Immunology Today October 1985

Genes, structures and function of T lymphocyte antigen receptors from Mitchell Kronenberg, Bernard Malissen and Herman N. Eisen

Our knowledge of the genes encoding the T-cell antigen receptors has expanded dramatically in the last year. Much of the new data was presented at a recent E M B O workshop*. The participants attempted to integrate the structural and genetic information with some of the immunological properties of T lymphocytes. A. Williams (Oxford) reviewed the structure of the immunoglobulin (Ig) -homology unit, which is the basic structural building block of Igs, T-cell antigen receptors, at least one domain of the MHC-encoded class I and class II polypeptides, fl2-microglobulin, Thy- 1, Lyt 2 and the receptor for polymeric immuno~0bulins, He described the structure of M R C - O X 2 , a protein of unknown function expressed on brain, thymocytes and some other tissues. M R C - O X 2 is composed of two Ig homology units, one similar to variable and one similar to constant regions. Williams asserted that Ig homology units are not best recognized by statistical analysis of overall sequence similarity, but can instead be identified by the presence of conserved amino acids, such as those around the two cysteine residues found in every domain, and by the presence of alternating hydrophobic and hydrophilic amino acids that give rise to a characteristic pattern of fl strands alternating with short but variable length sequences of amino acids with marked propensity to form reversed turns. He stressed the likelihood of a common armestor for this duplications and diverged to give rise to the contemporary members of the Ig gene superfamily. Williams presented the amino acid sequence of an abundant neuronal glycoprotein from squid that shares several biochemical properties with Thy-1. Although the protein sequenced is not an obvious homologue of the Thy-1 molecule, it nevertheless bears some resemblance to part of an Ig homology unit. *At the Centre d'Immunologie de MarseilleLuminy, April 29-May 3, 1985.

The ligand(s) recognized by the T ceil antigen receptor M. Steinmetz (Basel) discussed the molecular biology of the M H C encoded molecules and the evolutionary forces leading to a polymorphic rather than a polygenic repertoire of MHC-cncoded molecules. Variability may arise from point mutations, recombination, homologous but unequal crossing over, as well as gene conversion, as described for some of the K b mutants. The K and I regions have now been linked on a group of overlapping cosmids that encompass some 600 kilobases. This molecular

analysis enabled Steinmetz to identify a second hot spot of recombination in the M H C , located between the H - 2 K and A~ genes. A hot spot in the E Ogene had been described previously. A major portion of the M H C genes of several inbred mouse strains have now been characterized by restriction enzyme mapping of either cosmid clones or germline DNA. Some regions of the M H C , including both coding and large stretches of noncoding sequences, have more restriction fragment length polymorphisms and are therefore more variable; while other regions tend to be conserved. The cause(s) of this uneven variability have not been determined. After reviewing the recent data showing that mouse fibroblasts transfected with the genes coding for Ia molecules readily stimulate self-restricted and allospecific T cells, B. Malissen (MarseilleLuminy) described a series of exonshuffling experiments performed on I-E molecules. Analysis of the resulting chimeric molecules indicates that both Continued on

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Mast cell differentiation and heterogeneity from A. Dean Befus, John Bienenstock and Judah A. Denburg Major differences between mast cells from different tissues and species have been known for at least 20 years but have been rigorously studied only recently. A recent meeting* focused on the ontogeny and differentiation of mast cells, their functional characteristics and the clinical and biological significance of their heterogeneity. In-vitro and in-vivo studies have defined

two mast cell types in the rat. (L. Enerback, Goteborg). The type represented in the peritoneal cavity also appears to be distributed throughout connective tissues. The other, so-called intestinal mucosal mast cell, is found in the intestinal lamina propria and epithelium, and in the mesenteric lymph nodes; it proliferates rapidly in response *A meeting was held at the Millcroft Inn, Alton, Ontario, Canada, 3-6 February, 1985.

to helminthic infection of the intestine. Formaldehyde fixation inhibits the binding of dyes to the intestinal mucosal mast cell but not to the peritoneal mast cell, and specific fixatives or elaborate staining procedures must be used to stain intestinal mucosal mast cells in the rat. These differences in sensitivity to formaldehyde fixation appear to relate to the glycosaminoglycan content of the mast cell. In the rat, this sensitivity to different fixation procedures accurately predicts functional distinctions among Continued on p.

