i Molecular biology of immune diseases and- the immune response

f u ¢ 'aM T. Nepm

The 1990 Miami Bio/ Technoiogy Winter Symposia* attracted an energetic mix of basic academic and applied biotechnol- ogy scientists. Recognizing that the time interval between fundamental immune logical discoveries and their commercial development is growing shorter and shorter, this mee#ng represented an un- usual merging of basic immunological concepts and biotechnology applications. These two areas of interest converged w ~ a focus on the identification of can- didate molecules criti~l in immune- mediated and autoimmune disease.

Thymic select;on studies in transgenic mice

Several speakers illustrated new understanding in the details of the molecular interactions that shape the specificity of the immune response. H. Von Boehmer (Basel) demon- strated the presence of critical intra- thymic decision-making crossroads, where T cells in mice transgenic for an anti-H-Y T-cell receptor (I-CR) were positively selected in female mice and negatively selected in male mice. Cells carrying the transgenic receptor for H-Y recc~gnized in the context of H-2D b were efficiently induced to dif- ferentiate in the thymus of TCR trans- genie mice before, or sometimes even after, negative selection eveni:. I_abelling experiments with DNA pre- cursors ~howed that, in el3 TCR trans- genic mice, the differentiation steps involved in positive selection did not include cell division.

Dennis Loh (St Louis) discussed a related set of experiments in which mice transgenic for a TCR conferring alloreactivity against L d were ana- lyzed. Mature T cells using this trans- gene expressed both the CD8 molecule and the alloreactive TCR, detected with an anti-clonotypic anti- body. Negative selection was docu- mented in strains that expressed an L d

*The Biofrechnology Winter Symposium on qhe Mole~.ular Biology of Immune Diseases and the Immune Response' was held in Miami on 21-26 January 1990. 150

molecule; remarkably, positi,,e selec- tion, that is peripheral expression of CD8 + clonotype-positive cells, oc- curred only in strains that carried at, H-2K b locus in the absence of an L a gene. These data suggest that the K b molecule is itself the positive select- ing element necessary in these mice to lead to maturation and expression of the transgene on mature CD8 + T cells.

By breeding transgenic mice into K b mutant animals, Loh demon- strated that most, but not all, of the K b allelic mutants lost the ability to positively select for expression of tl ~e transgene. These studies of T-cell maturation and transgenic mice powerfully illustrate the close mol- ecular interplay between the TCR, major histoco;~Fatibility complex (MHC) and antigen elements in T-cell repertoire development.

Analysis of TCR usage In non-transgenic models, using

enterotoxin 'su pera ntigens' or MIs al- lelic differences (J. Kappler, Denver), similar dramatic effects are seen, par- ticularly for negative selection, but also for more subtle cases of posi- tive selection. P. Marrack (Denver) illustrated promising technological advances using a quantitative pol- ymerase chain reaction (PCR) tech- nique, which is capable of moni- toring, indiwdual V~ use in human peripneral T cells. That this is a tech-

Immunology Today, Vol. 1 I, No. 5 1990

toire, antigen and the MHC. C. David (Rochester, MN) discussed the murine genetic model for suscep- tibility to collagen-induced arthritis. The MHC contribution to suscep- tibility maps .oredominantly to the I-A region of H-2Q haplotypes. However, some H-2q strains, such as SWR, are resistant to collagen-induced ar- thritis. Since SWR mice lack many TCR genes, backcrossing experiments were performed to show that suscep- tibility segregated with strains which carried both H-2q and a normal TCR genome. This implicates one or more of the ten TCR V~ genes missing in SWR mice in the recognition of the type II collagen epitope in the context of I-Aq.

