Mechanisms of self-tolerance

Immunology Today, VoL 9, No. I 1, 1988 --

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The study of immunological tolerance has long been dependent on complex biological assays to detect the presence or absence of antigen-reactive cells. This approach has furnished abundant information about the experimental conditions needed to induce tolerance, but it has often been difficult to decide between explanations based on clonal deletion, cell inactivation or active suppression. This phenomenological deadlock is now giving way, thanks to the application of new investigation tech= niques, particularly those involving antibodies against the T-cell receptor and transgenic animals. The state of the art in tolerance mechanisms was the main theme at a recent meeting of the European Network of Immunology Institutes'. The current rapid progress in the study of clonal deletion depends largely on monoclonal antibodies that can identify T-cell receptors of known specificities. This allows a clear distinction to be made between clonal deletion and other possible tolerance mechanisms. Clonal deletion H. R. MacDonald (Ludwig Institute, Lausanne) reviewed the deletion of V~6-positive T cells in mice expressing the MIsa gene, concentrating on the effect on various subpopulations of thymocytes. As with the prototype (Vl~17a and I-E) system, thymocytes expressing low levels of V~6 (mainly CD4 + CD8 + cells) were unaffected by Mlsa expression, while those with high V~6 expression (mainly CD4 ÷ CD8- and CD4- CD8 + cells) were deleted. The rare but conceptually problematic CD4- CD8- cells expressing the ~13 T-cell receptor were not affected by V~6 deletion in MIsa mice, suggesting that they are not subject to the same tolerization mechanisms as most cells. The anatomical location of clonal deletion was examined by MacDonald in the V~6 system, and by W. van Ewijk (Erasmus University, Rotterdam) using V~17a in transgenic mice that express I-E molecules on various components of the thymus. The consensus result is that self*The 3rd ENIImeetingwasheldin lie desEmbiez, France,from31May-4.June1988

Mechanisms of self-tolerance ..... from NickCrispe reactive T-cell receptors are present in the thymic cortex and at the cortico-medullary junction, and are selectively lost from the inner cortex. This anatomical work is particularly informative when compared with the studies of B. Kyewski (German Cancer Research Center, Heidelberg) on the potential of thymocytes to form aggregates with I-A-negative macrophages, thymus epithelial cells or I-A-positive dendritic cells. These three categories of cell interactions appear sequentially in the regenerating thymus. Antibody against CD3, administered post-natally, did not inhibit the formation of aggregates with I-Anegative macrophages but abrogated thymocyte-epitheliai cell and thymocyte-dendritic cell interactions. The thymocytes in all these types of aggregates were predominantly CD4 + CD8 +. Since I-A-positive dendritic cells are found mainly in the thymic medulla, Kyewski concludes that developing CD4+ CD8 + thymocytes need their antigen receptors to form interactions with cortica! epithelia! cells (although not with I-A-negative macrophages), and retain the CD4+ CD8 + phenotype when they transit to the medulla and encounter I-Apositive dendritic cells. This correlation between thymocyte phenotype, anatomical location and clonal elimination strongly supports the idea that self-tolerance by clonal deletion occurs during interactions between CD4 ÷ CD8 + thymocytes and I-A-positive dendritic cells, which they first encounter at the cortico-medullary junction. In apparent contrast, transgenic mice with a deletion in the EoLtransgene promoter affecting expression in the thymus ('Ay mice'), which are I-E positive on cortical epithelium and mostly negative in the medulla (van Ewijk), nevertheless delete V~17a-positive T cells from the peripheral repertoire. However, these Ay mice express high levels of I-E on a very small number of cells in the medulla. Could these few cells account for the clonal deletion observed? In support of this expla-