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282 Mast cell--contd from p. 281

mast cell populations. This has not been studied adequately in other species. Factors i n m a s t cell o n t o g e n y T h e ontogeny of the mast cell and delineation of factors which stimulate growth and differentiation of mast cell subpopulations or related granulocytes has become a pivotal topic in understanding heterogeneity. M u r i n e mast cell lines dependent on interleukin 3 (IL-3), primarily a product of activated T cells, have been studied extensively (J. Schrader and colleagues, Melbourne). This cultured mast cell is considered by some to be analogous to the rat intestinal mucosal mast cell. Their precursors are common in the murine intestinal mucosa and bone marrow. Indeed, serum IL-3 levels rise transiently in mice with some parasitic infections or graft-versus-host-like reactions. Immunodetection of mouse IL-3 should enhance our understanding of the role of the mast cell/IL-3 system in-vivo (J. Ihle, Frederick; Schrader). T h e precursors of intestinal mast cells in the mouse are locally more common during nematode infection or graftversus-host reactions and migrate from the intestine through the mesenteric lymph nodes, thoracic duct and back into the intestinal mucosa where they complete their development (D. GuyGrand, Paris). The presence of an Lyt 2 + T cell is essential for elaboration of a factor, presumably IL-3, which induces intestinal mast cell proliferation in the mouse. Studies in mast cell deficient ( W / W v) and mast cell replete ( + / + ) littermates suggest that there may be another factor which influences precursor localization in the intestine. Studies on rats infected with the nematode Nippostrongylus brasiliensis (D. Haig and E. Jarrett, Glasgow) have shown that mesenteric lymph node cultures produce a worm antigendependent factor which stimulates mast cell growth and differentiation in vitro from bone marrow precursors. The interesting feature of mast cells which proliferate u n d e r these circumstances is that they contain the rat mast cell protease II ( R M C P - I I ) , which is found only within rat intestinal mucosal and bone marrow mast cells, and not within peritoneal or dermal mast cells which contain a protease termed R M C P - I . Granulated leukocytes from the rat intestinal epithelium bear the phenotypic markers O X 8 and W3/13 (G. Mayrhofer, Adelaide) which are not found on intestinal mast cells; the latter, as well as bone marrow or peritoneal mast cells, probably bear the surface marker OX7. These observations ques-

tion the relationship of intra-epithelial granulated lymphocytes to mast cells. Mayrhofer's studies stress that, at least with present information, there is more value to anatomic localization studies than to surface phenotype analysis in classification of mast cell subtypes. Y. Kitamura (Osaka), using histochemical techniques in the mouse as markers of mast cells analogous to rat intestinal mast cells or peritoneal mast cells, reported adoptive transfer studies which suggest that these two mast cell types may differentiate from one type to the other, depending upon environmental conditions. While a variety of murine retroviruses induce transformation and IL-3 dependency in murine mast cells, Abelson virus is an exception, transforming the cells but allowing them to become autonomous and independent of IL-3. They do not produce IL-3 in an autocrine fashion and the mechanism of their transformation and IL-3independence is unknown (Ihle). H. Ginsburg (Haifa) described studies on mouse mast cells grown on monolayers of embryonic skin fibroblasts, in which contact of fibroblasts and maturing mast cells was apparently necessary for mast cell maturation. Fibroblast supernatants or supernatants from fibrosarcoma cell lines could not substitute for direct contact on the fibroblast monolayer in this culture system. Studies of the ontogeny of h u m a n mast cells have lagged behind those in rodents. T. Ishizaka (Baltimore), using cultured cord blood mononuclear cells in the presence of IL-2-depleted conditioned medium from phytohemagglutinin (PHA)-stimulated lymphocytes, has recovered highly enriched populations of h u m a n polymorphonuclear basophils which bear the characteristics of peripheral blood basophils. These include high affinity IgE receptors, glycosaminoglycans and intact functional pathways of mediator release. Basophil precursors are nonadherent, n o n - B / n o n - T cells~ Peculiarly, labelled arachidonic acid could not be incorporated into metabolites in these cultured cells; this requires further clarification since such cells may represent only partially differentiated h u m a n basophils. A common basophil/eosinophil progenitor, responding to separate leukemic T-cell derived factors which may control terminal differentiation, may, be different from a mast cell progenitor (J. Denburg, Hamilton). The latter gives rise to metaehromatic cell colonies which lack the eosinophil/ basophil major basic protein (MBP) or

