Immunology Today, voL 7, No. 10, 1986
the veto cell-mediated inactivation of anti-H-43 a CTLp. This explanation is supported by the fact4 that the (anti-H-43 a) CTLp of H-43 b responder mice are primed by immunization with H-2 compatible H-43 a donor spleen cells carrying additional minor-H-alloantigens (e.g. H-Y antigen in the case of female H-43 b responder mice)4. This priming probably occurs via the process of linked recognition. If CTLp in the thymus are much more vulnerable to the veto mechanism than those in the spleen, due either to the unique thymic environment or to their cellu-
rostrum lar immaturity, the above rescue from the CTLp tolerance would not take place in the thymus. Consequently, the developing anti-self CTLp would be continuously eliminated in the thymus, and this, in conjunction with the concept of 'positive selection '6, may account in part for the massive cell death occurring in the thymus.
Hiromichi Ishikawa, Tomoo Hino, Hidehito Kato, Hiroko Suzuki and Kazuhisa Saito
Departmentof Microbiology,KeioUniversity Schoolof Medicine,Shinjuku-ku,Tokyo160, Japan
References 1 Muraoka, S., Ehman, D.L and Miller, R.G. (1984) Eur. J. Immunol. 14, 1010-1016 2 Ishikawa, H., Kubota, E., Suzuki, H. et a/. (1985)J. Immunol. 134, 2953-2959 3 Ishikawa, H., Suzuki, H., Hino, T. etaL (1985)2. Immunol. 135, 3681-3685 4 Ishikawa, H., Hino, T., Kato, H. etaL (1986) J. Immunol. 137 (in press) 5 Schwarz, R.H. (1986)Adv. ImmunoL 38, 31-201 6 Jerne, N.K. (1971 ) Eur. J. Immunol. 1, 1-9
HIV infection:facts and hypotheses The etiological agent of the acquired immunodeficiency syndrome (AIDS) was first isolated in 1983 and called lymphadenopathy associated virus (LAY)I. Other isolates of similar viruses have been named HTLV-III or ARV2'3. Numerous studies of their biological and molecular characteristics have confirmed that they are all different isolates of the same virus for which the name human immunodeficiency virus (HIV) has recently been proposed by an intemational committee 4. The current understanding of HIV's biological properties, supported by epidemiological and clinical observations, enables David Klatzmann and John Gluckman to propose a general model for its pathogenicity: a complex pathway of interaction between host and virus properties controls the stepwise evolution from primary infection to disease. Very low amounts of HIV infectious ~articles can be found in the serum, sperm, saliva, tears, or any other body fluid of an infected individual. This is probably why HIV is a poorly contagious virus and is almost exclusively transmitted via sexual contact or blood products. Indeed, except for massive injection of contaminated fluids (e.g. transfusion of plasma and factor VIII), we think that infection probably occurs more efficiently when infected lymphocytes are transmitted, each of them capable of producing thousands of viral particles. This also implies that allospecific T lymphocytes will be the first victims. Such a mode of infection has already been documented for the ovine lentivirus visna whose experimental infection is more easily obtained by injection of infected cells (G. Querat, unpublished). Of particular interest is the recent observation that the number of lymphocytes in the sperm is elevated in individuals with a history of repeated sexually transmitted diseasess. After exposure to HIV some individuals may resist infection, since 30% of the regular sex partners of infected patients do not show any evidence of infection 6. Animals models offer two possible explanations for such resistance:
Laboratoired'lmmunologieN~phrologiqueet Transplantation H(~pital, La Pitie-Salp~triere,75634ParisCedex13, France ~) 1986, Elsevier Science Publishers B.V., Amsterdam 0167 4919/86/$02 O0
D. Klatzmannand J.C. Gluckman (1) Sustained experimental bovine leukemia virus infection can be achieved by transmission of infected blood, but clearly depends on the inoculum size (A. Burny, unpublished). (2) After challenge with high doses of feline leukemia virus, approximately 30% of the cats resist infection without any evidence of specific cellular immune response, another 40% demonstrate a specific cellular and humoral response leading to immune protection, leaving only 30% to develop signs of chronic infection 7. With respect to these findings, it is possible that either an immune response or a classical non-specific clearance mechanism may protect against primary challenge with HIV.