(~) 19~8. Elsevier Science Publishers Lid° UK. 0167-4919/881502O0

nation, P. Matzinger (Basel Institute for Immunology~ showed that a few hundred thymic (or peripheral) dendritic cells were sufficient to tolerize in foetal thymus organ culture. Evidently, transgenic systems are not always 'cleaner' than more traditional cellular experiments. In this case, the current gene:ation of promoter-manipulated Ee transgenic mice does not effectively challenge the growing consensus that clonal deletion is the business of medullary dendritic cells. Differentiation pathways Until very recently there was no agreement on the sequence of phenotypes displayed by thymocytes in transit from CD4- CD5-dull CD8precursors to mature medullary-type cells. Several lines of evidence converged at the meeting to give the outline of a workable consensus. In his overall introduction to the thymus session, J.J.T. Owen (Department of Anatomy, Birmingham) raised the thorny question of the function of interleukin 2 (IL-2) in by ing data on the expression of IL-2 mRNA. There is a sharp peak in message synthesis at foetal day 15, precisely in step with the peak expression of Tac (the 55 kDa chain of the IL-2 receptor). In line with a role for IL-2 in differentiation, I.N. Crispe (NIMR, London) reviewed data showing that the earliest precursor thymocytes do not express Tac, but their progeny pass through a Tac-positive stage before acquiring CD4 and CDS. Against this line of argument, various questioners raised the inconsistency of antiTac blocking experiments in foetal thymus organ culture, and the apparent absence of Tac from rat thymocytes. With all these conflicting data, the role of the IL-2 receptor in early T-cell differentiation seems likely to hold immunologists' interest for some time. The next step after CD4- CD8cells seems most likely to be functionally immature CD4-CD8 ÷ cells, which appear in mouse foetal ontogeny at day 16, express the • ,,

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precursor and cortical cell marker J1 ld, and do not express detectable levels of antigen receptors. Similar cells in the rat were studied by T. Hunig (Univ. Munchen), using monoclonal antibodies OX~4 (which separates cortical cells from both precursors and mature cells) and R73 (a novel reagent that recognizes a constant epitope of the rat T-cell receptor). Immature CD4- CD8 + cells in the rat are OX44-; these cells become CD4 ÷ CD8 ÷ after overnight culture. They are large cells, most of which express very low (but clearly above zero) levels of R73. A few, however, express higher R73 and during 48 hours in culture the overall level increases. (Beyond 48 hours the problem of the selective expansion of minor contaminants renders the interpretation of such experiments very difficult.) Most strikingly, crosslinking of the T-cell receptor on CD4- CD8 ÷ OX44- cells inhibits their expression of CD4. While the interpretation of this effect is not yet clear, it seems currently the most promising system in which to study T-cell differentiation in vitro. The place of CD4 ÷ CD8 ÷ cells in differentiation was studied in the V~6 system by MacDonald, and in the V~17a system by D. Pardoll (NIAID, Bethesda). Both workers noted that the clonal deletion of T-cell receptors reactive with two MHC ciass-ii-associated specificities, MIsa and I-E, affects CD8 ÷ as well as CD4 + mature T cells. One obvious interpretation is that negative selection affects the precursors of CD8 ÷ T cells at a stage when they express CD4, and that at this stage all T-cell receptors are screened for self class I and self class II specificity. To test this, mice were injected with anti-CD4 monoclonal antibody, in an attempt to abrogate negative selection. In the V~17a system, C57.BR mice (V~17a ÷ genotype, I-Ek, V~17a- on peripheral T cells) were lethally irradiated and reconstituted with syngeneic bone marrow. Between 9-25 days after irradiation, 2 mg of purified anti-CD4 was injected twice daily. In the thymus all CD4 ÷ cells were antibody coated; CD4 ÷ CD8- cells were ablated, but CD4 ÷ CD8 ÷ cells survived. In the CD4- CD8 ÷ cell subset, Vl~17a expression appeared in treated mice. In the VI~6 system essentially similar results were obtained, except that 330 CD4 ÷ cell elimination was incom-