Charcot-Leyden crystal protein. These observations are consistent with the demonstration that mast cells in biopsy specimens from patients with urticaria pigmentosa, a localized proliferation of mast cells in the skin, lack M B P (G. Gleich, Rochester). Differentiation events in h u m a n hemopoietic stem cells are random (stochastic) processes, and differentiation factors, presumably present in the microenvironment in vivo, act upon stochastically committed cells to produce a given cell lineage (M. Ogawa, Charleston). B. Stadler and A. de Weck (Bern) have separated a hemopoietic IL-3-1ike factor from h u m a n mitogen-stimulated peripheral blood mononuclear cells and a h u m a n urinary bladder carcinoma cell line. This factor stimulates the proliferation of a subclone of a murine, IL-3 dependent mast cell line, as well as h u m a n bone marrow mast cells, but not basophils, in suspension cultures. Thus, h u m a n basophils and certain mast cells may represent distinct lineages and account for some aspects of mast cell heterogeneity in tissues. The ultimate fate of h u m a n peripheral blood basophils is still not clear; neither is the ontogenic relationship between basophils and mast cells, although most evidence suggests that basophils and mast cells represent distinct cell lineages. B i o c h e m i s t r y a n d histology H e p a r i n is the predominant proteoglycan in rat peritoneal mast cells but is absent from rat intestinal mucosal mast cells. From collaborative investigations (with T. Lee, D. Befus;J. Bienenstock, Calgary, Hamilton; Haig) K. Austen and R. Stevens (Boston) presented evidence that the predominant proteoglycan from isolated fi'om cultured rat intestinal mast cells is not chondroitin sulphate E as in the mouse cultured bone marrow mast cells, but seems to be a unique proteoglycan, chondroitin sulphate di-B. Although this remains to be confirmed, the amounts of this unique proteoglyean present in isolated and cultured cells differ, and other sulphated macromolecules within these cells that may represent proteoglycan remain to be identified. Interestingly, the rat basophilic leukemia (RBL) cell line which has been widely employed to study mast cell/basophil properties, also appears to contain chondroitin sulphate di-B. Moreover, R B L cells also contain the serine protease, R M C P - I I , hitherto thought to be restricted to the intestinal mucosal a n d cultured bone marrow mast cell. This new information about proteoglycan content and protease subtype in R B L cells from the rat suggests

