Immunology Today December 1985

Immunogenetics of endocrine disorders from G. S. Eisenbarth There is much evidence that type I diabetes (also termed insulin-dependent diabetes mellitus) of man, the Biobreeding (BB) rat and the Non-Obese Diabetic (NOD) mouse results from autoimmune pancreatic beta cell destruction and that the most common forms ofthyrold dysfunction (hypothyroidism associated with several different forms of 'thyroidltis' and the hyperthyroidism of Graves' disease) result from the interaction of autoantibodies or autoreactive T lymphocytes with thyroid follicular cells. The contribution of genes in the major histocompatlbility complex (MHC) to the development of autoimmune beta cell destruction and thyroid autolmmunity was the focus of a recent meeting. * For all of the organ-specific autoimmune diseases discussed (except type I polyendocrine syndrome), a gene or genes within the M H C contribute to disease susceptibility, i n the diabetes-prone BB rat multiple genes, including a gene within the M H C are 'necessary' but not sufficient to produce diabetes. In the breeding studies presented by E. Colle (Montreal) and G. S. Eisenbarth (Boston), diabetes developed only when the animals inherited at least one R Tl-u bearing chromosome (the histocompatibility type of the diabetes prone BB rat). Furthermore, diabetes developed in ten times more F~ animals homozygous for R 77-u than heterozygotes. Although the BB diabetogenic gene within the M H C is unknown, it is likely to lie within the class II region. Breeding studies indicate that a class I locus recombinant (PVG.R8 rats, A", B u, D u) and BBN chromosomes differing by class I restriction fragment patterns retain diabetogenicity. A striking finding by Colic and coworkers in their sequential breeding studies (F 3 and beyond) was that within litters with more than one diabetic animal, diabetic animals were either heterozygous for R Tl-u (e. g., u/x where x is the type of the non-BB parent) or homozygous (u/u). In both the Montreal and Boston studies a gene for severe circulating T-cell lymphopenia (90% or *A meeting on the immunogenetics of type I d i a b e t e s a n d autoimmune thyroid disease was held in St. John's, Newfoundland, Canada, 24-27 August 1985.

greater decrease in W3/25 ÷, OX19 ÷, O X 8 ÷ T cells) and immunodeficiency segregated independently of the M H C but with development of diabetes. The Boston studies suggest that the T lymphopenia is determined by a 'simple' recessive gene, while the Montreal studies suggest that more than one gene may influence the severity of the lymphopenia. The circulating T lymphopenia is present in these animals from birth despite normal thymic T cell counts. Type I diabetes develops in approximately 80% of female and 20% of male N O D mice, an inbred strain bred by

Makino and Tochino in J a p a n and now widely available. In crosses of N O D mice with C 3 H mice, F 1 animals do not develop diabetes. In F 2 or backcross animals diabetes occurred only in animals homozygous for the N O D mouse M H C (M. Hattori and R. A. Jackson, Boston). The N O D mouse is particularly interesting in that it fails to express surface I - E and produces no messenger R N A hybridizing with an I - E alpha probe. Thus if I - E contributes to diabetes in these mice it can do so only by its absence. In m a n the H L A alleles DR3 and DR4 are associated with development of type I diabetes. Approximately 95% of individuals with type I diabetes express DR3, DR4 or both compared with 40% of the general population. Studies presented by N. Maclaren (Gainesville) and S. Baekkesksov (Gentofte) indicate that restriction fragment length analysis with class II gene probes will further refine the M H C gene association in man. In particular a 3.7 kb BamHI fragment hybridizing with an H L A - D Q b e t a probe is more common in DR4 diabetogenic chromosomes than controls. The Continued on p. 346