Immunology Today, Vol. 9, No. ~1, 1988

plete, and surviving CD4 + cells, as one 13 locus is productively rewell as CD8 + cells began to express arranged, the other is not. However, both a loci are in-frame. In gene V~6. Two conclusions follow from transfer experiments, 13 and one these studies. First, CD4 functions as gene transferred both specificity for an accessory molecule in the clonal ovalbumin plus I-A k, and the crossdeletion of self class-II-reactive cells. reaction on I-As. The other ~ gene Second, the precursors of CD4- was translatable and gave surface CD8 + cells are subject to negative ~13 T-cell receptor expression, but selection at a CD4 + CD8 + differen- without a detectable specificity. In tiation stage. These experiments triple transfectants, only the ~ chain finally solve the longstanding prob- used by the donor T-cell clone was lem of the place of CD4 + CD8 + cells expressed with 13on the cell surface. in differentiation. They are indeed The overall conclusion is that allelic the precursors of mature cells, but exclusion of T-cell receptor oL chains they are also the principal targets for may sometimes be a consequence of clonal deletion. The question re- selective association with 13. A mains whether the extensive cell second T-cell clone was found with death in this population is largely two in-frame oL chain genes, but so accounted for by clonal deletion, or far there is no equivalent example whether other selective processes for 13. Proponents of the theory that V~ are also at work. and V~ form an interaction surface, both components of which are vital T-cell receptors: still a few surprises Several groups have been working for s!~ecificity, might be uneasy towards the production of soluble about the close associations of sevmolecules containing T-cell receptor eral VI3 regions with I-E or MIsa specibinding sites. Such molecules might ficity with little apparent regard for be used in studies of receptor-ligand V~,. A still more striking case of V~ affinity, and if crystallization were autonomy was provided by A. Livingpossible the structure of the anti- stone (Stanford) looking at T-cell gen-MHC recognition site would be clones specific for a defined epitope of great interest. Previous attempts of sperm-whale myoglobin in the have been based on building con- context of I-E. Here, four indepenstructs where the variable (V) regions dently isolated T-cell clones all used of T-cell receptor (~ and 113chains are V~8.2, D~2.1, J~2.7 with only a substituted for VH and VL regions of few semiconservative substitutions immunogiobulin. Such constructs at the VD or DJ junctions. Two clones are poorly expressed and assembled, were V~,I, one was V~,4 and one a problem that is usually attributed was undefined but different again. to profound structural differences So is the ~ chain expendable for between VI~ and VH or VL. (V~,-CH some I-E-restricted specificities? And constructs will form chimaeric mol- if so, how does this fit in with the ecules with Ig light chains.) To get idea of an overall geometry for T-cell around this problem, B. Malissen receptor-peptide-MHC interactions? (CIML, Marseilles) made constructs Insight on problems of this kind involving the membrane-proximal, seems unlikely until we know the constant (C)-Iike domains of the T- crystallographic structure of a T-cell cell receptor chains as well as the V receptor. regions, and exchanging the transmembrane region for Ig C domains. Accessory molecules: just glue, or more? It is widely accepted that one prinIn cell lines doubly transfected with a V,~C,,CK and a V~C~CK construct, re- cipal function of the accessory molcipient cells expressed a clonotypic ecules CD4 and CD8 is to bind to marker characteristic of the donor conserved sites on MHC class II or T-cell line and dependent on both class I molecules during antigen recand 13. A secreted molecule has been ognition. But are they there just to obtained, and evidence of binding to increase affinity, or do they particithe specific antigen (H-2K b) is being pate in some more sophisticated sigsought. nalling process? To tackle this probMalissen also spoke about mech~ lem, two groups have been using anisms of allelic exclusion in T cells, recombinant Ly2 (mouse CD8 c~) using a T-cell clone specific for oval- molecules. R. Zamoyska {University bumin in the context of I-Ak, and College, London) described the cellucross-reactive on I-AS. In this clone, lar distribution of the truncated ~'