While this showed the importance of permissive genomic TCR genes, the relevance of reperto:re selection was also detected iq similar backcross experiments: strains which carried susceptible MHE and TCR genes were nevertheless resistant to collagen-induced arthritis if minor lymphocyte stimulating (MIs)-la anti- gens were present with a permiss- ive MHC haplotype. Since MIs-1 a- bearing strains have previously been shown to clonally delete T cells carry- ing V68.1 and V~6, this suggests that very specific repertoire influences control restricted responses needed for susceptibility in th~s model. These studies clearly demonstrate both the specificity and the complexity of the multigenic influences that can influence MHC-linked disease susceptibility.

nology whose time has come was emphasized in the presentation by L. Steinman (Stanford), who used quantitative PCR analysis for V, use to detect very restricted o!igoclonali~ of TCR analyzed in brain autopsy specimens from patients with mul- tiple sclerosis. The search for restric- ted TCR use at the site of an autoimmune lesion has now become the next fervent objective for nu- merous laboratories on the trail of mechanisms of autoimmune acti- vation.

As this search !n human auto- immune disease heats up, one par- ticular animal model now presents a fairly complete picture of likely interactions between TCR reper-

Regulation of gene expression Other topics at the conference

continued this theme of relating new developments in mol~.:ular mechan- isms to immunologic31 dJsedse, for example the growing mderstanding of the role of genetic regulatory el- ements in immunologically relevant gene clusters. Rapid progress was re- viewed not only for regulation of TCR and MHC genes, but also for CD3, perforin and immunoglobulin clenetic control elements. T. Mak (Toronto) discussed a number of regulatory, and developmental changes induced in mice transgenic for a functional TCR gene, C~4. Developmental changes were seen in the form of increased numbers of thymic and peripheral T cells; dendritic epithelial cells were found expressing the transgenic C~4 and endogenous a TCR transcripts, while the adult T-cell distribution of

(~) 1990, Elsevier Science Publishers Ltd. UK. 0167--4919/90/$02.00

Immunology Today, Vol. 11, .~..',:J. 5 1990

TCR ~ and 13 chain genes was rela- tively unchanged.

R. Klausner (Bethesci_t) discussed regulation of T-cell activation medi- ated by the relatively obscure mem- bers of the CD3 complex, the Z;-q chains. Mutagenesis and transfection studies were performed to generate CD3 complexes containing modified TI chains, and compared to cells ex- pressing either a normal O1 hetero- dimer or a i;~ homodimer. Receptors containing -q were shown to couple receptor occupancy with phos- phatidylinositol hydrolysis. Analysis of a T-cell line deficient in ~ ex- pression was correlated with a resist- ance to apoptosis, which was reversed by regaining -q expression. These data potentially indicate a key receptor-associated molecular inter- mediate in the T-cell selection and developmental pathway.

Another area where studies of gene regulation have provided sig- nificant new insight into immuno- logical mechanisms is in the area of the immunoglobulin switch recom- binases. U. Storb (Chicago) summar- ized a se,'ies of observations in mice transgenic for K and heavy chain transge.n.e~ Mutations arising in ex- pressed immunoglobulins in the ma- ture B lymphocy[e~ carried variable region, but not constant region, mu- tat:,ons. Since Ig gene rearrangement is suppressed in these transgenic ani- mals, presumably regulated by feed- back inhibition from the transgene product, this indicates that ongoing rearrangement is not required for the somatic mutation mechanism, and

also that the genomic site for the Ig genes may not be critical for the hy- permutation process to occur. In mice with multiple tandem transgenes, different copies carried different mutations.

More traditional gene regulation studies dealing directly with mechan- isms of immunological mediators were also reported at the me,~ting. E. Podack (Miami) reported that there are a number of putative regulatory elements in the human perforin promoter region which may be im- portant for constitutive expression of pe,'-forin in natural killer (NK) ceils, in contrast to inducible expression in cytotoxic T lymphocyte (CTL) precur- sors. B. Mach (Geneva) reported the characterization of the 'RF-X' DNA bindinq protein. This nuclear protein binds to a consensus regulatory el- ement within the upstream regu!at- Dry region of MHC class II genes: sequencing of the RF-X gene and truncation experiments identified a DNA binding domain of 180 amino acids, related to other helix-loop- helix regulatory proteins. Patients with a congenital class-II deficiency and primary immunodeficiency (bare lymphocyte syndrome) were pre- viously thought to lack expression of the RF-X protein; however, it now appears that this is not the case, but rather that the:, may lack some cofac- tor or interacting regulatory element which participates in RF-X binding to DNA.

