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CD8+ T lymphocytes in intracellular microbial infections
ImmunologyToday,VoL 9, No. 6, 1988
is very different from using them to target toxins or drugs. In fact, whereas the latter approach only allows a transient cytotoxic effect, the targeting of antigens exploits all the potential of the immune response itself. What are the effects that might follow antigen targeting? T -'~l!sare well equipped with a variety of toxins and poisons: antigen-specific class ii-restricted T cells can kill tumor cells that are presenting the antigen and also (in some instances) innocent bystander tumor cells. Such bystander lysis may solve the problem generated by the appearance of Id-negative or class II-negative tumor variants; however there is little evidence that bystander lysis might be operative in vivo. One system that by definition is operative in vivo is delayed-type hypersensitivity, which is mediated by class II-restricted T cells and which, in some cases, correlates better than cytotoxicity with the rejection of grafts or tumors. A second useful in-vivo effect is the generation of T-cell help. There are examples where the response against weakly immunogenic determinants can be elicited only if another (helper) determinant is corecognized on the same cell 3.4. It is possible that a similar . . . . . . . effect of 'intramolecular help' may be elicited by target-
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The Basel Institute for Immunology was founded and is supported by F. Hoffmann LaRoche& Co., Ltd, Basel, Switzerland.
References
10chi, A., Worton, K.S.,Woods, G., Gravelle,M. and Kitagami, K. (1987) Eur. J. ImmunoL 17, 1645 2 Lanzavecchia,A., Abrignani, S., Scheidegger,D. etaL (1988) J. Exp. Med. 167, 345 3 Keene,J.A. and Forman,J. (1982~J. Exp. Med. 155, 768 4 Mitchison,N.A. (1983) Transplant. Proc. 15, 2121
+ T lymphocytesin intracellular microbial infections
Current theories on infections with intracelMar bacteria, protozoa, and fungi support the notion that MHC class II-restricted CD4+ T cells are activated and that resistance depends exclusively on this T-cell subset. Here, Stefan Kaufmann summarizes recent evidence that in these infections MHC class I-restricted CD8+ T ceils are also activated, and participate in protection; they appear to lyse infected target cells and produce gammainterferon in vitro. The possible role of these CD8+ T cells during intracellular microbial infections is discussed. A major challenge for the immune system is to combat bacterial, parasitic, mycotic and viral infections. While many infectious agents can be aoequately dealt with by antibody-mediated mechanisms, others have chosen an intracellular habitat which protects them quite efficiently from humoral host defence 1.2 (see inset opposite). During intracellular replication, antigenic moieties of these pathogens appear on the surface of infected host cells which can then be recognized by T lymphocytes. In this way infected host cells can be discriminated from uninfected cells and appropriate effector mechanisms aimed at the eradication of infection can be initiated. For this purpose, T cells have two principally different mechanisms, namely their activation and ability to lyse infected target cells3. Cytolysis seems to be adequate for the defence against viral infections, whereas activation is thought to be more appropriate for defence against nonviral intracellular pathogens. In this review, recent
168
ing to the tumor cell a foreign antigen that would serve as a helper determinant to generate a cytotoxic response against tumor-specific antigens. The two kinds of responses - against targeted antigen and against tumorspecific antigen - would allow the tumor little chance to escape the immune response by generating negative variants. Nature has obviously put a lot of time and effort into developing optimal strategies for the immune system. If we can exploit these strategies it would be a pity not to try.
Department of Medical Microbiology and Immunology, Universityof Ufm, ObererEselsberg,D-7900 UIm, FRG.
Stefan H.E. Kaufmann t~lnir~anr ;c ~,,~,,,~, r~r~enn+~rl e-, ,~,.-,^t-÷ ~LIIC]L - h . + ~..~LUI~LIt.. ..,,~.^h.4-; . . Iil~.llCiii. . i~.-,.v,,,,.,,,.e ,~ to ,)LJ~j~j~aL isms may also play a role in the defence against intracellular bacteria, fungi and protozoa. For simplicity the term intracellular microbes will be used for these pathogens to distinguish them from viral agents.
Different types of antigen presentation In contrast to antibodies which bind free antigen directly, T cells recognize foreign antigen in conjunction with self structures encoded by the major histocompatibility complex (MHC) on the surface of host cells. Foreign antigens can associate with either MHC class II (HLA-DR, DP, DQ in man, H-21A, IE in mice) or class I (HLA-A, B, C in man and H-2K, D, L in mice) structures. Virtually all host cells express MHC c!ass I molecules and hence can present antigens recognized in their context. It is generally assumed, however, that antigen association with class I structures is restricted to proteins that are newly synthesized within the cell. With respect tb infectious agents, only products of viral genes qualify as 'endogenous antigens'4. In contrast, antigens that are taken up via endocytosis are termed 'exogenous antigens'. These include antigens from bacterial, protozoan and fungal agents residing within the phagolysosome of an infected host cell. These exogenous antigens are degraded by lysosomal enzymes, and class II molecules can act as shuttle molecules which pass antigenic peptides to the cell surface. 1988, Elsevier Publications, Cambridge
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ImmunologyToday,Vol.9, No. 6, 1988
Intracellular pathogens A group of pathogenic bacteria, fungi and protozoa have developed means that allow them to live within cells of their host. But besides their common predilection for an intracellular habitat, these pathogens vary markedly. Intracellular pathogens can be divided into two groups according to the type of host cell they target: one group preferentially uses professional phagocytes (more precisely, cells of the mononuclear phagocyte system), whereas the others primarily inhabit nonprofessional phagocytes (i.e. one or more types of host cells other than mononuclear phagocytes or granulocytes). This separation is not only convenient but also reflects the preferred use of different evasion mechanisms. However, this division is not stringent and intracellular pathogens should rather be seen as a spectrum with these two types as extremes. Professional phagocytes are particularly well equipped to engulf, kill, and degrade invading microbes. Therefore, selection of these host cells means emphasis on intracellular survival strategies rather than on entry mechanisms. Microbial entry can rely on conventional phagocytosis and the pathogen may j-st modulate this process. However, the inside of a macrophage represents an aggressive milieu and hence requires potent evasion mechanisms. These include inhibition of phagosome-lysosome fusion, resistance to reactive oxy-
gen metabolites and lysosomal enzymes. Once activated by lymphokines, however, the intracellular milieu of an infected macrophage becomes increasingly hostile and many - although not all pathogens may finally die. Pathogens of this type include the etiologic agents of tuberculosis, Mycobacteriurn tuberculosis and M. boris; M. leprae, the cause of leprosy; M. lepraernurium, the agent of rat leprosy; Listeria monocytogenes which is increasingly recognized as being responsible for many food-borne diseases; Legionella pneumophila which causes a severe pneumonia called Legionnaires disease; and Leishmania spp. the agents of different forms of leishmaniasis. The second type of intracellular microbe primarily inhabits nonprofessional phagocytes and hence is forced to use its own entry mechanism which often requires energy expenditure by the pathogen. Once inside a nonprofessional phagocyte, the milieu is less hostile than in a professional phagocyte. Therefore, similar but less refined evasion mechanisms may be employed by these pathogens. Many microorganisms belong to this group including Chlamydia sp., Rickettsia sp., --~':" plasmodia a,.u --'~ Trypano,,,,',,a,,a soma sp. Mentioned in this review are Rickettsia spp., the agents of different forms of typhus, which readily enter and replicate in endothelial cells; the lymphocyte-inhabiting protozoan, Theileria parva,
Class II molecules are expressed by a limited number of host cells - primarily B lymphocytes, dendritic cells and mononuclear phagocytes - and only these cells should be able to present microbial antigens adequately. Accordingly, in the case of class I presentation the type of the infectious agent is limiting, whereas in class II presentation it is the type of infected host cell. The two modes of antigen presentation have their counterpart in two phenotypically distinct T-cell subsetss. T cells expressing the CD4 molecule (T4 in man, L3T4 in mice) recognize antigen in association with MHC class II molecules, whereas CD8 + T cells (T8 in man, Lyt2 in mice) are specific for antigen in the context of MHC class I structures. It has been suggested that the segregation into two distinct T-cell subsets reflects the different requirements for defence against intracellular viral and microbial infections 1,3. Class I-restricted cytolytic T cells (CTL) are appropriate for defence of viral infections since, due to the variety of host cells that can be infected by
which causes a fatal cattle disease called East Coast fever; and mouse malaria plasmodia, which at different stages of their cycle inhabit hepatocytes and erythrocytes. It should be emphasized, however, that the separation into two types of intracellular pathogens oversimplifies reality. A single organism often preferentially, although not exclusively, uses one type of host cell. At one pole Rickettsia sp., which normally infects nonprofessional phagocytes, is also found in macrophages. At the other pole, M. leprae not only inhabits macrophages but also Schwann cells. L. monocytogenes, a widely used model organism for facultative intracellular infections, can also enter endothelial cells and it appears that for these targets only, the pathogen employs an active penetration mechanism. Intracellular microbial pathogens can be divided into facultative pathogens (able to live both inside and outside the host cell) or obligate pathogens (which only survive inside the cell). Inhabitants of both professional and nonprofessional phagocytes comprise either type of pathogen. The tubercle and leprosy bacilli are both mycobacteria which preferentially replicate in professional phagocytes with the former being a facultative and the laker an obligate intracellular pathogen. It is possible that the preferred choice of host cell, as well as the ability to survive extracellularly, have great impact on the success of a given immune mechanism.