Immunology Today, Vol. 9, No. 11, 1988 i,ii

chain of CD8 (which lacks a cytoplasmic domain), versus the full length oL chain. Both a and oL' associate with the 113(Ly3) chain but peripheral T cells express almost entirely ~ while early thymocytes express equal amounts of ~ and OL'. Native OLchain could confer CD8 function, but an oL'-Iike mutant chain could not. Apart from native c~, the only other functional construct included extracellular and transmembrane oL, coupled to the cytoplasmic domain of CD4. This suggests that the cytoplasmic domain of Ly2 is important in function, but that the intracellular part of CD4 can substitute. In contrast, J. Gabert (ClML, Marseilles) described CD8 ~ chain constructs in which the transmembrane and cytoplasmic domains had been replaced with the corresponding parts of an MHC class I molecule; on transfection, these molecules functioned as intact Ly2. These apparently conflicting results give support to the idea that there is more to CD8 function than simply increasing the overall avidity of cell interactions, but do not allow us to determine if the prevalence of the truncated (oV) form of the Ly2 chain is functionally important in early differentiation.

Peripheral T-cell inactivation

..... ..... nuw many sig nals are neeaea to activate a mature T cell? And what are the consequences if antigen receptors are ligated in the absence of other signals? These problems were examined in several presentations on the inactivation of post-thymic T cells. In vivo, stable tolerance to allogeneic or semi-allogeneic bone marrow cells could be established without irradiation of the host, using anti-CD4 plus anti-CD8 antibodies (H. Waldmann, Univ. Cambridge). Similarly, anti-CD4 administered with protein antigens gives longlasting T-cell tolerance. Both these effects could be interpreted if presentation of allogeneic class II or antigen plus class II, in the absence of a signal though the CD4 molecule, caused cell inactivation. The cell types on which antigen is displayed may also determine the outcome of recognition. On this topic, H-G. Rammensee (Max-Pianck Institut, TCibingen) discussed the involvement of CD4 in abrogation of the veto effect. Veto is a transient phenomenon following in-vivo pri-

news a,d{ea!uresming with live antigenic cells, in which host MHC class-l-reactive T cells are inactivated by some types of donor ceils, particularly donor CD8 + cells. Veto can be over-ridden by the activation of host cells reactive with donor class II, but is restored by the co-injection of anti-CD4. Lack of appropriate presentation of antigen to CD4 + cells may also account for the tolerance of CSa-deficient mice to their own pro-CSa sequestered in hepatocytes, since they respond •readily to exogenous CSa (from the serum of nondeficient mice) (B. Stockinger, Basel Institute for Immunology). Information about the cell biological consequences of receptormediated T-cell inactivation was reported by R. Schwartz (NIAID, Bethesda). A T-cell clone exposed to antigen plus class II either on heavily fixed antigen-presenting cells, or in liposomes spread on glass, or even to anti-CD3 coated on a culture dish, becomes refractory for an extended period to appropriate stimulation by antigen on normal antigenpresenting ce!ls. The abortive activation process is accompanied by a rise in intracellular Ca2÷ concentration, but no polyphosphoinositide breakdown (signal 1 in the absence of signal 2?). As might be predicted, brief exposure to a calcium ionophore alone mimics the inappropriate presentation of antigen. However, the abortive activation signal could be converted to a stimu-

latory one by the addition of histoincompatible macrophages, arguing for a costimulating signal that could be provided without a cognate interaction (unless, of course, the macrophages take up class II antigens from the liposomes!). None of the widely available interleukins would substitute, but this system may be an important assay for each new one.

Is the thymus solved? With a convincing way of identifying cells subject to clonal deletion, a good working hypothesis for the role of CD4 + CD8 + cells, and a developing consensus on cell lineages, how much longer are we going to be interested in the thymus? One key question that remains is the mechanism of the influence of the thymus on T-cell restriction specificity, a phenomenon attributed by several speakers to 'positive selection', albeit with vociferous heckling from the floor. The technological 'New Wave' has not yet swept away this problem, but monoclonal antibodies that define r-cell receptor restriction specificity cannot be far away. Perhaps they already exist unrecognized, in someone's liquid nitrogen tank? Given the pace of recent advance, we may well find out at the next ENII. ', I. N. Crispe is at the National Institute for Medical Research, Mill Hill, London NW71AA, UK. . . . . . . . . . . . . . . . . . . . . . . . .

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