Immunology Today, voL 5, No. 8, 1984 accomplished at the meeting on mast cell heterogeneity which will be held in early 1985 in Canada. O n e current example where species differences may be seriously misleading is in the definition of the cultured mast cell from murine bone marrow. For various reasons outlined by Jarrett and Haig, these cultured murine mast cells have been tentatively labeled 'mucosal'. However, to a large extent this is based upon cross-species analogies with rat, but not with mouse peritoneal mast cells. In the absence of homogeneic data, together with the lack of data on direct comparisons between cultured 'mucosal' and cultured 'non-mucosal' mast cells, it is difficult to assess the significance of the differences described. Such comparisons of distinct mast cell subtypes in culture are essential to assess the effects of the culture procedures, but have not yet been performed. After such investigations it may be clear that the mast cells grown from rat mesenteric lymph node by ourselves 9 and more recently by Haig andJarrett, and known to be analogous to the mast cell in the rat intestinal mucosa, may not be analogous to the mast cells cultured from murine bone marrow. O n e widely cited difference between mast cell subtypes, namely the T-cell dependency of the 'atypical or mucosal' type has never been properly substantiated as being restricted to this mast cell type. It is clear that proliferation of 'mucosal' mast cells is T-cell dependent ~0 and that in the absence of thymic (T-cell) influence, numbers are subnormal. However, in such T-cell deficient animals both mast dell populations are present. T h e T-cell dependency of the typical mast cell subtype has only been studied in nonproliferative conditions. The essential study, yet to be done, is to establish under conditions where 'typical, non-mucosal' mast cell proliferation is occurring, whether such proliferation is T-cell dependent. If this is the case, the arguments relating T-cell dependency to 'atypical' mast cells lose their significance and must be reassessd. It is important that the concepts surrounding mast cell heterogeneity not be restricted to two subtypes, 'mucosal and non-mucosal' because heterogeneity is certain to be more extensive than this and may involve not only related lineages, but encompass cell size 1, phase of cell cycle 11, and recent cellular secretory activity. At one extreme, heterogeneity may be fully site specific as micro-environmental factors regulate the phenotypic expression of different portions of the genome. W e are optimistic that the review by Jarrett and Haig, together with the reservations we have raised, will

219 aid in an efficient advancement of knowledge in this field and in important therapeutic developments for allergic and other diseases. D. BEFUS T. LEE J. DENBURG J. BIENENSTOCK Host Resistance Programme, Departments of Pathology and Medicine, McMaster University, Hamilton, Ontario, Canada, L 8 N 3Z5.

References 1 Sehulman, E. 8., Kagey-Sobotka, A., MacGlashan, D. W. Jr., Adkinson, N. F. Jr., Peters, S. P., Sehleimer, R. P. and Lichtcnstein, L. M. (1983)dr. Immunol. 131, 1936-1941 2 Befus, A. D., Goodacre, R., Dyck, N. and Bienenstoek,J. (1984) Fed. Proc. 43, 1973

Molecules in the immunoglobulin superfamily SIR, In a recent review (Immunol. Today 1984, Vol. 5, 133) Marchalonis et aL analysed the similarities between major histocompatibility ( M H C ) antigens, Thy-1 antigen and immunoglobnlins (Igs) and concluded that there was little evidence for evolutionary relationships between these molecules and that the data for a T h y - l : I g relationship was particularly unconvincing. In m y view this conclusion is incorrect and was arrived at largely because the comparisons were made without taking into account the structural features and conserved sequence patterns of Ig domains. Below, I discuss the issues mainly with reference to ht'.,~Thy-1 data although the same arguments could be made for the homology with Ig of the M H C antigens 1'2, the T-cell receptor 3'4 and the poly-Ig receptor 5. Homologies with Igs can only sensibly be made at the level of single Ig domains which consist of a sequence of about 100 amino acids. In these domains the sequence is folded into anti-parallel/3strands that form two/3-sheets that are held together by inpointing hydrophobic residues and a conserved disulphide bond 6'7. This is shown in Fig. 1 where the E-strands are labelled with the letters along the sequence and are marked on domain structures as they are seen by x-ray crystallography. The V-domain and C - d o m a i n folding patterns are shown and it can be seen that these share a common core structure made up offlstrands A , B , E and G , F , C but that they differ in the middle of the domains where the V-domains have an extra loop of sequence (labelled/3-strands C ' and C").

