in promoting IgA and IgG responses than were adult T cells (33). The relative incapacity of newborn B cells to generate IgG and IgA responses has been confirmed in many laboratories (for review, see (341). Similar results are obtained whether the polyclonal activator used is entirely (PWM). partially (NOctrrdicr water-soluble mitogen: NWSM). or not (Epstein-Barr virus: EBV) dependent upon T cells. The IgM responses of newborn B cells are somewhat lower than those of adults in most studies. with the greatest differences observed with PWM and the least with EBV. Adult values for IgG and IgA responses are achieved between 2 and 5 years. Andersson et trl. (35) studied the maturation of IgG subclass responses using EBV and PWM as stimulants. With adult B cells EBV induces responses in the relative order IgG3 > IgGl >> IgG4 > IgG2. while PWM stimulates primarily IgGI and IgG,.3 With either activator. maturation of IgGI and IgG3 isotype responses occurs more rapidly than IgG2 and IgG4. IgG antibodies to most carbohydrate antigens are of the lgG2 class. These observations may be relevant to the poor responses of infants to polysaccharide antigens. There are striking parallels between the phenotypes and functions of B cells from neonates and immunodeficient adults. The “immature” phenotype of u + cells in IgA-deficient patients was alluded to earlier (26). The majority of CY+ and y+ of athymic nude mice or of germ-free mice are also p+ After thymus transplantation or exposure to a conventional environment, respectively. most y + and

B-CELL

ONTOGENY

AND PATHOGENESIS

II

IX+ B cells express only the one isotype. The immature phenotype probably reflects the failure of B cells to be activated, and might result from the absence of normal signals or abnormal responses to these signals (36). The studies of Pereira et al. (37) have drawn attention to the similarity of functional deficiencies shared by B cells from patients with common varied immunodeficiency and from neonates. When T-cell help or suppression is not limiting, these B cells usually produce some IgM but little or no IgG (37). To say that B lymphocytes from immunodeficient patients are immature unfortunately does not shed much light on the nature of their molecular defects. A major impediment to progress in this area is the fact that the polyclonal activators used to induce B-cell functions, possibly excepting EBV, address a minor subset of previously activated, relatively large, low-density lymphocytes which do not express sIgD (38-42). What studies in many laboratories have defined as intrinsic abnormalities of B cells may simply reflect the absence from the circulating pool of that small but critical subset. Development of irr \~itro methods to induce differentiation as well as proliferation of the small resting B lymphocyte pool should be a major research objective for all of human cellular immunology, but is particularly critical to the field of immunodeficiency diseases. ACKNOWLEDGMENTS 1 am grateful to Dr. Donna Hummell for her critical review and discussion of this paper. and Mrs. Kristina Roth for expert editorial assistance and typing.

REFERENCES I. 2. 3. 4.

Gwen. J. J. T.. Copper, M. D.. and Raff. M. C., h’<~tlrre (Lo~/oN) 249. 361. 1974. Raff. M. C.. Megson. M.. Gwen. J. J. T., and Cooper, M. D.. Nrrflrrr (Londo,t) 259, 224. 1976. Gathings, W. E.. Lawton. A. R.. and Cooper. M. D.. E~rr. J. Irr~rnrotol. 7. X04. 1977. Hayward. A. R.. Simons, M. A.. Lawjton. A. R.. Mage. R. CJ..and Cooper. M. D.. J. Esp. Met/. 148, 1367, 1978. 5. Cooper. M. D.. Lawton. A. R.. and Bockman. D. E.. itr/nc/ 2, 791. 1971. 6. Siegal. F. P.. Pernis, B., and Kunkel. H. G.. Etrr. J. fv7~7rrnrd. 1, 482. 1971. 7. Cooper. M. D.. and Lawton. A. R.. An7cr. J. Pd701. 69, 513. 1972. 8. Preud’homme. J. L.. Griscelli, C.. and Seligmann. M.. Clirr. Ivrt~7rtr7ol. lr77rntrnopt72/7o/. 1, 141. 1973. Y. Pearl. E. R., Vogler. L. B.. Okos. A. J.. Crist. W. M.. Lawton. A. R.. and Cooper. M. D.. J. Imrr7rrn~d. 120, 1169, 1978. IO. Waldmann. A.. C/in. Imu77tnd. /m,77rrnopc7rho/. 40. Z-36. 1986. I I. Cooper. M. D., J. C/in. Ir77muno/. 1. 8 I. 1981, 12. Hieter. P. A., Korsmeyer. S., Waldmann. T. A.. and Leder. P.. Nntrrrc> (L.wrd~n) 290, 368, 1981. 13. Schwaber. J.. Molgaard. H.. Orkin. S. H.. Gould. H. J.. and Rosen. F, S.. Ntrtlrre (Lo&J~) 304, 3.55, 1983. 14. Tonegawa. S.. h’trturr (Londo~7) 302. 575, 1983. IS. Siu. G.. Clark. S. P.. Yoshikai. Y.. Malissen. M.. Yanagi. Y.. Strauss, E.. Mak. T. W.. and Hood. L.. Cc,// 37, 393. 1984. 16. Ritchie. K. A., Brinster. R. L.. and Storb. U., Ntrtrrre (Lond0,7) 312, 517. 1984. 17. Riley. S. C.. Brock. E. J.. and Kuehl. W. M.. Nrtrrrre (Lontforr) 289, 804. 1981. 18. Conley. M. E.. J. Immlrnol. 134, 3070. 1985. 19. Jerne. N. K.. Ann. In7mtnol. (Puris) 125C, 373, 1974. 20. Bottomly. K.. Janeway. C. A.. Jr.. Mathieson. B. J.. and Mosier. D. E.. Eur. J. Imti7~7d. 10. 159. 1980.