Tropismof HIV Like most viruses HIV has preferential target cells in which it replicates after entering the organism. It was indeed the remarkable selective CD4 ÷ lymphocyte defect noted in AiDS patients8-1° that led us to investigate whether HIV displayed a selective tropism for CD4 ÷ lymphocytes: in vivo, HIV was only recovered from CD4 ÷ lymphocytes of infected patients, and only normal CD4 ÷ and not CD8 ÷ cells could be successfully infected in vitro 11. While HTLV-I, the first human retrovirus to be discovered, has also been claimed to display a CD4 + lymphotropism on the basis that HTLV-I infected cells are often CD4 +, recent experiments have demonstrated that CD8 ÷ lymphocytes are also infected and transformed by the virus 12. However, it has subsequently been noted that HIV can be present, or even replicate in various other cell types. Viral particles have thus been observed in normal B lymphocytes and cells of the monocytemacrophage lineage 13 , and HIV has been shown to actually replicate in some EBV-transformed B lymphoblastoid cell lines14 and cell lines of monocytic origin 1 2 1' 5 . Similarly, viral antigens or particles have been found in follicular dendritic cells of the lymphnode reticulum 16-18 and in cells of the brain 19, that are either passenger lymphocytes and monocytes, or microglial cells (Ref. 20, and R. Vazeux, unpublished)(Table 1).
291
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-rostrum Such tropism can well account for the clinical signs of HIV infection: predominantly CD4 ÷ immune deficiency, lymphadenopathy, brain lesions, and lymphoma of B cell origin. It remains to be determined whether Kaposi's sarcoma as well as some intestinal and lung lesions are due to direct viral infection or are merely signs of opportunistic infections or tumors. Cell susceptibility to viruses can be controlled either at the genomic level by regulatory sequences, as described for HTLV-121, or at the membrane level through the interaction between a specific receptor and viral proteins, or at both levels. In the case of HIV, transfection experiments in various cell types, including animal cells in which HIV cannot replicate, have not shown any cell specificity for its regulatory sequences 22'23. On the contrary, there is now strong evidence that the CD4 molecule is (part of) an HIV-specific receptor. Indeed, monoclonal antibodies directed against different epitopes of the CD4 molecule can prevent HIV infection of CD4 + lymphocytes24. Similarly, they block penetration of, and fusion induced by, pseudotypes constructed with vesicular somatitis virus inserted in an HIV envelope2s. Recently, co-precipitation experiments have shown that HIV binding to CD4 + lymphocytes involves a close association between the viral envelope glycoprotein (gp 1 10) and the CD4 molecule 26. However, the exact nature of the HIV receptor may be more complex. Recent experiments have shown that transfection of the CD4 gene leads to virus binding in CD4 + transfected human cell lines (Hela, Raji) or murine fibroblasts27. However, the latter27 as well as human/ murine CD4 ÷ T-lymphocytes hybrids28 cannot sustain subsequent virus replication. Furthermore, there is some evidence that, at a given time, only a fraction of normal CD4 + lymphocytes can be infected by HIV, since no more than 5-10% of these lymphocytes express cytoplasmic viral antigens or are affected by HIV's cytopathic effect at the peak of virus replication in vitro 24. Table 1. Specificity of the cellular tropism of HIV Cell types and organs
Presence of Replication b in antigen or normal transformed viral cells cells particles a
Human: T lymphocytes: CD4 + + + + CD8 + _ _ B lymphocytes + + monocyte/macrophages + + + follicular dendritic cells + ? microglial brain cells + + ? Animal: chimpanzee, baboon or + + ? Rhesus lymphocytes other primate ? lymphocytes rodent lymphocytes or fibroblasts CD4-transfected cells: human + murine CD4+ murine/human T cell hybrids aHIV particles and/or antigens have been respectivelydetected by electron microscopy or immunohistological methods. bVirus replication has been determined by reversetranscriptase assaysin cell cultures, or by viral RNA detection by in situ hybridization.