viruses, the MHC restriction element required must have a similarly wide distribution. In addition, lysis of infected host cells before assembly of viral progeny effectively blocks viral replication. Furthermore, newly synthesized viral antigens can associate with class I molecules. In contrast, microbial antigens should fail to associate with class I molecules and hence the host depends on CD4÷ class II-restricted T cells to combat these infections. Nonactivated mononuclear phagocytes provide the major habitat for many of these pathogens. These cells express class II molecules and can be activated by lymphokines to gain antimicrobial effector functions. Thus, two apparently satisfactory mechanisms for combating different types of intracellular pathogens exist. However, a number of questions remain unanswered. Many microbes infect host cells other than mononuclear phagocytes, and these cells may not express MHC class II antigens nor become microbicidal after lymphokine stimulation. Furthermore, mononuclear phagocytes vary
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Immunology Today, Vol. 9, No. 6, 1988
in their antimicrobial potential and some may not be adequately equipped for intracellular killing. It is hard to envisage how, under these circumstances, CD4 ÷ helper T cells alone are sufficient for effective protection. Rather, one must assume that some other as yet unknown factor is required. Several lines of evidence suggest that CD8 ÷ class I-restricted CTL play a more important role in the resistance to microbial pathogens than hitherto thought. As a corollary, exogenous antigens should have the potential to associate with class I MHC molecules (see Fig. 1). CD$+ T cells and antimicrobial protection Zinkernagel and co-workers 6 were the first who analysed genetic restriction of cell-mediated antimicrobial resistance. They found that adoptive protection against murine listeriosis is class II restricted and this study has been taken as strong support for the notion that acquired resistance to intracellular bacteria is a function solely of helper T lymphocytes. However, Cheers and Sandrin 7 later provided evidence to the contrary by showing that protection against this pathogen is class I restricted. Earlier studies performed in the rat had also indicated resistance to listeriosis to be class I restricted 8. Other studies have shown that the T cells relevant to antilisterial protection belong to both the CD4 ÷ and CD8 + T-cell subsets9-12. In antilisterial resistance, it has been suggested that CD8 + T cells control granuloma formation at the site of bacterial implantation 13. Together, these findings are consistent with the notion that not only CD4 ÷ class II-restricted, but also CD8 + class I-restricted T lymphocytes are involved in the acquisition of antilisterial resistance. This idea is not restricted to listeriosis but also seems to hold true for several other intracellular microbes 14-19. Although in the different systems, and depending on the stage and severity of the disease, the relative contribution of CD4 + and CD8 ÷ T cells to resistance varies, some effect of both subsets is commonly found. Recent studies by different groups also provide strong evidence for a protective role for CD8 + T cells in murine malaria2O-22.
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Cytolytic activities of CD8÷ T lymphocytes In viral infections the best analysed function of CD8 + T cells is their capacity to lyse virus-infected target cells in vitro: so do CD8 ÷ T cells arising during intracellular microbial infections function similarly? CTL specific for microbial antigens have now been identified in several systems. CD8 ÷ T lymphocytes from mice with listeriosis express high cytolytic activity towards L. monocytogenesinfected bone marrow macrophages23. 24. As is the case in most viral infections, restimulation in vitro is required for the expression of optimum killer activity. Similarly, CD8 + T cells from mice primed with Mycobacterium leprae, M. tuberculosis or M. bovis specifically lyse bone marrow macrophages primed with the homologous agent 25,26. Although in these studies bulk cultures of T cells did not show stringent MHC restriction, on the clonal level class I-restricted T cells could be identified. T cells from mice infected with Rickettsia sp. can also specifically lyse target cells infected with the homologous organism271 Lysis was found to be H-2 restricted; unfortunately neither the phenotype nor the class restriction of these T cells has been determined. Theileria parva,
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Fig.1. Simplifiedschemeof antigenprocessing.Thesolidlinesindicatethe two generallyacceptedroutesof exogenousand endogenousantigens;the dotted line indicatesthe putativewayof an exogenousantigento the classI MHCgene product. which inhabits T lymphocytes of cattle, activates CD8 ÷ T cells capable of lysing infected T cells in an antigenspecific and class I-restricted way 28. Collectively, these data show that antigen-specific, class I-restricted CD8 ÷ CTL are generated during infection with a variety of intracellular microbes. Other functions of CD8÷ CTL CD8 + T-cell lines with reactivity to L. monocytogenes, M. leprae or M. tuberculosis fail to secrete interleukin 2 (IL-2); however, many lines produce gamma-interferon (IFN-~) after costimulation with antigen plus accessory cells and exogenous IL-2 (Refs 24-26). Also, CD8 ÷ T cells from mice infected with M. boris and challenged with purified protein derivative produce IFN-~/(Ref. 29). This cytokine is thought to be the major mediator of macrophage activation, and IFN-~,-containing T-cell supernatants can indeed activate tuberculostatic macrophage functions. Adoptive transfer of CD8 + T cells partially protects mice against malaria, and protection is reversed by administration of IFN-'y-neuLralizing monoclonal antibodies 2°. Taken together, these findings argue for a direct or indirect role for IFN-'y in the protection afforded by CD8 ÷ T lymphocytes. Mycobacteria-reactive CD8 ÷ T cells seem to have an additional mechanism of resistance. Coculture of CD8 ÷ T cells with M. bovis-infected bone marrow macrophages induces tuberculostasis apparently independent from IFN-~, secretion and probably dependent on target cell lysis26. CD4 ÷ T cells with reactivity to intracellular microbes have also been shown to secrete IFN-,yand they do so in the absence of exogenous IL-2 (Refs 30-32). Furthermore, CD4 ÷ T-cell lines specific for these pathogens are often cytolytic for infected target cells provided the latter express class II molecules 33. It therefore appears that at least with respect to IFN-~/secretion and target cell lysis the two T-cell populations possess a similar potential and that the expression of biological functions must be regulated differently: class I-restricted CD8 ÷ T cells can interact with virtually all host cells but often require IL-2 from CD4 ÷ T cells for expression of their functions. In contrast, class II-restricted CD4 ÷ T cells which can supply their own IL-2 are limited to class II-expressing host cells.