Monoclonal antibodies against mycobacterial antigens from Howard D. Engers, Barry R. Bloom and Tore Godal The World Health Organization (WHO) scientific working groups on the Immunology of Leprosy (IMMLEP) and Tuberculosis (IMMTUB) recently organized two international workshops designed to characterize the specificity and reaction patterns of approximately 55 murine monoclonal antibodies (mAbs) which had been raised against Mycobacterium leprae or Mycobacterium tuberculosis and submitted to the IMMLEP and IMMTUB monoclonal antibody banks. The I M M L E P and I M M T U B workshops were organized such that aliquots of the mAbs were coded and sent (including some duplicates) to the participating laboratories for independent analysis by a variety of techniques in order to determine the specificity of the mAbs and the nature and molecular weight(s)

of the corresponding antigens (Table I). In the I M M T U B workshop, the mAbs were also used as probes to screen a M. tuberculosis derived recombinant DNA library I for the expression of mycobacteria-specific protein antigens (Table If). Continued on p. 347

© 1985,ElsevierScience Publishe~B.V., Amsterdam 0167 49191851502.00

Immunology Today, vol. 6, No. 12, 1985

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m o r e f u n d a m e n t a l u n d e r s t a n d i n g of these illnesses a n d hopefully selective m e t h o d s for abrogating the a u t o i m m u n e responses are needed. It is a testimony to the seriousness of several of the diseases that trials of i m m u n o t h e r a p y u s i n g cyclosporin A to influence the course of type 1 diabetes a n d G r a v e s ' eye disease are u n d e r w a y . A n o t h e r testimony to the severity o f type 1 diabetes is that organi-

zations such as the J u v e n i l e Diabetes F o u n d a t i o n provide a relatively large n u m b e r of post-doctoral fellowships, career development awards, and research grants to investigators throughout the world a n d welcome applications f r o m i m m u n o l o g i s t s attacking a portion or the whole of the puzzle of o r g a n specific a u t o i m m u n i t y . As evidenced by the N e w f o u n d l a n d meeting, there are

Monoclonal--contd from p. 345 A series of monoclonal antibodies reactive with a broad r a n g e of different M. leprae-derived antigens are now available (Table IA): (1)3//. leprae-specific m A b s reacting with four distinct M. leprae protein molecules ( m A b s 8, 18, 21, 23, 24). T h e genes coding for these proteins, plus an additional 28 k D a protein, have been identified recently u s i n g the m A b s as probes 2. (2) M. leprae-specific m A b s reactive with the phenolic glycolipid 1 which is u n i q u e to M. leprae ( m A b s 6, 9, a n d 10). O n e of these m A b s has been u s e d to identify phenolic glycolipid 1 a n t i g e n in the s e r u m , saliva or urine of leprosy patients 3. (3) Several cross-reative m A b s which recognize carbohydrate/lipid molecules or a 5 5 - 6 5 k D a m u l t i b a n d protein which apparently expresses both specific a n d cross-reactive epitopes. T h e results of the I M M T U B workshop (Table I B) concerning the M . tuberculosis-related m A b s can be s u m m a r i z e d as follows: (1) A n u m b e r of m A b s exhibiting a specificity pattern restricted to the M. tuberculosis complex (i.e., reacting with M . tuberculosis, M . boris B C G a n d M. africanium) were d e m o n s t r a t e d to react with three distinctM, tuberculosis proteins ( m A b s 1, 4, 15, 20, 21, 23 a n d 27). H o w e v e r , no strain-specific m A b s have been identified to date. (2) A series of m A b s exhibited limited cross reactivity, reacting with a wide r a n g e of molecular weight molecules r a n g i n g from 71 k D a d o w n to 12 kDa. Four m A b s b o u n d to a 19 k D a molecule, whereas a n o t h e r three gave a strong reaction with several c o m p o n e n t s ( m u l t i b a n d ) in the 65 k D a region. (3) Several broadly cross-reactive m A b s were difficult to characterize with regard to the actual molecular weight(s) of the antigen(s) in question. T w o m A b s reacted with a 23 k D a molecule a n d a n o t h e r two showed m u l t i b a n d activity. T h e antigens recognized by the m A b s listed in T a b l e I were identified as proteins based on their sensitivity to treatm e n t with proteolytic e n z y m e s such as subtilisin. In several cases, however, the