12

.4LEXANDEK

cerraf.

H..

Proc,.

NO//.

,AoI~/.

22. 23.

No\\al. khan.

G. J. V.. ,.1,//r//. /<(,I,. P.. Gilliarn. A. (‘.. and

74.

Ahney. vlrl,rcd.

E. R.. (‘ooper. M. 120, 2041 IY7X.

25, 26.

Perlmuttcr. Conley.

M.

27. 2X.

Shimizu. Levitt.

79.

Geha.

30.

rc’.v/. 64. 385. lY7Y Vogler. L. B.. and Lawton.

3 I,

Lawton. 104.

A. P. and Gilbert. E.. and Cooper.

A.. D.. R..

and Haber.

.S( i. (ISA //,/,,rrtriol. Tucker. D..

M.

81.

N..

Self.

Pllami.

LAWTON

1846.

IYX4.

I. 3;. lYX7. P. W.. /-(,c/. I’r01

Kearnev.

W.. I%,(,. ,Vtrt/. A~oc!. D.. 1%‘. Eng/. J. iZfct/.

S.. Far-ah. A.

R..

D.. ./. (‘/i/r.

and

Cooper.

IYX5. ;md

.Sc,i. l:.yi\ 295. 4YS.

I?irkhou\c.

81, 71XY. IYXI.

(r~~.c~.\r. 72. E. E.. and

I~~r~~rrr~f~~/. /1//r,:q!.

S. A..

2652.

A. K..

E. Schneeberger.

Clirr.

K. S.. Royal.

44.

J. F.. I*aw.ton.

Honjo. T.. (‘rl// 36, X01. 19x4. P.. Kich. K.. and Cooper. M.

Hpslop.

A. R..

R.

5. 13.5.

M.

II..

C/i,r.

K. M.

TV... I

IYX4.

1650.

19X3.

Rosen,

E S.. .I. C‘lirr.

30x. 1976. Hayward.

34.

Miiller.

35. 36.

Andersson. Gathingh.

37. 3X.

Pereira. Stevens.

39. 40.

Lucivero. Kuritani.

41, 42.

Ito. S.. and Martiner-Maza.

A. R..

G. (Ed.). W.

and

Lawton.

fr~~/trlrn~~l.

U.. Bird. A. G.. E.. Kubagawa.

R. S., Webster. R. H.. Mac).

.4. K.. J. Iu~rnrrr~ol. R~,I.. and H..

57.

//tr/t~rl~~rj/.

I/rir,rll~0~(11/10/.

Britton. S., I/,~,,rrr/r~j/. Kc,i,. 57, 5, 19x1. and Cooper. M. D.. /~?~~rrrr/rol. RcI,. 57. 107. IYX I,

A. D. B.. and E.. and Thiele.

A, and

121 7. lY77.

19x1.

Platt-Mollh, C. J., .Sctr&.

G.. Lab ton. 4. R.. and Cooper. T.. and Cooper. M. D.. J. t:vp. Lawton. 0..

119.

M. MEL/.

T.A.E.. E/lr. .I. ~~~I!IzI!)Io/. 12, 540. .I. l/~r/,~r/~l~~/. 14. 449. 1981.

D.. C/i/i. E.:rp. I/~r/trr~~~r~l. 155, IShl. 19x1.

R.. J. /u/~u/~~Io/. 133. 1891. IYXJ. Britton. S.. .I. t\o. ,Ifcc/. 157. 1X08.

19X3.

Irr-

1YX.C.

lY72.

33.

l/r/

45,

IX?.

1981.

1982.

I.