Immunology Today, Vol. 9, No. 6, 1988
Can antigens of intracellular microbes associate with classI? The observation that intracellular microbes - a source of exogenous antigens - induce class I-restricted CD8 -~ T cells stands in contrast to the widely held view that antigen association with class I is a feature of endogenous antigens only. In support of this assumption it has been shown that although infectious virus is commonly required for the induction of virus-specific CD8 + T cells, virus-specific killer cell activation by noninfectious antigen preparations does occur 34-37. Recently Braciale et al. 37 compared the ability of infectious and noninfectious virus preparations to induce class I-restricted CD8 + CTL and to prime target cells for killing. In accordance with the current view, most T cells stimulated by noninfectious virus preparations belonged to the class II-restricted CD4 + set, while T cells stimulated by infectious virus were primarily class I restricted. However, a small proportion of class I-restricted CTL was activated by noninfectious virus. Staerz et al. 38 successfully activated class I-restricted CD8 + CTL using unfractionated ovalbumin, a classic example of an exogenous antigen. Studies by Townsend and co-workers 39 using the influenza virus nucleoprotein have shown that antigenic peptides are sufficient for target cell recognition. Indeed, the three-dimensional st~-ucture of a class I antigen is consistent with direct peptide binding 4°. These latter findings suggest that endogenous proteins are degraded and peptide fragments bind to class I molecules. Elegant as they are, however, they do not answer the questionsof whether, how, and where exogenous antigens can associate with class I molecules. A trivial explanation for the association of microbial antigens with class I (see Refs 23-26) would be that the crude antigen preparations used already contained antigenic peptide fragments which could directly associate with class I rnol~.~'~'1~.,. . . .v,.,,..=.;+~' . '+"' pnor processing -:~' ~,,;,,,,o, ~ ~" to *~',,,~ . . .~tuu,~-J:-.. v,-~ Townsend eta/. 38. While peptides could well be present in preparations of killed microbes, it appears unlikely that they represent a relevant fraction of viable organisms. Viable intracellular bacteria possess potent evasion mechanisms which allow them to survive inside host cells, and intracellular replication may particularly favour the production of proteins or peptides that gain access to the endogenous pathway or bind directly with class I molecules. It has been postulated that a specialized cell exists with the unique capacity to associate exogenous antigens with class I molecules41. We found that bone marrow macrophages were an excellent source for CTL •
targets 23-26. These cells represent a homogeneous population of quiescent macrophages expressing class I but not class II antigens with good phagocytic activity and hence may well represent such a specialized cell33. Clearly these assumptions need further analysis before any decision about their validity can be drawn. CD8+ T cells with broad target reactivity As indicated above, CD8 + CTL from L. monocytogenes and M. tuberculosis/M, bovis immune mice comprise several T cells capable of lysing macrophages independent of their H-2 haplotype provided that the macrophages had been primed with the relevant microbial antigen 24.26,42. Using a L. monocytogenes-specific T-cell clone, evidence could be presented that antigen-specific, apparently non-MHC-restricted killing is nonetheless mediated by the T-cell receptor 42. While the underlying mechanisms remain obscure it may be interesting to note that CTL with even broader reactivity pattern have recently been described in microbial systems (Table 1). Thus, the adoptive immune response of mice to Bacteroids fragilis infection is mediated by CD8 + T cells of apparently antigen-specific, non-MHC-restricted character 43. In experimental listerioses of rats, CD8 + T cells have been identified which differ from typical natural killer (NK) cells, but lyse syngeneic fibroblasts in the absence of antigen44. Recently, Dale and Beachey 4s have described human CD8 + T cells which become cytolytic for human heart cells after activation by peptides of streptococcal M protein. Because myocardial cells and streptococcal M proteins share antigenic epitopes one could suspect that these T cells are specific for a crossreactive entity. Interestingly, these cells apparently lack MHC restriction. NK/LAK (lymphokine activated killer) cells are triggered during infections with intracellular pathogens, and host cells infected with certain :_J.
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Table I. Differenttypesof killerce{Isin intracellularmicrobialinfections MHCrestriction
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Immunology Today, Vol. 9, No. 6, 7988
Evidence from human studies Evidence that CD8 + CTL participate in immunity to intracellular bacterial pathogens in humans is still rare. However, immunohistological analysis of lesions has shown that CD8 + T lymphocytes are present in tuberculin-positive individuals at the site of purified protein derivative challenge as well as in lesions of leprosy patients s3-ss. In lepromatous lesions higher numbers of CD8 + T cells are found than in tuberculoid lesions. Because lepromatous leprosy is accompanied by CD8 + suppressor T cells one could argue that the CD8 + T cells seen are of this rather than of cytolytic type. It is important, therefore, that Modlin eta/. ss recently found that CD8 + T cells in tuberculoid leprosy lesions express a putative differentiation marker of CTL, whereas CD8 + T cells in lepromatous lesions lack this antigen. Recently, evidence for the presence of antigen-reactive CD8 + T cells in tuberculosis patients and in malaria patients has been provided s6.sT. Rare as they are, these obervations provide the first hints of the participation of CD8 + T cells in human immune response to intracellular microbes.
although other factors by themselves or in synergy with IFN-~/may be involved; class II-restricted CD4 + T cells are thought to be the major producers of these lymphokines58. However, activation of cells devoid of class II molecules may exclusively depend on class Irestricted CD8 + T lymphocytes since only they can see microbial antigens on these cells. This may be the case in malaria 2o.
Lysis. Many host cells are not adequately equipped for intracellular killing even after lymphokine activation. This is generally true for many nonprofessional phagocytes and in fact the selection of unarmed cells serving as 'trojan horses' would represent an obvious goal for an intracellular pathogen. It is even true for some professional phagocytes that are insufficiently armed for killing of particularly resistant pathogens. As a rule tissue macrophages seem to be less prepared for intracellular killing than monocytes. In these situations target-cell lysis may become an unavoidable step for the host. Both T-cell subsets possess cytolytic activity although CD8 + T cells have a broader target spectrum. Mere destruction of the cellular habitat may already be harmful for Possible in.vivo role of CrL Caution is needed in discussing a possible in-viva role obligate intracellular microbes. Also, for many facultative of CTL in microbial infections: these cells cannot be intracellular pathogens, existence outside the cell may be isolated from other host responses, in particular those more difficult due to the possibility of dissolution by lysosomal enzymes. Finally, certain microbes can be killed mediated by helper T cells. Furthermore, the cellular habitat of these microorganisms is quite heterogeneous, by NK/LAK cells directly48-s°. At the same time, tissue destruction is an inevitable ranging from cells with a low to those with a high corollary of lysis. The stronger the immune response, and antibacterial potential. I shall attempt to dissect this complex interaction of cellular activation and cytolysis, the more important and essential the target cell, the and discuss what might take place in a granulomatous worse the consequences for the host. Schwann cells provide a major habitat for M. leprae but are not harmed lesion as an example of an important micromilieu of significantly. Nevertheless, Schwann cell destruction rephost-pathogen interaction (Fig. 2). resents a major pathomechanism in leprosy. We have recently found that Schwann cells presenting M. leprae Activation. The activation of antimicrobial mechanisms in professional nhanocvtes hy T-rpll Ivmnhnl(in~,¢ ic ~^,~,11 antigens in association with class f molecules can be known 1. Also, in nonprofessional phagocytes intra- killed by mycobacteria-reactive CD8 + CTL indicating that cellular defence mechanisms against certain pathogens, destruction of Schwann cells harbouring M. leprae contributes to nerve damage in leprosy (U. Steinhoff and including rickettsiae, chlamydiae and malaria plasmodia, S.H.E. Kaufmann, submitted). .......... can be a~ivated 2,2o,s8. IFN-,y is an important mediator, Another threat for the host is microbial dissemination through target cell lysis, particularly for intracellular Q Directmicrobicidy Q Tissuedamage pathogens which survive or replicate in the extracellular environment before entering new host cells. Thus secondary sites of the body may be colonized and --- J,,':-'x~'-:~--, e infection of other individuals facilitated. t' e0", f ~ ~'.j ,, / Coordinated lysis and activation. Microbial release following host cell lysis can be made beneficial by coordinating cytolysis with adequate effector systems. In this way V"-'N', microbes become accessible to humoral effector ~ Transmission mechanisms. Also, lysis of infected host cells with low antimicrobial potential allows microbial transmission from a protective niche to a more aggressive cell. Elimination of intracellular pathogens may then be completed by highly competent professional phagocytes such as granulocytes or monocytes attracted to and Fig.2. A modelof T-cellfunctionsin antimicrobialresistance.(I) T cellsactivateantimicrobial activated at the site of lysis under the influence of macrophagefunctions via interleukins.Although this step is primarilybeneficial,activated inflammatory stimuli and T-cell lymphokines. It is conmacrophagesalsosecretevariouscompoundsthat may harm surroundingtissue.(2) Lysisof ceivable that these mechanisms will only function to the infectedhost cellsaffectsintracellularmicrobesmoreor lessdirectly.(3) Lysisof infectedhost benefit of the host under highly coordinated conditions cellsleadsto tissuedestruction.(4)Lysisof infectedhostcellsallowsmicrobialdissemination.(5) which in situ may exist in granulomatous lesions. Coordinatedlysisof infectedhostcellsandsubsequentactivationof potentphagocytesallows microbialtransmissionfroma protectiveto an aggressivesurrounding.For furtherdetailssee Granuloma. A granuloma is composed of mononuclear text. phagocytes at different developmental stages -includ•
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ImmunologyToday, VoL 9, No. 6, 1988
ing epitheloid cells, multinucleated giant cells and immigrant monocytes - as well as of CD4 + and CD8 + T lymphocytes. Granulomatous lesions represent the histologic focus for the defence against many intracellular microbes, perhaps best exemplified in tuberculosis of the lung. In tuberculous granulomas, tubercle bacilli are found inside macrophages apparently unable to destroy their inhabitants. While these cells may already fulfill an important role in protection by containing the pathogens within discrete foci and preventing them from dissemination, in the long run, they are insufficient for microbial elimination and present a protective niche for persistent microbial pathogens. In this situation, lysis could become a necessary prerequisite for microbial elimination. Discharge of microbes into the unpleasant environment of a hypoxic necrotic granulomatous center may directly lead to growth inhibition and hence be beneficial. On the other hand, discharge into the blood or the bronchioalveolar system will allow microbial dissemination to secondary tissue sites or shedding to other individuals. These harmful sequelae, as well as tissue destruction, predominate in caseous granulomas lacking phagocytes adequately equipped for intracellular killing. In contrast, in productive granulomas monocytes with high antibacterial potential are present. Mycobacteria released through cytolysis are then engulfed by these phagocytes which fulfill the final steps of microbial elimination after lymphokine activation. Concluding remarks
The scenario described is highly speculative. However, it illustrates the complexity of the host-pathogen relationship in intracellular microbial infections where a single cell and a single mechanism may both benefit and threaten the host. Further studies are required to elucidate the conditions leading to the activation of CD8 + CTL by exogenous antigens as weii as their effector role in microbial infections. Both basic and applied research may benefit from such studies. References
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16 Pedrazzini,T., Hug, K. and Louis,J.A. (1987)J. ImmunoL
139, 2032-2037 17 Pavlow, H., Hogarth, M., McKenzie, I.F.C.and Cheers, C. (1982) Cell. ImmunoL 71,127-138 18 Titus, R.G., Milon, G., Marchal, G. et aL (1987) Eur. J. Immunol. 17, 1429-1433 19 Hussein,S., Curtis, J., Griffith, S. and Turk, J.L. (1987) Cell. Immunol. 105, 423-431 20 Schofield, L., Villarquiran, J., Ferreira,A. et aL (1987) Nature 330, 664-666 21 Weiss,W.R., Sedegah, M., Beaudoin, R.L.etal. (1988) Proc. Natl Acad. Sci. USA 85, 573-576 22 Mogil, R.J., Patton, C.L. and Green, D.R. (1987)J. Immunol. 138, 1933-1939 23 Kaufmann, S.H.E., Hug, E. and De Libero, G. (1986)J. Exp.