3 Befus, A. D., Pearce, F. L., Gauldie, J., Horsewood, P. and Bienenstock, J. (1982) J. ImmunoL 128, 2475-2480 4 Befus, A. D., Pearce, F. L., Goodacre, R. and Bienenstoek, J. (1982) in In-vivo Immunology (Nieuwenhuis, P., van den Brock, A. A. and Hanna, M. G. Jr., eds), 521-527 5 Becker, B., Chung, K. F., McDonald, D., Frick O. L. and Gold, W. M. (1984) Fed. Proc. 43, 1935 6 Barrett, K. E. and Pearce, F. L. (1983) Int. Arch. AllergyAppL Immunol. 72, 234-238 7 Siraganian,-R. P. and Hazard, K. A. (1979) J. Immunol. 122, 1719-1725 8 Barrett, K. E., Leung, K. B. P. and Pearce, F. L. (1983) Br. J. Phammcol. 78, 58 9 Denburg, J. A., Befus, A. D. and Bienenstock, J. (1980) Immunology 41,195-202 10 Ruitenberg, E. J. and Elgersma, A. (1976) Nature (London) 264, 258-260 11 Meyer, C., Wahl, L. M., Stadler, B. M. and Sirang-anian, R. P. (1983)J. Immunol. 131, 911-914 Within the Ig domains conserved p a t t e r n s of sequence are seen and these occur particularly in the/3-strand segments 7. Some of these conserved patterns are c o m m o n to V and C domains while others are specific for each domain type (Refs 7, 8; see below). To argue convincingly that a sequence is related in evolution to Ig domains the following points should be taken into account: (1) The comparisons should be made over segments of sequence of about 100 amino acids. (2) These segments must contain cysteine residues that could form the conserved disulphide b o n d - b e t w e e n /3strands B and F and ultimately this linkage should be established. (3) Secondary structure predictions should be consistent with fl-strands along the sequence as for V and C domains and ideally the circular dichroism spectrum of the molecule should indicate a high content offl-structure and little a-helix. (4) Sequence similarities that fit with the conserved patterns o f Ig domains should be observed. (5) The sequence similarities should be statistically significant. Marchalonis et al. dealt only with point (5) and used statistical analyses based on amino acid compositions and sequence. Analysis of amino acid compositions is inadequate given that the level of identity is only 20-30% and that the sequences involved vary from about 100 amino acids (/32-microgtobulin and T h y - 1 ) to 755 amino acids (poly-Ig receptor). Comparison at the level of domain segments is impossible with this method unless the bizarre procedure of converting sequence to amino acid composition is contemplated. In their statistical analysis based on sequences 1V~,~.~_halonis et al. did find

Immunology Today, voL 5, No. 8, 1984

220

,

A

V

B

,, S-S,

C

C'

C*D

,,,

E

F

F

6"

",, ',, "',, S-S

IZ

A

IB

C

D

Fig. 2. Alignments of V-domain sequences (2V L and 2VH) through beta strands D, E and F in comparison with sequences that are V-like (T receptor, Thy-1 and Poly-Ig R(I) and (IV)) and others that show no convincing similarity to Ig domains (bovine superoxide dismutase and bovine ribonudease).

G

/" E

/B-sfrands abng V and IZ domuins D

V-domain OH

D E

C-domain COOH Fig. 1. Structure of Ig V and C domains At the top a schematic diagram shows the positions offl-strands indicated by letters along a V and C Ig domain along with the position of the conserved disulphide bond. Below this are shown diagrams of the a-carbon backbone ofa V and C domain from an Ig light chain. (From Ref. 16.)

The Ig V-domain sequences and rat Thy-1 sequence can be found in Ref. 9 and the T receptor and poly-Ig receptor sequences are from Refs 3 and 5 respectively. Bovine ribonuclease sequence and structure is presented in Ref. 17 and ribonuclease Cys 26 is aligned with the Cys of/J-strand F as suggested by Marehalonis, J. J. et aL, Immunol. Today Vol. 5, 133-142. An alternative alignment with one gap is also made to give an optimal alignment with NEWM V u. The bovine superoxide dismutase sequence is from Ref. 11 and is aligned on the basis of matching structurally equivalent /]-strand segments to the appropriate V-domain ones and adjusting to give the best obvious identities. The boxes are placed around residues for which three out of the eight sequences in the V-like family are the same and for any position in the other three sequences that matches them. To the right are shown the number of residues in positions that are constant amongst the V-like sequences (8/8 positions) and also the number of boxed residues for each sequence. The numbers of boxed residues for the dis-similar sequences are not directly comparable to those for the V-like ones since the dis-similar sequences were not used to identify boxed residues. A more comparable number is shown in brackets and was computed by adding each dis-similar sequence to the V-like set and counting any position where three out of the nine residues were the same. Residues

at 8/8 positions R H VF~D L G N V T A D K N G V A I V D ~ _ D

P L I S I IBR

KETAAAKFERQHM

T M V V H E K P DSuperoxidedismutase0

TSAASSSN

Boxed residues 3(6) 4(7)

M ribonuclease

KET A AAKF E RQHMD--~S~SAASS G I P GVP

I S A

K

G T S

L R T G D

~]G r r ~ f l ~ ~ l ~ l ~ o 0

rr~o

~N

NQMMKS Y'C

T W D

A TYYCQQYN

I

4(5)

S V~NEW

4

15

D V. EU

4

18 17

K E qEf~.~TJ'l" ' P

DI~T

N T A ¥ , EILISISILIR S E D TIA F'Y~'C A GGY G I VHEU

sNR F L ~ - ~ '131" A s

THRHR~--EJ.NIQP

TI~I~IT

KIL~T

D~TL~L

'WF

v

s

Q P s E P R D SiA ViY F C

Y I t