To explain these findings, we propose that HIV cell infection is controlled by two different mechanisms: binding, which clearly depends on virus interaction with CD4; and penetration, which is controlled either by another molecule (not specific for CD4 ÷ lymphocytes) or by the availability (i.e. number, configuration, and mobility) of the CD4 molecule at the cell membrane. The results29 we obtained using a binding assay with fluorescein-isothiocyonate labelled HIV (HIV-FITC) are in line with this hypothesis. Indeed, while others have observed the binding of purified HIV to approximately 100% of CD4 ÷ lymphocytes using an indirect staining with purified human anti-HIV immunoglobulins 3°, HIVFITC stains only 10% of CD4 + lymphocytes. Since this assay has been shown to be specific, and because preincubation of CD4 ÷ lymphocytes with heatinactivated HIV-FITC before HIV infection blocks further virus replication 29, we think that this test detects the HIV high affinity binding, which is the only binding to result in virus penetration into the cell. Using this technique we also showed that 'HIV receptors' could be functionally regulated at the cell membrane. We used various agonist or antagonist stimuli of cell activation, including mitogens, monoclonal antibodies and drugs, to analyse the modifications of HIV binding independently of any change in the expression of the CD4 molecule. Functional interaction with early activation events, such as preincubation with cyclosporin A, a drug known to inhibit T-lymphocyte activation, can modify HIV receptor expression so as to render the cells unable to bind HIV (Table 2)31,32. As alternative models for HIV receptors emerge from all these findings (Fig. 1). First, CD4 is the HIV receptor on CD4 expressing cells. The virus penetration is controlled by the availability (number, mobility, configuration) of CD4 at the cell membrane. However, cells normally negative for CD4 expression can occasionally be infected provided they express a CD4-1ike molecule or a phagocitic capability facilitated by opsonization through Fc receptors. Secondly, CD4 mediates HIV binding to CD4 + cells, but another molecule, which does not necessarily interact with the viral proteins, is part Of the 'functional receptor' which is required for virus penetration. Such a two-step infection (binding and penetration) has already been described for the endocytosis of West Nile virus in macrophages 33. In any case, signals which interfere with cellular activation might either 'freeze' the membrane or modify the relationship of the receptor components. The infected cell HIV is a lentivirus34. Retroviruses of this subgroup cause slowly developing non-tumorigenic diseases and replicate through complementary DNA (cDNA) intermediates that can be found either as unintegrated circular or linear double strands, or as integrated cDNA in the host's genome (provirus form). One of the main characteristics of lentiviruses is that their replication is restricted, which results in latent cell infection. For example, monocytes, the preferential target of visna, are latently infected without expressing viral proteins but their activation and differentiation to macrophages leads to virus replication 3s. As a consequence, the latently infected cells remain invisible to the immune system and persist. Similarly, we have already
ImmunologyToday,vol. 7, No. 10, 1986
rostr.m
-
Table 2. Inhibition of HIV binding on normal CD4 +
T-lymphocytes and subsequent replication after culture Cell pretreatment a HIV-FITCbinding b HIV replication c -
-I-
+
CD4 antibodies a
-
-
- (transient) + +
- (transient) Not tested Not tested
PHA PMA Con A CD3 antibody CD2 antibodies
+ -
Not tested -
Cyclosporin A
-
-
aBefore HIV-FITC incubation, cells were preincubated for 30 min. to 1 h with: saturating concentration of CD4 monoclonal antibodies optimal mitogenic concentration of PHA, PMA or Con A mitogenic concentration of anti-CD3 saturating concentration of anti-CD2. Only antibodies directed to CD2 epitope 1 induce the inhibition of binding and replication 500 ng/ml of Cyclosporin A for 1 h blsothiocyanate-labelled, heat-inactivated, virus (HIV-FITC)was incubated for 1 h with the target ceils, before the percentage of positively labelled virus was determined by direct immunofluorescence29. cVirus replication was determined as described1.