Meal.164, 363-368 24 De Libero, G. and Kaufmann, S.H.E.(1986)J. Immunol. 137, 2688-2694 25 Chiplunkar, S., De Libero, G. and Kaufmann, S.H.E.(1986) Infect. Immun. 54, 793-797 26 De Libero, G., Flesch, I. and Kaufmann S.H.E.(1988) Eur. J. Immunol. 18, 59-66 27 Rollwagen,F.M., Dasch, G.A. and Jerrells,T.R.(1986) J. Immunol. 136, 1418-1421 28 Morrison, W.I., Goddeeris, B.M., Teale,A.J. etal. (1986) Immunol. Today 7, 211-216 29 Sonnenfeld, G., Mandel, A.D. and Merigan, T.C. (1979) Immunology 36, 883-890 30 Kaufmann, S.H.E.and Hahn, H. (1982)J. Exp. Med. 155, 1754-1765 31 Kaufmann, S.H.E.(1984) Cell. Immunol. 83, 215-220 32 Kaufmann, S.H.E.and Flesch,I. (1986)Infect. Immun. 54, 291-296 33 Kaufmann, S.H.E., Hug, E., V~th, U. and De Libero, G. (1987) Eur. J. Immunol. 17, 237-246 34 Koszinowski,U., Gething, M.J. and Waterfield, M. (1977) Nature 267, 160-163 35 Yamada, A., Young, J.F.and Ennis,F.A. (1985)J. Exp. Med. 162, 1720-1725 36 Yamada,A., Ziese, M.R., Young, J.F.,Yamada, Y.K., and Ennis, F.A. (1985)J. Exp. Med. 162,663-674 37 Braciale,T.J., Morrison~L.A., Sweetser, M.T. etal. (1987) Immunol. Rev. 98, 95-114 38 StaerT,U.D., Karasuyama,H. and Garner, A.M. (1987) Nature 329, 449-451 39 Townsend, A.R.M., Rothbard,J., Gotch, F.M. etal. (1986) Cell 44, 959-968 40 Bjorkman, P.J.,Saper, M.A., Samraoui,B. etal. (1987) Nature 329, 506-512 41 Bevan,M.J. (I 986) Nature 325, 192-194 42 Kaufmann, S.H.E., Rodewald, H.G., Hug, E. and De Libero, G. J. Imm,~noL (in press) 43 Shapiro,M.F., Onderdonk, A.B., Kasper, Di. and Finberg, R.VV.(1982)J. Exp. Med. 154, 1188-1197 44 Chen-Woan, M., McGregor, D.D. and Forsum, U. (1981) J. ImmunoL 127, 2325-2330 45 Dale, J.B. and Beachey, E.H.(1987)J. Exp. Med. 166, 1825-1835 46 Carl, M. and Dasch, G.A. (1986)J. Immunol. 136, 2654-2661 47 Klimpel, G.R., Niesel, D.W. and Klimpel, K.D. (1986) J. ImmunoL 163, 1081-1086 48 Blanchard, D.K., Stewart II, W.E., Klein, T.W., Friedman, H., and Djeu, J.Y. (1987)J. Immunol. 139, 551-556 49 Nencioni, L., Villa, L., Boraschi, D. Berti, B. and Tagliabue, A. (1983)J. Immunol. 130, 903-907 50 Murphy, J.VV.and McDaniel, D.O. (1982)J. ImmunoL 128, 1577-1583 51 Lowell, G.H., MacDermott, R.P.,Summers, P.L.etaL (1980) J. ImmunoL 125, 2778 52 Hersey,P. and Bolhuis, R. (1985)Immunol. TodayS, 223-239
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53 Beck,J.S., Morley,S.M., Gibbs,J.H. etaL (1986) Clin. Exp. Immunol. 64, 484-494 54 Van Voorhis,W.C., Kaplan,G., Sarno, E.N.etal. (1982)New EngL J. Med. 307, 1593-1597 55 Modlin, R.L, Melancon-Kaplan, J., Pirmez,C. etaL Proc. Natl Acad. Sci. USA (in press)
56 Rossi,G.A., Balbi, B. and Manca, F. (1987)Am. Rev. Respir. Dis. 136, 575-579 57 Sinigaglia,F., Matile, H. and Pink,J.R.L.(1987)Eur. J. Immunol. 17, 187-192 58 Adams, D.O. and Hamilton, T.A. (1984)Annu. Rev. Immunol. 2,283-318
MHC classII expressionby the gut epithelium The intestinal epithelium represents a barrier between luminal antigen and the underlying local immune system. Expression of MHC class II antigens - normally associated with cells of defined immunological function - and the alteration in the pattern of expression of these antigens found in certain diseases, has aroused considerable interest. In this article, Paul Bland reviews what is known of class II molecules on the intestinal epithelium and discusses their relevance in the wider context of immune regulation in the gut. Immune responses to food antigens and to intestinal pathogens are important both in protection of the host and in the pathogenesis of gastrointestinal diseases. Because of the need for rapid induction and regulation of appropriate immune responses to food and microbial antigens in the gut, it is likely that these processes take place at, or close to, the site of absorption. Analysis of the cellular and molecular mechanisms of antigen handling within the gut are therefore vital to our understanding of the aetiopathogenesis of gut disease and to the rational development of prophylactic regimes. Although the cellular circuitry within Peyer's patches is i i~,~l,F,~..k+~,,,.,ll,, ; . . . . I. ,A,,.J : ~ ~ L . . . . . . I _±~ ul,u~..luuI.EUl)" IIIVUIVI~U III LII~ r~cJUlaUOrl of responses to orally presented antigens, the tissue at the interface between luminal antigen and the local immune system the epithelium of the small intestine - is also a possible participant in the induction and regulation of the immune response. Since 1978, when class II molecules of the major histocompatibility complex (MHC) were first found in the small intestinal epithelium, a greater awareness has developed that the epithelium may be intimately involved in selective antigen handling and in the control of appropriate immune responses.
Constitutive expression of epithelial class II molecules Expression of MHC class II molecules by the intestinal epithelium was first described in the guinea-pig by Wiman and colleagues ~and subsequently in the mouse 2, the rat3 and humans4. A common gross distribution is found in all species. Thus, class II molecules are expressed by the small intestinal epithelium only, large bowel epithelium being negative, or only faintly positive, in healthy tissues~9. The tissue distribution (Fig. 1) is restricted to the upper two-thirds of the villus: only fully differentiated absorptive epithelial cells (enterocytes) constitutively express class II molecules. They are not expressed by mucus-secreting goblet cells. 174
Department of Veterinary Medicine, University of Bristol, Langford House, Langford,BristolBS187DU, UK.