Immunology Today, vol. 6, No. 10, 1985 that these cells may be transformed intestinal mucosal mast cells. An alternative explanation is that basophils contain what otherwise appears to be a mast cell marker. If true, this would suggest a close relationship between these mast cells and basophils. Some caution must be exercised in extrapolating results from studies of transformed cells which have been maintained in culture for such long periods of time. In the rat, the intestinal mucosal mast cell type has been identified only in the intestine and draining mesenteric lymph nodes. Mast cells throughout the respiratory tract, including the trachea, bronchial tree and parenchyma are analogous to the peritoneal mast cell, except in the lamina propria underlying the cartilaginous portion of the trachea, where there are two histochemically distinct mast cell types (T. Goto, Tokushima; Befus and Bienenstock). Whether the cell type histochemically analogous to intestinal mucosal mast cells is also functionally similar remains to be established. Bleomycin treatment of normal and nude rats, in doses which regularly produce lung fibrosis, stimulated a massive increase in mast cell numbers. In keeping with this, mast cells isolated from the fibrotic lung parenchyma of bleomycin-treated rats have histochemical and functional properties analogous to peritoneal mast cells in the same animals. In the lungs and intestines of monkeys there are two histochemically distinct populations and the histamine content of cells from the lung and intestine varies (K. Barrett and D. Metcalfe, Bethesda). Two mast cell populations distinguished by formaldehyde sensitivity can also be identified in h u m a n lung and intestine (Befus), although their functional differences remain unknown. However, in h u m a n nasal and lung biopsies before and after challenge with specific antigen there was no ultrastructural evidence for mast cell heterogeneity (M. Kaliner, Bethesda), nor did functional and biochemical studies of isolated h u m a n lung and intestinal mast cells reveal subsets of mast cells (L. Lichtenstein, Baltimore). Lung mast cells resembled those from the intestine, and differed from peripheral blood h u m a n basophils in histamine release and certain aspects of arachidonic acid metabolism. In contrast, H. Otsuka (Hamilton), Befus, D e n b u r g and J. Dolovich (Hamilton) reported that among the histochemically distinct mast cell populations in h u m a n nasal mucosa, only those which were sensitive to formaldehyde appeared to be responsive to the topical steroid, bedometasone diproprionate, and to correlate with patient

283

Nomenclature Meeting participants agreed that a recommendation for an appropriate nomenclature is not yet possible, although workers in this field should abandon the use of such general and potentially confusing descriptive terms as 'mucosal', 'connective tissue', 'typical' or 'atypical' mast cells and 'P cells' in favour of an indication of derivation and as full a description as possible in terms of organ source, proteoglycan content and other characteristics. A suggestion to employ the terms 'mast celllike' or 'basophil-like', using the suffLx to denote Kkely relationship, was debated, but not uniformly adopted. It was agreed that the use of cultured cells to define mast cell types must be cautious, owing to effects of culture, such as acquisition or loss of factor dependency, and selection of certain cell types. T h e issue o f ' t h y m u s dependency' is i m p o r t a n t b u t difficult to resolve since some of the factors important for promotion of mast cell growth, such as IL-3, are not produced only by T cells and may be responsible for proliferation and/or maturation. The thymic dependence or independence of peritoneal mast cells is not yet dear. Nevertheless, two types of mast cells are distinguishable in rats on the basis of staining, fixation, functional attributes and factor dependency. T h e bone marrow derived mast cell seems phenotypically similar to the intestinal mucosalmast cell, but not to that found in the peritoneal cavity. As for heterogeneity in other species, including man, the evidence is, at best, inconclusive. There is no good evidence yet for two or more types of mast cells in humans. All investigators, when writing about mast cells or basophlls, should delineate the following: (1) species of origin; (2) whether the cells have been cultured and, if so, culture conditions; (3) organ source; (4) proteoglycan content, where it is chemically defined (in this regard 'heparincontaining cells' as a description was considered acceptable; (5) where possible, response to secretagogues or, more specifically, secretion of leukotrienes and prostaglandins in response to anti-IgE or antigen; (6) histochemistry, especially the conditions of both fixation and staining. A meeting may be held during the 6th International Congress in Toronto next year to consider a nomenclature (contactJ. Bienenstock for details). symptoms. Denburg further discussed evidence for the contribution of hemopoietic progenitors to tissue accumulation of basophils and, possibly, nasal mucosal mast cells: circulating basophil/eosinophil colony-forming cells are raised in atopic individuals generally but decreased during the ragweed pollen season in ragweed-allergic subjects, at a time when nasal mast cell counts rise significantly. Interestingly, the metachromatic cells in such hemopoietic colonies are, like steroid-responsive nasal mast cells, sensitive to formaldehyde. E. Goetzl (San Francisco) reported that substance P and somatostatin can be detected in various mast cell/basophil preparations as well as in other cells of the myelopoietic lineage. These far reaching observations will stimulate much investigation. A distinct protease released from intestinal mucosal mast cells during anaphylactic reactions and parasite infection in rats is a powerful marker of intestinal mast cell activity (H. Miller, Edinburgh) and the existence of similarly distinct proteases in h u m a n mast cell populations is an exciting possibility with obvious diagnostic implications. In the rat, corticosteroid