demonstrated that activation of infected lymphocytes is necessary for HIV replication 36. So far, all lymphocyte stimuli tested - mitogens (PHA, ConA, WGA), soluble antigens extracted from bacteria, viruses, fungi, or allogeneic cells - result in virus replication37: When initiated, HIV replication is very efficient because of the powerful viral cis- and trans-acting regulatory elements 38~o. These two properties, latency and powerful activation, may be differently expressed according to the infected cell types. For example, comparison of virus replication in CD4 + lymphocytes and CD4 + transformed cell lines has shown that, with respect to the percentage of infected cells, replication is more efficient in normal lymphocytes 41. Such a heterogeneity can also be noted among the cells of continuous lines, in most of which the virus is replicating without any additional stimulation while a small percentage latently harbors the virus47.
CD4 is the only I spec f c receptor I
I
O t h e r molecules p a r t i c i p o t e in the receptor
Virus
Virus
CD4 + cell
CO4- cell
]
Virus
Virus
CD 4 ~"cell
Penetration in o CD4* Penetration in cell requires adequate other cells con binding between viral use either CD4proteins and CD 4 like epitopes or other non specific receptors like FC
Other cell
Penetration of H I V in CD4" lymphocytes requires both molecules
Penetration in other cells con use eitherone of these two specific binding proteins with or without other non specific molecules
Fig.1. Alternative hypotheses for HIV receptor. bodies can protect non-infected CD4 lymphocytes from such fusion process ~. The question remains: why does the cell die? Activation of a viral or cellular cytotoxic protein has been postulated 4s,46. Moreover, it has recently been proposed that this cell damage could be the result of the interaction between viral and CD4 protein on the membrane of the same cell47. Alternatively, it could be due to intracytoplasmic interaction of viral protein and CD447.48 which also explains why CD4 is no more detectable at the cell membrane during virus replication, while specific CD4 mRNA is present and even up-regulated 48. However, all these mechanisms are still hypothetical and the only evidence of a cytopathic effect in vivo is the description of giant cells in the brain of infected individuals2°.
Soluble proteins The follow-up of CD4+-enriched cultured cells after infection shows the progressive disappearance of CD4 from the membrane of most cells, although only 5-10% of them express cytoplasmic viral antigens 1~. This rather indirect effect of viral replication can be explained by the masking of the CD4 molecule by free soluble viral proteins. These proteins are difficult to detect, probably because the epitopes that are usually recognized are those involved in the interaction. Free viral proteins are produced in larger amounts than the viral RNA available for encapsidation, due to the fact that HIV activating elements (trans-activating factors) act, at least partly, at the translational level 49.
The effects of viral replication
These are shown in Fig. 2. Cytopathogenicity The first biological property of HIV which distinguished it from other previously described human retroviruses was its strong cytopathic effect 36. At the time, or soon before reverse transcriptase activity becomes detectable in culture supernatants, giant multinucleated cells can be observed 11. This cell fusion process probably depends on the interaction between the viral proteins (gp 110) expressed at the membrane of virus producing cells and the CD4 protein expressed by other - not necessarily infected - cells. Indeed we have shown that the addition of normal CD4 + MOLT-4 cells to infected CEM cells results in rapid appearance of giant cells42, and others have recently observed that anti-CD4 monoclonal anti-
Normal uninfecfed CD4 + cells Activation
®/.~ "~ ®
/~
J f~.~..,~:..~.~-,.."~
o [nfected cells
~'~ celhl
particles ' -
\'-.-J )
j
® "~J.,
®
'~,J®
""
"~'~Virot soluble
®
__~CO)
Cytopothic e f f e c t plus possible fusion to non-infected cells
Inhibition of T lymphocytes
~...
proteins
"
\\\
"~" ( ~
celIP°lycl°nalactlvationB
-%
Destruction of normal cells coated with
virol proteinl
Fig.2. The effects of viral replication.