Paul Bland Expression at the light microscope level in isolated enterocytes appears to be restricted to the basolateral enterocyte membrane 1°. Electron microscopy confirms this distribution in the mouse 2.11for both class I and class II antigens, suggesting that the mobility of MHC antigens in the enterocyte membrane is restricted by the junctional complex. More recently, however, microvillus staining has been reported in human tissue6. Although granular staining in the supranuclear region has been noted both in tissue sections9 and in isolated enterocytes 1°, the ultrastructural localization of enterocyte class II molecules on specific organelles has, unfortunately, not been reported. The appearance under light microscopy (Fig. la and b), however, is consistent with the intracellular accumulation of class II molecules within the vesicular endosome compartment in the apical cytoplasm of the enterocyte. This interpretation corresponds with the situation in B cells in which the distribution of class II molecules overlaps in the endosome compartment with the recycling transferrin receptor, allowing the uncoupling of the class II invariant chain to take place before expression at the cell membrane ~2. Nothing is known of the recycling of class II molecules within enterocytes, but it seems likely that if they have an antigen receptor function within the epithelium, their restricted distribution in the plasma membrane would allow for binding and release of the antigenic ligand in an endosomebasolateral membrane direction only (see below).
Induced expression of epithelial class II molecules. Although class II molecules are expressed constitutively at low density in the normal distribution described above, the distribution and intensity of expression can be altered by immune parameters~ Thus, in Trichinella spiralis-induced inflammation 13 and graft-versus-host disease (GVHD)3,13 expression is intensified and, moreover, de novo expression occurs in immature crypt epithelial cells (Fig. lc and d). Similarly, those gastrointestinal disorders associated with primary or secondary immune defects show changes from the normal epithelial class II expression. In both coeliac disease and dermatitis herpetiformis, where villus atrophy and crypt hyperplasia are classical features, both residual immature enterocytes and crypt cells express class II (Refs 14,15). Moreover, the surface epithelium in untreated coeliac patients expresses the products of the HLA-DP and DQ loci in addition to DR which is expressed constitutively 16. ~ ) 1988, Elsevier Publications, Cambridge
0 1 6 7 - 4919188/$02,00
is very different from using them to target toxins or drugs. In fact, whereas the latter approach only allows a transient cytotoxic effect, the targeting of antigens exploits all the potential of the immune response itself. What are the effects that might follow antigen targeting? T -'~l!sare well equipped with a variety of toxins and poisons: antigen-specific class ii-restricted T cells can kill tumor cells that are presenting the antigen and also (in some instances) innocent bystander tumor cells. Such bystander lysis may solve the problem generated by the appearance of Id-negative or class II-negative tumor variants; however there is little evidence that bystander lysis might be operative in vivo. One system that by definition is operative in vivo is delayed-type hypersensitivity, which is mediated by class II-restricted T cells and which, in some cases, correlates better than cytotoxicity with the rejection of grafts or tumors. A second useful in-vivo effect is the generation of T-cell help. There are examples where the response against weakly immunogenic determinants can be elicited only if another (helper) determinant is corecognized on the same cell 3.4. It is possible that a similar . . . . . . . effect of 'intramolecular help' may be elicited by target-
CD8
The Basel Institute for Immunology was founded and is supported by F. Hoffmann LaRoche& Co., Ltd, Basel, Switzerland.
References
10chi, A., Worton, K.S.,Woods, G., Gravelle,M. and Kitagami, K. (1987) Eur. J. ImmunoL 17, 1645 2 Lanzavecchia,A., Abrignani, S., Scheidegger,D. etaL (1988) J. Exp. Med. 167, 345 3 Keene,J.A. and Forman,J. (1982~J. Exp. Med. 155, 768 4 Mitchison,N.A. (1983) Transplant. Proc. 15, 2121
+ T lymphocytesin intracellular microbial infections
Current theories on infections with intracelMar bacteria, protozoa, and fungi support the notion that MHC class II-restricted CD4+ T cells are activated and that resistance depends exclusively on this T-cell subset. Here, Stefan Kaufmann summarizes recent evidence that in these infections MHC class I-restricted CD8+ T ceils are also activated, and participate in protection; they appear to lyse infected target cells and produce gammainterferon in vitro. The possible role of these CD8+ T cells during intracellular microbial infections is discussed. A major challenge for the immune system is to combat bacterial, parasitic, mycotic and viral infections. While many infectious agents can be aoequately dealt with by antibody-mediated mechanisms, others have chosen an intracellular habitat which protects them quite efficiently from humoral host defence 1.2 (see inset opposite). During intracellular replication, antigenic moieties of these pathogens appear on the surface of infected host cells which can then be recognized by T lymphocytes. In this way infected host cells can be discriminated from uninfected cells and appropriate effector mechanisms aimed at the eradication of infection can be initiated. For this purpose, T cells have two principally different mechanisms, namely their activation and ability to lyse infected target cells3. Cytolysis seems to be adequate for the defence against viral infections, whereas activation is thought to be more appropriate for defence against nonviral intracellular pathogens. In this review, recent
168
ing to the tumor cell a foreign antigen that would serve as a helper determinant to generate a cytotoxic response against tumor-specific antigens. The two kinds of responses - against targeted antigen and against tumorspecific antigen - would allow the tumor little chance to escape the immune response by generating negative variants. Nature has obviously put a lot of time and effort into developing optimal strategies for the immune system. If we can exploit these strategies it would be a pity not to try.
Department of Medical Microbiology and Immunology, Universityof Ufm, ObererEselsberg,D-7900 UIm, FRG.
Stefan H.E. Kaufmann t~lnir~anr ;c ~,,~,,,~, r~r~enn+~rl e-, ,~,.-,^t-÷ ~LIIC]L - h . + ~..~LUI~LIt.. ..,,~.^h.4-; . . Iil~.llCiii. . i~.-,.v,,,,.,,,.e ,~ to ,)LJ~j~j~aL isms may also play a role in the defence against intracellular bacteria, fungi and protozoa. For simplicity the term intracellular microbes will be used for these pathogens to distinguish them from viral agents.
Different types of antigen presentation In contrast to antibodies which bind free antigen directly, T cells recognize foreign antigen in conjunction with self structures encoded by the major histocompatibility complex (MHC) on the surface of host cells. Foreign antigens can associate with either MHC class II (HLA-DR, DP, DQ in man, H-21A, IE in mice) or class I (HLA-A, B, C in man and H-2K, D, L in mice) structures. Virtually all host cells express MHC c!ass I molecules and hence can present antigens recognized in their context. It is generally assumed, however, that antigen association with class I structures is restricted to proteins that are newly synthesized within the cell. With respect tb infectious agents, only products of viral genes qualify as 'endogenous antigens'4. In contrast, antigens that are taken up via endocytosis are termed 'exogenous antigens'. These include antigens from bacterial, protozoan and fungal agents residing within the phagolysosome of an infected host cell. These exogenous antigens are degraded by lysosomal enzymes, and class II molecules can act as shuttle molecules which pass antigenic peptides to the cell surface. 1988, Elsevier Publications, Cambridge
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ImmunologyToday,Vol.9, No. 6, 1988
Intracellular pathogens A group of pathogenic bacteria, fungi and protozoa have developed means that allow them to live within cells of their host. But besides their common predilection for an intracellular habitat, these pathogens vary markedly. Intracellular pathogens can be divided into two groups according to the type of host cell they target: one group preferentially uses professional phagocytes (more precisely, cells of the mononuclear phagocyte system), whereas the others primarily inhabit nonprofessional phagocytes (i.e. one or more types of host cells other than mononuclear phagocytes or granulocytes). This separation is not only convenient but also reflects the preferred use of different evasion mechanisms. However, this division is not stringent and intracellular pathogens should rather be seen as a spectrum with these two types as extremes. Professional phagocytes are particularly well equipped to engulf, kill, and degrade invading microbes. Therefore, selection of these host cells means emphasis on intracellular survival strategies rather than on entry mechanisms. Microbial entry can rely on conventional phagocytosis and the pathogen may j-st modulate this process. However, the inside of a macrophage represents an aggressive milieu and hence requires potent evasion mechanisms. These include inhibition of phagosome-lysosome fusion, resistance to reactive oxy-
gen metabolites and lysosomal enzymes. Once activated by lymphokines, however, the intracellular milieu of an infected macrophage becomes increasingly hostile and many - although not all pathogens may finally die. Pathogens of this type include the etiologic agents of tuberculosis, Mycobacteriurn tuberculosis and M. boris; M. leprae, the cause of leprosy; M. lepraernurium, the agent of rat leprosy; Listeria monocytogenes which is increasingly recognized as being responsible for many food-borne diseases; Legionella pneumophila which causes a severe pneumonia called Legionnaires disease; and Leishmania spp. the agents of different forms of leishmaniasis. The second type of intracellular microbe primarily inhabits nonprofessional phagocytes and hence is forced to use its own entry mechanism which often requires energy expenditure by the pathogen. Once inside a nonprofessional phagocyte, the milieu is less hostile than in a professional phagocyte. Therefore, similar but less refined evasion mechanisms may be employed by these pathogens. Many microorganisms belong to this group including Chlamydia sp., Rickettsia sp., --~':" plasmodia a,.u --'~ Trypano,,,,',,a,,a soma sp. Mentioned in this review are Rickettsia spp., the agents of different forms of typhus, which readily enter and replicate in endothelial cells; the lymphocyte-inhabiting protozoan, Theileria parva,