Hybridoma technology The importance of natural regulat-

or,/functions involved in mechanisms of immune maturation and develop- ment were also highlighted in the plenary award presentation by Cesar Milstein (MRC, Cambridge). Miistein reminded the conference attendees l of the intricate interplay between el- ~, ements involved in immunoglobulin ~ rearrangements and somatic mutation ~ that occur during the course of B-cell ~' maturation and selection. He spoke ! directly to the biotechnology interest I of the audience with a word of i

caution regarding recombinant and 1 bacterial expression systems which i exploit the germ-line repertoire only. He urged the audience to analyze and use natural mechanisms of regulation i

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and selection rather than asking sur- I rogates like Es.,-herichia coil to re- capitulate such intricate biological I mechanisms in the production of ! specific monoclona! antibodies. J

It was clear from this meeting that ! detailed analysis of molecular recog- i nition and gene regulation in immu- ! noiogy are very rapidly providing significant new insights into mech- ,! anisms of immune response and 1 disease. This meeting highlighted t

numerous rapid developments in this i area - many more than reviewed in ~! this brief report - and helped point 1 the way for new directions in both I basic studies and pha,maceutical i development, f

. . . . ,

Gerald Nepom is at the Immunology and Dia- betes Program, Virginia Mason Research Center, 1000 Seneca St, Seattle, WA 98101, USA

Chemotactic properties of interleukin 1

The recent review b,/F.S, di Giovine and G.W. Duff (Immunol. Tuday, 1990, !, 13-20) represents a nice over- view of the biology of interleukin 1 (IL-1). We would like to comment on the chemotactic properties of IL-1 for T cells. The evidence supporting this concept is based on the work of Miossec et al. 1. Some years ago we tried to repeat these results to h~ve a standard in our experiments on T-cell migration. However, we were unable to demonstrate any chemotactic ac- tivity of IL- 1 for T cells, using recombi- nant IL-le and IL-113 (Genzyme).

Since then two groups have re- ported that IL- 1 was not chemotactic for T cells. Yamaki etaL 2 showed in a rat system that IL-1 was not chemo- tactic for rat T cells, and Zachariae et ai. 3 reported that recombinant IL-1 had no chemotactic properties for human T cells in their in-vitro exper- iments. They stated that it remains to be proved whether the chemotactic response in the work of Miossec et aL 1 can be traced to IL-1, even if IL-1 was highly purified. A clue to these contradictory results may be found in the work of Larsen et al. 4, who suggested that IL-1 induces produc- tion of IL-8, which has been proven to be chemotactic for T cells. Therefore, in our opinion, the concept of IL-1

1990. Elsevier Science Publishers Ltd. UK 0167--4919i90/$02 00

being chemotactic for T cells still re- mains questionable.

Liesbeth H.M. van der Raaij-Helmer Dick M. Boorsma

Department of Dermatology, Free University Hospital, De Boelelaan I 117, 1081 HV

Amsterdam, The Ne[herJ~ nds.

References 1 Miossec, P., Yu, C. and Ziff, M. (1984) J. Immunol. 133, 2007-2011 2 Yamaki, Y., Uede, T., Shijubo, N. et aL (1988) J. Immunol 140, 4388-4396 3 Zachariae, C., Ternowitz, T., Larsen, C.G. et aL (1988) Arch. DermatoL Res. 280, 354-357 4 Larsen, C.G., Anderson, A.O., Appela, E. et aL (1989) Science 243, 1464-1466

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