treatment produces a dramatic decrease in intestinal mast cells within 24 h and a precipitous decline in intestinal R M C P - I I levels, with no obvious change in peritoneal mast cells. The mechanisms underlying these acute changes and the fate of mast cells and protease remain unknown, although these represent fundamentally important questions should glucocorticoid treatment in m a n have similar mast cell subtype-specific effects, as suggested by the studies of h u m a n nasal mast cells discussed above. M. Beaven (Bethesda) and coworkers showed that mast cells from the peritoneal cavity of rats separated by elutriation differ in cell size, histamine content and synthetic activity. These differences appear to relate to maturational changes and must be analysed in the context of comparisons between peritoneal and intestinal mucosal mast cells. S. Wasserman and D. M a r q u a r d t (San Diego) suggested that in rodent mast cell populations there may be interactions in which corticosteroids or other drugs alter adenosine binding in cultured cells. Such investigations would provide new and interesting ways to analyze bio-

Immunology Today, vol. 6, No. 10, 1985

284 chemical and functional aspects of mast cell heterogeneity. I. Pecht (Rehovot) described a method in which two plasma m e m b r a n e components of mast cell origin, namely the Fc receptor for IgE and the cromolyn-binding protein, could be manipulated for study. Somehow these two m e m b r a n e glycoproteins interact to form an ion conducting component in the bilayer. T h e use ofmonoclonal antibodies to the cromolyn-binding protein, together with in-vitro manipulation in bilayers, promises to uncover new and significant aspects of mast cell biology and mechanisms underlying heterogeneity in secretagogue and antiallergic responsiveness. A. Soil (Los Angeles) reviewed the complex series of interactions associated with histamine release and acid secretion in the canine fundic mueosa. The regulatory processes underlying

Genes, structures--conM from p. 281 the serological and T-cell recognition sites of I-E map to the polymorphic residues located inside the/~1 domain of E~. In collaboration with N. Shastri, Malissen and colleagues demonstrated that Ia-expressing mouse fibroblasts (L cells) are compromised in their ability to process some native proteins such as hen egg lysozyme. They compared the ability of Ia-positive L cell transfectants and splenic antigen presenting ceils to stimulate a cloned T-cell line with either native lysozyme or Tll, a tryptic peptide derived from lysozyme that contains the antigenic determinant recognized by the clone. The splenic antigen-presenting cells could effectively present both molecules to the T-cell clone. I-A transfected fibroblasts, however, could present only the peptide, not the native molecule. These results are similar to those obtained with antigen-presenting cells that have been pretreated with either lysosomotropic drugs or aldehydes; such cells are unable to present native protein antigens although they can present peptides derived from these antigens. Collectively these results suggest that many antigens need to be processed before they can stimulate T cells, and that drugtreated antigen-presenting cells or I-A transfected fibroblasts are less capable of carrying out this processing. J. Klein (Tfibingen) presented experiments aimed at questioning the necessity for antigen processing. He described the production of synthetic lipid vesicles with inserted Ia molecules. Protein amigens can be coupled covalently to the liposomes. These liposomes were then shown to stimulate the proliferation of cloned T cells and T-cell hybridomas in

gastric function, the role of histamine and the ontogenetic and functional relationships between histamine-containing cells in the gastric mucosa and mast cell subtypes is a promising area of investigation. Similarly, the report by A. Froese (Winnipeg) and co-workers of a unique receptor for IgE on rat basophilic leukemia cells induced by mycoplasma infection, promises to provide a new tool to manipulate gene expression in mast cells/basophils and to investigate heterogeneity and functional properties of different surface glycoproteins.