Class II molecules are expressed by a limited number of host cells - primarily B lymphocytes, dendritic cells and mononuclear phagocytes - and only these cells should be able to present microbial antigens adequately. Accordingly, in the case of class I presentation the type of the infectious agent is limiting, whereas in class II presentation it is the type of infected host cell. The two modes of antigen presentation have their counterpart in two phenotypically distinct T-cell subsetss. T cells expressing the CD4 molecule (T4 in man, L3T4 in mice) recognize antigen in association with MHC class II molecules, whereas CD8 + T cells (T8 in man, Lyt2 in mice) are specific for antigen in the context of MHC class I structures. It has been suggested that the segregation into two distinct T-cell subsets reflects the different requirements for defence against intracellular viral and microbial infections 1,3. Class I-restricted cytolytic T cells (CTL) are appropriate for defence of viral infections since, due to the variety of host cells that can be infected by
which causes a fatal cattle disease called East Coast fever; and mouse malaria plasmodia, which at different stages of their cycle inhabit hepatocytes and erythrocytes. It should be emphasized, however, that the separation into two types of intracellular pathogens oversimplifies reality. A single organism often preferentially, although not exclusively, uses one type of host cell. At one pole Rickettsia sp., which normally infects nonprofessional phagocytes, is also found in macrophages. At the other pole, M. leprae not only inhabits macrophages but also Schwann cells. L. monocytogenes, a widely used model organism for facultative intracellular infections, can also enter endothelial cells and it appears that for these targets only, the pathogen employs an active penetration mechanism. Intracellular microbial pathogens can be divided into facultative pathogens (able to live both inside and outside the host cell) or obligate pathogens (which only survive inside the cell). Inhabitants of both professional and nonprofessional phagocytes comprise either type of pathogen. The tubercle and leprosy bacilli are both mycobacteria which preferentially replicate in professional phagocytes with the former being a facultative and the laker an obligate intracellular pathogen. It is possible that the preferred choice of host cell, as well as the ability to survive extracellularly, have great impact on the success of a given immune mechanism.
viruses, the MHC restriction element required must have a similarly wide distribution. In addition, lysis of infected host cells before assembly of viral progeny effectively blocks viral replication. Furthermore, newly synthesized viral antigens can associate with class I molecules. In contrast, microbial antigens should fail to associate with class I molecules and hence the host depends on CD4÷ class II-restricted T cells to combat these infections. Nonactivated mononuclear phagocytes provide the major habitat for many of these pathogens. These cells express class II molecules and can be activated by lymphokines to gain antimicrobial effector functions. Thus, two apparently satisfactory mechanisms for combating different types of intracellular pathogens exist. However, a number of questions remain unanswered. Many microbes infect host cells other than mononuclear phagocytes, and these cells may not express MHC class II antigens nor become microbicidal after lymphokine stimulation. Furthermore, mononuclear phagocytes vary
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in their antimicrobial potential and some may not be adequately equipped for intracellular killing. It is hard to envisage how, under these circumstances, CD4 ÷ helper T cells alone are sufficient for effective protection. Rather, one must assume that some other as yet unknown factor is required. Several lines of evidence suggest that CD8 ÷ class I-restricted CTL play a more important role in the resistance to microbial pathogens than hitherto thought. As a corollary, exogenous antigens should have the potential to associate with class I MHC molecules (see Fig. 1). CD$+ T cells and antimicrobial protection Zinkernagel and co-workers 6 were the first who analysed genetic restriction of cell-mediated antimicrobial resistance. They found that adoptive protection against murine listeriosis is class II restricted and this study has been taken as strong support for the notion that acquired resistance to intracellular bacteria is a function solely of helper T lymphocytes. However, Cheers and Sandrin 7 later provided evidence to the contrary by showing that protection against this pathogen is class I restricted. Earlier studies performed in the rat had also indicated resistance to listeriosis to be class I restricted 8. Other studies have shown that the T cells relevant to antilisterial protection belong to both the CD4 ÷ and CD8 + T-cell subsets9-12. In antilisterial resistance, it has been suggested that CD8 + T cells control granuloma formation at the site of bacterial implantation 13. Together, these findings are consistent with the notion that not only CD4 ÷ class II-restricted, but also CD8 + class I-restricted T lymphocytes are involved in the acquisition of antilisterial resistance. This idea is not restricted to listeriosis but also seems to hold true for several other intracellular microbes 14-19. Although in the different systems, and depending on the stage and severity of the disease, the relative contribution of CD4 + and CD8 ÷ T cells to resistance varies, some effect of both subsets is commonly found. Recent studies by different groups also provide strong evidence for a protective role for CD8 + T cells in murine malaria2O-22.
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Cytolytic activities of CD8÷ T lymphocytes In viral infections the best analysed function of CD8 + T cells is their capacity to lyse virus-infected target cells in vitro: so do CD8 ÷ T cells arising during intracellular microbial infections function similarly? CTL specific for microbial antigens have now been identified in several systems. CD8 ÷ T lymphocytes from mice with listeriosis express high cytolytic activity towards L. monocytogenesinfected bone marrow macrophages23. 24. As is the case in most viral infections, restimulation in vitro is required for the expression of optimum killer activity. Similarly, CD8 + T cells from mice primed with Mycobacterium leprae, M. tuberculosis or M. bovis specifically lyse bone marrow macrophages primed with the homologous agent 25,26. Although in these studies bulk cultures of T cells did not show stringent MHC restriction, on the clonal level class I-restricted T cells could be identified. T cells from mice infected with Rickettsia sp. can also specifically lyse target cells infected with the homologous organism271 Lysis was found to be H-2 restricted; unfortunately neither the phenotype nor the class restriction of these T cells has been determined. Theileria parva,
~Class Anhgen
Exogenous Pathway/0
]I ÷
>Class II f Class I +Anhgen
~ Class l
v
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Fig.1. Simplifiedschemeof antigenprocessing.Thesolidlinesindicatethe two generallyacceptedroutesof exogenousand endogenousantigens;the dotted line indicatesthe putativewayof an exogenousantigento the classI MHCgene product. which inhabits T lymphocytes of cattle, activates CD8 ÷ T cells capable of lysing infected T cells in an antigenspecific and class I-restricted way 28. Collectively, these data show that antigen-specific, class I-restricted CD8 ÷ CTL are generated during infection with a variety of intracellular microbes. Other functions of CD8÷ CTL CD8 + T-cell lines with reactivity to L. monocytogenes, M. leprae or M. tuberculosis fail to secrete interleukin 2 (IL-2); however, many lines produce gamma-interferon (IFN-~) after costimulation with antigen plus accessory cells and exogenous IL-2 (Refs 24-26). Also, CD8 ÷ T cells from mice infected with M. boris and challenged with purified protein derivative produce IFN-~/(Ref. 29). This cytokine is thought to be the major mediator of macrophage activation, and IFN-~,-containing T-cell supernatants can indeed activate tuberculostatic macrophage functions. Adoptive transfer of CD8 + T cells partially protects mice against malaria, and protection is reversed by administration of IFN-'y-neuLralizing monoclonal antibodies 2°. Taken together, these findings argue for a direct or indirect role for IFN-'y in the protection afforded by CD8 ÷ T lymphocytes. Mycobacteria-reactive CD8 ÷ T cells seem to have an additional mechanism of resistance. Coculture of CD8 ÷ T cells with M. bovis-infected bone marrow macrophages induces tuberculostasis apparently independent from IFN-~, secretion and probably dependent on target cell lysis26. CD4 ÷ T cells with reactivity to intracellular microbes have also been shown to secrete IFN-,yand they do so in the absence of exogenous IL-2 (Refs 30-32). Furthermore, CD4 ÷ T-cell lines specific for these pathogens are often cytolytic for infected target cells provided the latter express class II molecules 33. It therefore appears that at least with respect to IFN-~/secretion and target cell lysis the two T-cell populations possess a similar potential and that the expression of biological functions must be regulated differently: class I-restricted CD8 ÷ T cells can interact with virtually all host cells but often require IL-2 from CD4 ÷ T cells for expression of their functions. In contrast, class II-restricted CD4 ÷ T cells which can supply their own IL-2 are limited to class II-expressing host cells.