Table I. Characterization of monoclonal antibodies to mycobacteria Monoclonal IgG Mycobacterial antibody subclass specificitya designation (code nos. in brackets) A IMMLEP workshop,June 1984 (14) MC-4027 IgG1 M. leprae (8) MC-2404 IgGl M. leprae (24) MC-9215 IgGl M. leprae (3) MC-5205 IgG2a cross-reactive (5) MC-0401 IgGi cross-reactive (7) MC-2009 IgG1 cross-reactive (15) MC-4220 IgG2b cross-reactive (18) MC-58~8 IgGl M. leprae (12) MC-3607 IgGa (M. leprae)d (21) MC-8026 IgG1 M. leprae (23) MC-8908 IgGl M. leprae (1) MC-2924 IgG3 cross-reactive (2) MC-4311 IgM cross-reactive (4) MC-6225 IgG3 cross-reactive (13) MC-3906 IgG3 cross-reactive (22) MC-8610 IgM cross-reactive (6) MC-1433 IgGl M. leprae (9) MC-2817 IgM M. leprae (10) MC-3131 IgG1 M. leprae (B) IMM TUB workshop,June 1985 (11) 51-A IgGza L-cross-reactive (13) WTB-78 IgG1 L-cross-reactive (31) SA2D5H4 ? L-cross-reactive (33) ML II H9 I g G ~ L-cross-reactive (7) F 29-29 IgG2a cross-reactive (15) WTB-72 IgGl M.t complex (21) HY T 28 IgG M.t. complex (23) WTB-71 IgG2b M.t. complex (17) SAI D2 D1 IgGl cross-reactive (29) TBI B2 H2 I g G 3 cross-reactive (9) MT107 F5/C5 IgM cross-reactive (32) SA2 AIA5 IgM cross-reactive (10) F29-47 IgM L-cross-reactive (12)HY T6 IgG L-cross-reactive (16) 66 IgG2a L-cross-reactive (19) WTB-23 IgG1 L-cross-reactive (27) TB-C-13 IgGza M.t. complex (1) F 23-49 IgG2a M.t. complex (4) F 24-2 IgM M.t. complex (20) WTB-68 IgG1 M.t. complex (3) SA 12 IgG2a L-cross-reactive (30) TBI A II C3 IgA M.t. avium

m a n y i m m u n o l o g i c a l similarities linking these various disorders a n d it is likely that knowledge gained in a n y single model or disease will be widely applicable.

n-i GeorgeS. Eisenbarth is at theJoslin Diabetes Center, Boston, Massachusetts 02215, USA.

Molecular weight b (ND = not done)

Nature of antigen c

Ref.

200 kDa 55;65 kDa 55;65 kDa 55;65 kDa 55;65 kDa 55;65 kDa 55;65 kDa 36 kDa 35 kDa 18 kDa 12 kDa unknown unknown unknown unknown unknown ND ND ND

protein proteins proteins proteins proteins proteins protems protein protem protein protein

6 7 7 7

unknown unknown unknown unknown Phenolic glycolipid Phenolic glycolipid Phenolic glycolipid

71 kDa 65 kDa e 65 kDa e 65 kDa e 40 kDa 38 kDa f 38 kDa f 38 kDa f 23 kDa 23 kDa

protein protein protein protein protein protein protein protein protein protein?