293
Immunology Today, vol. 7, No. 10, 1986
-ros/rum
will help to delineate the properties of each viral component. It is obvious that the ultimate result of viral replication, which seems to occur in 10 -3 to 10 -5 lymphocytes s~, will be the release of numerous viable viral particles, infecting additional cells, expanding the virus reservoir (Fig. 2). The immune responseto HIV Humoral response
Antibody responses observed in HIV-infected individuals indicate that all of HIV's gene products are immunogenic, but the predictive value of each pattern (antibodies directed either at the gag and/or the env products) has not yet been determined. Apparently conflicting results have been reported regarding neutralizing antibodies 42,52,s3. But, if the conclusions of the authors are different, their results are similar, showing that when they exist, neutralizing antibodies are present in very low concentrations. Genetic variability
Fig.3. Relationship between destruction of the follicular dendritic network and the presence of CD8+ infiltrates of germinative centres in lymph nodes of HIV-infected patients. (a) Disrupted follicular dendritic cells stained by a CVK anti-HIV antibody 66. (b) Double-labelling technique showing infected FDC (in red) and CD8+ cells (in blue) at their contact. (c) An activated CD8+ cell presenting a cytotoxic granule, in close contact with a destroyed FDC, nevertheless characterized by its desmosome. (Pictures kindly provided by C. Parravicini; see Ref 18 and 62.)
294
This large release of free soluble proteins after cell lysis might also be responsible for various effects on the immune system, such as a decrease in lymphocyte proliferation capabilities 1ts0, a polyclonal immunoglobulin secretion by B lymphocytes, or - on,the contrary - a depressed responsiveness to polyclonal B-cell activators such as PWM, SAC, and EBVs°. In this respect, the availability of recombinant proteins or synthetic peptides
One of the most striking properties of HIV is its important genetic variability, especially in the env and F proteins 54's5. Such variability might indeed represent a potential selective advantage of mutations in these regions and could represent an "escape' mechanism from the immune response as described for other lentiviruses (EIAV) 56. Follow-up of the virus in a single patient using restriction mapping and sequencing has not shown any important genomic shift 57. In contrast, comparing the restriction map patterns of two isolates, one from an infected blood donor, the other from his recipient, showed an individual adaptation of the virus; although both isolates resembled each other more than two non-related isolates would, they were still clearly different s8. It is also important to note that, in spite of the fact that some individuals are potentially contaminated by hundreds of different viruses, only one can be isolated from their lymphocytes. To explain these findings, we propose thatthis genetic variability enables the virus to express - early after initial infection - those epitopes to which the host is a low responder, making clear that once an optimally adapted virus emerges, little change in its 'antigenic make-up' is expected to be observed. Such a 'low-high responder' immune mechanism has been documented for the transplantation antigens, and it is interesting to note here that HIV env and/or F proteins possibly share some homology with MHC class II proteins 59 '60 . Molecular analysis of different viral isolates has clearly demonstrated that some conserved regions are shared by all isolates s4'55, which might be crucial for the conservation of some of the basic viral properties, i.e. the CD4 tropism. Again, the availability of synthetic peptides or recombinant proteins expressed in a different immunogenic context may help to develop a response not observed during natural infection. Cellular response
The early augmentation of the number of CD8 ÷ lymphocytes is suggestive of a specific proliferative response against HIV infected cells. Recently HIV-specific HLA-class I-restricted cytotoxic T cells and lymphokineactivated killer cells have been described 61. However,
For technical reasons we are unable to reproduce this figure in colour. See the October issue of Immunology Today for full colour illustration,
Immunology Today, voL 7, No. 10, 1986