Immunology Today, Vol. 9, No. 6, 1988
Can antigens of intracellular microbes associate with classI? The observation that intracellular microbes - a source of exogenous antigens - induce class I-restricted CD8 -~ T cells stands in contrast to the widely held view that antigen association with class I is a feature of endogenous antigens only. In support of this assumption it has been shown that although infectious virus is commonly required for the induction of virus-specific CD8 + T cells, virus-specific killer cell activation by noninfectious antigen preparations does occur 34-37. Recently Braciale et al. 37 compared the ability of infectious and noninfectious virus preparations to induce class I-restricted CD8 + CTL and to prime target cells for killing. In accordance with the current view, most T cells stimulated by noninfectious virus preparations belonged to the class II-restricted CD4 + set, while T cells stimulated by infectious virus were primarily class I restricted. However, a small proportion of class I-restricted CTL was activated by noninfectious virus. Staerz et al. 38 successfully activated class I-restricted CD8 + CTL using unfractionated ovalbumin, a classic example of an exogenous antigen. Studies by Townsend and co-workers 39 using the influenza virus nucleoprotein have shown that antigenic peptides are sufficient for target cell recognition. Indeed, the three-dimensional st~-ucture of a class I antigen is consistent with direct peptide binding 4°. These latter findings suggest that endogenous proteins are degraded and peptide fragments bind to class I molecules. Elegant as they are, however, they do not answer the questionsof whether, how, and where exogenous antigens can associate with class I molecules. A trivial explanation for the association of microbial antigens with class I (see Refs 23-26) would be that the crude antigen preparations used already contained antigenic peptide fragments which could directly associate with class I rnol~.~'~'1~.,. . . .v,.,,..=.;+~' . '+"' pnor processing -:~' ~,,;,,,,o, ~ ~" to *~',,,~ . . .~tuu,~-J:-.. v,-~ Townsend eta/. 38. While peptides could well be present in preparations of killed microbes, it appears unlikely that they represent a relevant fraction of viable organisms. Viable intracellular bacteria possess potent evasion mechanisms which allow them to survive inside host cells, and intracellular replication may particularly favour the production of proteins or peptides that gain access to the endogenous pathway or bind directly with class I molecules. It has been postulated that a specialized cell exists with the unique capacity to associate exogenous antigens with class I molecules41. We found that bone marrow macrophages were an excellent source for CTL •
targets 23-26. These cells represent a homogeneous population of quiescent macrophages expressing class I but not class II antigens with good phagocytic activity and hence may well represent such a specialized cell33. Clearly these assumptions need further analysis before any decision about their validity can be drawn. CD8+ T cells with broad target reactivity As indicated above, CD8 + CTL from L. monocytogenes and M. tuberculosis/M, bovis immune mice comprise several T cells capable of lysing macrophages independent of their H-2 haplotype provided that the macrophages had been primed with the relevant microbial antigen 24.26,42. Using a L. monocytogenes-specific T-cell clone, evidence could be presented that antigen-specific, apparently non-MHC-restricted killing is nonetheless mediated by the T-cell receptor 42. While the underlying mechanisms remain obscure it may be interesting to note that CTL with even broader reactivity pattern have recently been described in microbial systems (Table 1). Thus, the adoptive immune response of mice to Bacteroids fragilis infection is mediated by CD8 + T cells of apparently antigen-specific, non-MHC-restricted character 43. In experimental listerioses of rats, CD8 + T cells have been identified which differ from typical natural killer (NK) cells, but lyse syngeneic fibroblasts in the absence of antigen44. Recently, Dale and Beachey 4s have described human CD8 + T cells which become cytolytic for human heart cells after activation by peptides of streptococcal M protein. Because myocardial cells and streptococcal M proteins share antigenic epitopes one could suspect that these T cells are specific for a crossreactive entity. Interestingly, these cells apparently lack MHC restriction. NK/LAK (lymphokine activated killer) cells are triggered during infections with intracellular pathogens, and host cells infected with certain :_J.
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by NK/LAK-like cells than their uninfected counterparts46-48. Finally, some reports have described direct microbial killing by NK/LAK cells49-s1. It appears, therefore, that a spectrum of killer cells is activated during infections with intracellular pathogens including antigen-specific, MHC-restricted CTL; antigenspecific, apparently non-MHC-restricted CTL; broad target cell killers; and NK/LAK cells. The recent demonstration that broad cytolytic activity is mediated by T cells that bear the ~/G rather than the oL~ T-cell receptor heterodimer and that are distinct from typical NK/LAK cells52 raises the question whether these CTL play a role in bacterial infections.
Table I. Differenttypesof killerce{Isin intracellularmicrobialinfections MHCrestriction
Antigenspecificity
Target
Microbe
Restricted
Specific
Infected hostcell
Non-(broad-)restricted Non-(broad-)restricted NK/LAK-like
Specific Broadreactivity NK/LAK-like
Infected hostcell Certain hostcell Infected hostcell
NK/LAK-like
NK/LAK-like
Organismdirectly
L. monocytogenes23, M. tuberculosis/M,bovis26, M. leprae,2s, R. tsutsugamushi/R,typhi27, T. parva28 L. monocytogenes42, M. tuberculosis/M,bovis26 L. monocytogenes44,groupA streptococci4~ L. pneumophila48, R. prowazecki/R,typhi46, Shigella flexneri47 S. typhimurium49,Cryptococcusneoformans~°, Shigellasp.~~
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Evidence from human studies Evidence that CD8 + CTL participate in immunity to intracellular bacterial pathogens in humans is still rare. However, immunohistological analysis of lesions has shown that CD8 + T lymphocytes are present in tuberculin-positive individuals at the site of purified protein derivative challenge as well as in lesions of leprosy patients s3-ss. In lepromatous lesions higher numbers of CD8 + T cells are found than in tuberculoid lesions. Because lepromatous leprosy is accompanied by CD8 + suppressor T cells one could argue that the CD8 + T cells seen are of this rather than of cytolytic type. It is important, therefore, that Modlin eta/. ss recently found that CD8 + T cells in tuberculoid leprosy lesions express a putative differentiation marker of CTL, whereas CD8 + T cells in lepromatous lesions lack this antigen. Recently, evidence for the presence of antigen-reactive CD8 + T cells in tuberculosis patients and in malaria patients has been provided s6.sT. Rare as they are, these obervations provide the first hints of the participation of CD8 + T cells in human immune response to intracellular microbes.
although other factors by themselves or in synergy with IFN-~/may be involved; class II-restricted CD4 + T cells are thought to be the major producers of these lymphokines58. However, activation of cells devoid of class II molecules may exclusively depend on class Irestricted CD8 + T lymphocytes since only they can see microbial antigens on these cells. This may be the case in malaria 2o.