7 8 9 7 9

unknown

6 10

-11 -7 6,8 11 -11 12 --

20-80 kDa protein? -20-80 kDa unknown -19gkDa protein 6,8 19gkDa protein 13 19gkDa protein -19gkDa protein -19 kDa protein -14h kDa protein 6,8 14h kDa protein 6,8 14h kDa protein 11 12 kDa protein 14 unknown unknown -a Mycobacterial specificity was tested on antigens prepared from up to 23 species of mycobacteria using ELISA, RIA, IFA and several immuno-blotting techniques (see Refs. 6, 8 and 12 for details). Categories of' specificity : M. leprae = Mycobacterium leprae ; cross-reactive = reacting with a majority of the 23 strains tested; L-cross-reactive = reactingwithalimitednumberof the23 strainstested; M.t. complex = reacting with M. tuberculosis, M. boris BCG and M. af'n'canium. b Molecular weights were determined by SDS polyacrylamide gel electrophoresis with different immunoblotting techniques (Ref. 6-8, 12-14). c Antigens were classifiedas protein in nature if they were sensitive to the proteolytic enzyme subtilisin. d M. lepraespecificwhen tested by RIA and IFA but cross-reactive by dot-blot analysis. e Antigen recognized by mAbs coded 13, 31 and 33. f Antigen recognized by mAbs coded 15, 21 and 23 (number 23 recognized an epitope different than the other two mAbs). g Antigen recognized by mAbs coded 10, 12, 16 and 19. h Antigen recognized by mAbs coded 1, 4 and 20,

Immunology Today, vol. 6, No. 12, 1985

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Table II. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA probes and monoclonal antibodies Reaction pattern

1

4

A B C D

+ +

-

E F G H I

.

.

J

.

.

K

.

.

L

+

M

.

Reaction with IMMTUB monodonal antibodies a 20 13 31 33i + + + +

+ +

. . . .

+ + +

. . . .

. . . .

ND + +

.

.

.

.

. +

.

+

.

+ .

.

+ .

27

. . . .

ND + + + +

. .

+ .

. . . .

11

Number of phage clones 1 2 1 22

-

-

+

-

78

-

+

2

+

+

.

4 1 25 3 2

1 2O

The IMMTUB mAbs listed are among those described in Table I. The remaining 24 mAbs did not react with any of the individual phage tested in this assay system. The assays were performed as described in Ref. 1, using the same recombinant DNA library. actual nature of the antigen could not be established. There was no apparent relationship between the antigens and/or the immunization schedules used to produce the m A b s and the characteristics of the m A b s as judged by their specificity or reaction patterns summarized in Table I. As an additional analysis in the I M M T U B workshop, the rnAbs were used as probes by two laboratories in order to detect recombinant phage clones expressing target antigens recognized by the m A b s (Table II). A library of M. tuberculosis genomic D N A fragments expressed in the Z gt 11 expression vector 1 was screened with a pool of the 33 m A b s submitted to the workshop. I n an initial screen of about 106 recombinant phage, 135 positive signals were detected. I n a subsequent screen using a selected pool of antibodies (1,4, 13, 20, 21, 23 and 27) 28 additional positive signals were identified. T h e phage corresponding to the positive signals then were plaque purified and tested for reactivity with the individual m A b s to determine which target antigen (epitope) was expressed. T h e resulting reactivity patterns can be arranged into 6 main groups (Table II). In two instances, a group of m A b s was observed to re,act with the same target antigen but not with the same epitopes on the molecule in question. I n s u m m a r y , the recombinant D N A results were in quite good agreement with the results presented in Table I, and demonstrate that the process of screening a recombinant D N A library can be a very efficient m e a n s of delineating the fine specificity patterns exhibited by parasitespecific monoclonal antibodies. It is hoped that this battery of mAbs, combined with the relevant recombinant D N A technology can now be applied to several aspects of immunodiagnosis and vaccine research for both leprosy and tuberculosis. Some possibilities are: (1)