rule, and phenotypic distinctions based on stage of cell cycle, state of maturation/development or activation/ recovery, and the influences of the microenvironment are ubiquitous. A n understanding of this heterogeneity will require answers to many questions about the ontogeny, stability and clinical relevance of functionally distinct mast cell populations. F~

Conclusion

A. Dean Befus is at the Gastrointestinal Research Group, Department of Microbiology and Infeetious Diseases, Health Sciences Centre, University of Calgary, Calgary, Alberta, T i N 4NI, Canada; John Bienenstock is at the Department of Pathology, McMaster University," andJudah A. Denburg is at theDepartment ofMedicine, M e , aster University, Hamilton, Ontario L8N 3Z5, Canada.

It became clear at this meeting that functional heterogeneity may be far more extensive t h a n we currently recognise. Its meaning, however, is unclear. Extensive heterogeneity is obvious because genotypic differences are the an antigen-specific, Ia-restricted manner in the absence of antigen-presenting cells. Thus Klein concluded that in spite of the existence of T-cell clones with a preference for partially digested antigens, the notion that class IIrestricted T cells are only capable of recognizing processed antigen is not generally valid.

The structure of the T cell antigen receptor The a/fl heterodimer that constitutes the T-cell antigen receptor is now a familiar structure to most immunologists. C. H a n n u m (Denver) described some of the technical problems that had to be overcome in order to achieve the first partial amino acid sequence of an achain polypeptide from a h u m a n T-ceil tumor. He also described an experiment from the Denver group which suggests that the fine specificity ofa T cell for both antigen and self-MHC can be correlated with the a/~ heterodimer. The DO- 11.10 T-cell hybridoma recognizes chicken ovalbumin @OVA) along with.I-A a and cross-reacts with cOVA plus I-A b, as well as having a weaker ailoreaction against I-A b. The cOVA peptide capable of stimulating this cell line includes amino acids 323-339 and its receptor can be bound by an anti-clonotypic monoclonal antibody. When a group of 160 T hybridomas from an animal primed wkh cOVA in the context of I-A a were screened for cross reactivity with cOVA plus I-A b, one positive hybridoma was found. This cell, like D0-11.10, is specific for c O V A and also bound the original D0-11.10 anti-clonotypic antibody. Therefore, specificity for an antigen and for an M H C restriction element

The proceedings of the meeting will be published by Raven Press and dedicated to Ellen Jarrett, who died in December 1984.

(I-A d, cross-reactive with I-A6), and the presence of serologic determinants on the a/f3 heterodimer rarely found even on cOVA-specific T ceils, were all correlated.~ L. Samelson (Bethesda) demonstrated that the murine a/f3 heterodimer is physically associated with other cell-surface molecules. With an anti-clonotypic antibody, he coprecipitated four polypeptides of 32, 25, 23 and 22 kDa under non-reducing conditions. This set of polypeptides bears some similarity to the T3 molecular complex that comodulates and coprecipitates wkh the a/fl heterodimer on h u m a n T cells. There are, however, significant differences between the mouse proteins and the subunks of T3. For example, the routine 32 kDa molecule is a dimer, yielding a single 16 kDa band on reduction and SDS gel electrophoresis, and the 23 kDa species migrates as a 27 kDa species on reduction. Samelson has shown that this 23 kDa molecule is phosphorylated at a serine amino acid as early as 10 min after antigen- or lectinmediated activation of a T-ceil hybridoma, suggesting that this phosphorylation could be part of the signaling process for T-cell activation. T cell antigen receptor genes The structure of the mouse and h u m a n germline/3 chain genes was reviewed by M. Kronenberg (Pasadena), T. M a k (Toronto) and T. Rabbitts (Cambridge, UK). In both species the gene organization is quite similar. There are two nearly identical and closely linked C/3 genes, each preceded by a cluster of J/3 gene segments. A D 0 gene segment can be found 5' of eachJ~ gene segment cluster. C o genes are re-