Lysis. Many host cells are not adequately equipped for intracellular killing even after lymphokine activation. This is generally true for many nonprofessional phagocytes and in fact the selection of unarmed cells serving as 'trojan horses' would represent an obvious goal for an intracellular pathogen. It is even true for some professional phagocytes that are insufficiently armed for killing of particularly resistant pathogens. As a rule tissue macrophages seem to be less prepared for intracellular killing than monocytes. In these situations target-cell lysis may become an unavoidable step for the host. Both T-cell subsets possess cytolytic activity although CD8 + T cells have a broader target spectrum. Mere destruction of the cellular habitat may already be harmful for Possible in.vivo role of CrL Caution is needed in discussing a possible in-viva role obligate intracellular microbes. Also, for many facultative of CTL in microbial infections: these cells cannot be intracellular pathogens, existence outside the cell may be isolated from other host responses, in particular those more difficult due to the possibility of dissolution by lysosomal enzymes. Finally, certain microbes can be killed mediated by helper T cells. Furthermore, the cellular habitat of these microorganisms is quite heterogeneous, by NK/LAK cells directly48-s°. At the same time, tissue destruction is an inevitable ranging from cells with a low to those with a high corollary of lysis. The stronger the immune response, and antibacterial potential. I shall attempt to dissect this complex interaction of cellular activation and cytolysis, the more important and essential the target cell, the and discuss what might take place in a granulomatous worse the consequences for the host. Schwann cells provide a major habitat for M. leprae but are not harmed lesion as an example of an important micromilieu of significantly. Nevertheless, Schwann cell destruction rephost-pathogen interaction (Fig. 2). resents a major pathomechanism in leprosy. We have recently found that Schwann cells presenting M. leprae Activation. The activation of antimicrobial mechanisms in professional nhanocvtes hy T-rpll Ivmnhnl(in~,¢ ic ~^,~,11 antigens in association with class f molecules can be known 1. Also, in nonprofessional phagocytes intra- killed by mycobacteria-reactive CD8 + CTL indicating that cellular defence mechanisms against certain pathogens, destruction of Schwann cells harbouring M. leprae contributes to nerve damage in leprosy (U. Steinhoff and including rickettsiae, chlamydiae and malaria plasmodia, S.H.E. Kaufmann, submitted). .......... can be a~ivated 2,2o,s8. IFN-,y is an important mediator, Another threat for the host is microbial dissemination through target cell lysis, particularly for intracellular Q Directmicrobicidy Q Tissuedamage pathogens which survive or replicate in the extracellular environment before entering new host cells. Thus secondary sites of the body may be colonized and --- J,,':-'x~'-:~--, e infection of other individuals facilitated. t' e0", f ~ ~'.j ,, / Coordinated lysis and activation. Microbial release following host cell lysis can be made beneficial by coordinating cytolysis with adequate effector systems. In this way V"-'N', microbes become accessible to humoral effector ~ Transmission mechanisms. Also, lysis of infected host cells with low antimicrobial potential allows microbial transmission from a protective niche to a more aggressive cell. Elimination of intracellular pathogens may then be completed by highly competent professional phagocytes such as granulocytes or monocytes attracted to and Fig.2. A modelof T-cellfunctionsin antimicrobialresistance.(I) T cellsactivateantimicrobial activated at the site of lysis under the influence of macrophagefunctions via interleukins.Although this step is primarilybeneficial,activated inflammatory stimuli and T-cell lymphokines. It is conmacrophagesalsosecretevariouscompoundsthat may harm surroundingtissue.(2) Lysisof ceivable that these mechanisms will only function to the infectedhost cellsaffectsintracellularmicrobesmoreor lessdirectly.(3) Lysisof infectedhost benefit of the host under highly coordinated conditions cellsleadsto tissuedestruction.(4)Lysisof infectedhostcellsallowsmicrobialdissemination.(5) which in situ may exist in granulomatous lesions. Coordinatedlysisof infectedhostcellsandsubsequentactivationof potentphagocytesallows microbialtransmissionfroma protectiveto an aggressivesurrounding.For furtherdetailssee Granuloma. A granuloma is composed of mononuclear text. phagocytes at different developmental stages -includ•
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ImmunologyToday, VoL 9, No. 6, 1988
ing epitheloid cells, multinucleated giant cells and immigrant monocytes - as well as of CD4 + and CD8 + T lymphocytes. Granulomatous lesions represent the histologic focus for the defence against many intracellular microbes, perhaps best exemplified in tuberculosis of the lung. In tuberculous granulomas, tubercle bacilli are found inside macrophages apparently unable to destroy their inhabitants. While these cells may already fulfill an important role in protection by containing the pathogens within discrete foci and preventing them from dissemination, in the long run, they are insufficient for microbial elimination and present a protective niche for persistent microbial pathogens. In this situation, lysis could become a necessary prerequisite for microbial elimination. Discharge of microbes into the unpleasant environment of a hypoxic necrotic granulomatous center may directly lead to growth inhibition and hence be beneficial. On the other hand, discharge into the blood or the bronchioalveolar system will allow microbial dissemination to secondary tissue sites or shedding to other individuals. These harmful sequelae, as well as tissue destruction, predominate in caseous granulomas lacking phagocytes adequately equipped for intracellular killing. In contrast, in productive granulomas monocytes with high antibacterial potential are present. Mycobacteria released through cytolysis are then engulfed by these phagocytes which fulfill the final steps of microbial elimination after lymphokine activation. Concluding remarks
The scenario described is highly speculative. However, it illustrates the complexity of the host-pathogen relationship in intracellular microbial infections where a single cell and a single mechanism may both benefit and threaten the host. Further studies are required to elucidate the conditions leading to the activation of CD8 + CTL by exogenous antigens as weii as their effector role in microbial infections. Both basic and applied research may benefit from such studies. References
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MHC classII expressionby the gut epithelium The intestinal epithelium represents a barrier between luminal antigen and the underlying local immune system. Expression of MHC class II antigens - normally associated with cells of defined immunological function - and the alteration in the pattern of expression of these antigens found in certain diseases, has aroused considerable interest. In this article, Paul Bland reviews what is known of class II molecules on the intestinal epithelium and discusses their relevance in the wider context of immune regulation in the gut. Immune responses to food antigens and to intestinal pathogens are important both in protection of the host and in the pathogenesis of gastrointestinal diseases. Because of the need for rapid induction and regulation of appropriate immune responses to food and microbial antigens in the gut, it is likely that these processes take place at, or close to, the site of absorption. Analysis of the cellular and molecular mechanisms of antigen handling within the gut are therefore vital to our understanding of the aetiopathogenesis of gut disease and to the rational development of prophylactic regimes. Although the cellular circuitry within Peyer's patches is i i~,~l,F,~..k+~,,,.,ll,, ; . . . . I. ,A,,.J : ~ ~ L . . . . . . I _±~ ul,u~..luuI.EUl)" IIIVUIVI~U III LII~ r~cJUlaUOrl of responses to orally presented antigens, the tissue at the interface between luminal antigen and the local immune system the epithelium of the small intestine - is also a possible participant in the induction and regulation of the immune response. Since 1978, when class II molecules of the major histocompatibility complex (MHC) were first found in the small intestinal epithelium, a greater awareness has developed that the epithelium may be intimately involved in selective antigen handling and in the control of appropriate immune responses.
Constitutive expression of epithelial class II molecules Expression of MHC class II molecules by the intestinal epithelium was first described in the guinea-pig by Wiman and colleagues ~and subsequently in the mouse 2, the rat3 and humans4. A common gross distribution is found in all species. Thus, class II molecules are expressed by the small intestinal epithelium only, large bowel epithelium being negative, or only faintly positive, in healthy tissues~9. The tissue distribution (Fig. 1) is restricted to the upper two-thirds of the villus: only fully differentiated absorptive epithelial cells (enterocytes) constitutively express class II molecules. They are not expressed by mucus-secreting goblet cells. 174
Department of Veterinary Medicine, University of Bristol, Langford House, Langford,BristolBS187DU, UK.
Paul Bland Expression at the light microscope level in isolated enterocytes appears to be restricted to the basolateral enterocyte membrane 1°. Electron microscopy confirms this distribution in the mouse 2.11for both class I and class II antigens, suggesting that the mobility of MHC antigens in the enterocyte membrane is restricted by the junctional complex. More recently, however, microvillus staining has been reported in human tissue6. Although granular staining in the supranuclear region has been noted both in tissue sections9 and in isolated enterocytes 1°, the ultrastructural localization of enterocyte class II molecules on specific organelles has, unfortunately, not been reported. The appearance under light microscopy (Fig. la and b), however, is consistent with the intracellular accumulation of class II molecules within the vesicular endosome compartment in the apical cytoplasm of the enterocyte. This interpretation corresponds with the situation in B cells in which the distribution of class II molecules overlaps in the endosome compartment with the recycling transferrin receptor, allowing the uncoupling of the class II invariant chain to take place before expression at the cell membrane ~2. Nothing is known of the recycling of class II molecules within enterocytes, but it seems likely that if they have an antigen receptor function within the epithelium, their restricted distribution in the plasma membrane would allow for binding and release of the antigenic ligand in an endosomebasolateral membrane direction only (see below).
Induced expression of epithelial class II molecules. Although class II molecules are expressed constitutively at low density in the normal distribution described above, the distribution and intensity of expression can be altered by immune parameters~ Thus, in Trichinella spiralis-induced inflammation 13 and graft-versus-host disease (GVHD)3,13 expression is intensified and, moreover, de novo expression occurs in immature crypt epithelial cells (Fig. lc and d). Similarly, those gastrointestinal disorders associated with primary or secondary immune defects show changes from the normal epithelial class II expression. In both coeliac disease and dermatitis herpetiformis, where villus atrophy and crypt hyperplasia are classical features, both residual immature enterocytes and crypt cells express class II (Refs 14,15). Moreover, the surface epithelium in untreated coeliac patients expresses the products of the HLA-DP and DQ loci in addition to DR which is expressed constitutively 16. ~ ) 1988, Elsevier Publications, Cambridge
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