taxonomic assignment of mycobacteria, based on D N A characteristics; (2) identification and isolation of genes coding for antigens presenting diagnostic and/or protective potential (indeed, three recent papers describe the use of m A b s in diagnostic assays for leprosy-specific antibodies/antigens ~-s) (3) large-scale production of such interesting antigens, together with synthetic peptides produced on the basis of the nueleotide sequences identified. This s u m m a r y emphasizes the fact that the international workshop approach can be a very effective m e a n s of obtaining a definitive, u n a n i m o u s characterization of a large n u m b e r of m A b s in a short period of time. M o r e detailed information can be found in the workshop reports (Refs. 15 and 16). T h e i M M L E P and I M M T U B steering committees propose to repeat such workshops when a sufficient n u m b e r of new interesting m A b s a n d / o r antigens have been assembled. Potential contributors of these reagents should contact the steering committee chairmen in order to be kept informed of recent developments. Subject to available reserves, samples of the m A b s or the recombinant D N A library described above are available to qualified investigators, u p o n receipt of a brief (1 page) s u m m a r y of the experiments to be carried out.

n-i Acknowledgements A large portion of the research described in this summary was supported by grants from the WHO/World Bank/UNDP Special Program for Research and Training in Tropical Diseases (IMMLEP) and from the W H O Vaccine Development Program (IMMTUB). It is a pleasure to acknowledge the uninhibited participation and collaboration of the scientists involved in these two workshops.

References 1 Young, R. A. et al., (1985) Proc. Natl Acad. &i. USA. 82, 2583-2587 2 Young, R. A. et al., (1985) Nature (London) 316, 450-452 3 Young,D. B. etal., (1985)J. Infect. Dis. 152, 1078-1081 4 Sinha, S. et al., (1985) Int, J. Lepro~ 53, 33-38 5 Klatser, P. R., de Wit, M. Y. L. and Kolk, A. H. J. (1985) Clin. Exp. Immunol. 62, 468-473 6 Kolk, A. H. J. et at., (1984) Clin. Exp. Immunol. 58, 511-521 7 Gillis, T. P. and Buchanan, T. M. (1982) Infect. hnrnun. 37, 172-178 8 Klaster, P. R., Van Rens, M. M. and Eggehe, T. A. (1984) Clin. Exp. ImmunoL 56, 537-544 9 Ivanyi, J. etal. (1983)Clin. Exp. Immunol. 52, 528-536 10 Young, D. B. et al. (1984) Infect. lmmun. 43, 183-188 11 Coates, A. R. M. et al., (1981) Lancet ii, 167-169 12 Young, D. B. et al., (1985) Clin. Exp. ImmunoL 60, 546-552 13 Schou, C. et aL, Acta Pathol. Microbiol. &and. Section C (in press) 14 Minden, P. et aL, (1984) Infect. Immun. 46, 519-525 15 Engers, H. D., Abe, M., Bloom B. R. Mehra, V., Britton, W., Buchanan, T. M., Khanolkar, S. R., Young, D. B., Closs, O., Gillis, T., Harboe, M., Ivanyi, J., Kolk, A. H. J. and Shepard, C. C. (1985) Infect. Immun. 48, 603-605 16 Engers, H. D., Houba, V., Bennedsen, J., Buchanan, T. M., Chaparas, S. D., Kadival, G., Closs, O., David, J. R,, Van Embden, J. D. A., Godal, T., Mustafa, S. A., Ivanyi, J., Young, D. B., Kanfman, S. H. E., Khomemko, A. G., Kolk, A. H. J., Kubin, M., Louis,J. A., Minden, P., Shinnick, T. M., Trnka, L. and Young, R. A. Inject lmmun. (in press)

Howard. D. Engers ( WHO- IR TC) is at the Dept. of Pathology, University of Geneva Medical School, Geneoa, Switzerland," Barry R. Bloom (Chairman, I M M L E P steering committee) is at the Albert Einstein College of Medicine, New York, iV. E, USA; and Tore Godal (Chairman, I M M T U B steering committee) is at The Norwegian Radium Hospital, Oslo, Norway.

Monoclonal antibodies against mycobacterial antigens.

The World Health Organization (WHO) scientific working groups on the Immunology of Leprosy (IMMLEP) and Tuberculosis (IMMTUB) recently organized two i...
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