rostru since latently infected cells do not express viral antigens, and because expression of these antigens is associated with the cell death, cytotoxic cells may not play a significant role in the control of HIV infection. On the contrary, they may participate in an 'autoimmune' reaction. Circumstantial evidence indicates that cytotoxic CD8 ÷ cells might be involved in the destruction of HIV-presenting follicular dendritic cells (FDC) of the lymph node reticulum 18. Sequential follow-up of lymph node biopsies from lymphadenopathy patients shows that at first the germinal center reticulum is enlarged and many viral antigens are present on the FDC, while later evolution of the disease is accompanied by penetration of CD8 ÷ cells into the germinal centers. Such cells are found in close contact with FDC, and their presence is correlated with destruction of the latter62. It can be hypothesized that the same mechanism leads to an autoimmune destruction of non-infected CD4 ÷ lymphocytes coated with soluble proteins specifically bound to the HIV receptor (Fig. 3)63. Immune status of the host Immune status of the infected host might be crucial for controlling immune response to HIV. The pre-existing mild immune defects that can be observed in healthy homosexuals before HIV infection 36, could tip the balance in favor of establishment of infection rather than virus control. The multiple viral, bacterial, or fungal infections observed in these subjects are probably the cause of such pre-existing immune abnormalities. In addition, they will contribute, after primary infection, to the permanent activation of the immune system, which enhances HIV dissemination. In this respect, activation of allospecific T cells might be crucial. The allogeneic sperm lymphocytes which can occasionally enter the organism during sexual intercourse may be responsible for a graft-versus-host-like reaction leading to immunodeficiency 64. We think that they also play a major role because they could activate already infected alloantigen-specific T-cells, leading to a more rapid viral dissemination. Genetic factors As in other viral infections, genetic factors could control the level of the immune response against HIV. A strong response might result in the elimination of a few contaminating viral particles, but also in an accelerated evolution towards immune deficiency by enhancing the autoimmune destruction of CD4 + cells. Except for the case of Kaposi's sarcoma, which is significantly associated with HLA-DR 5 (Ref. 65), there is currently no evidence of major histocompatibility gene association with HIV-related diseases. 'Suicide" of HIV specific cells The local recruitment of HIV specific CD4 + lymphocytes at the site of the immune reaction might result in their 'suicide' since they will be exposed to viral infection. Such a mechanism may explain the disappearance of antibodies specific for viral core protein observed during the progression of the disease. The persistence of antibodies specific for the envelope will then represent the stigma of early infection, before the variation of the envelope.
-
A model for the pathology of HIV infection We propose that progressive infection is a step-wise process, each step involving more cells and more extensive virus replication. The first step in infection represents the group of healthy carriers. Few CD4 + lymphocytes (being the target cells of primary infection) are infected. Virus replication occurs only after their activation, an event unlikely to happen since T-lymphocytes need to be specifically stimulated through its antigen receptor. A balance exists between the occasional stimulation of infected cells and their 'natural clearance', but each viral replication event increases the pool of infected cells and augments the probability that other cells than CD4 ÷ lymphocytes can be infected. Later, in some individuals, multiple infections or allostimulation will finally enhance viral replication. When macrophages are involved, the chance of virus replication probably increases because these cells are more often (not specifically) stimulated than lymphocytes. Progressive increase of viral replication will enhance the impairment of the immune system. B lymphocytes will be affected through their regulation by CD4 + cells but also by the effect of soluble viral proteins. A new balance is established, with virus replicating and attacking new cells on one side, an autoimmune reaction on the other, and immunodeficiency in progress. This fragile status quo might last until replication is suddenly enhanced when, for example, EBV-transformed cells become infected. They will be a reservoir for permanent and active replication, which will contribute to the final active destruction of the immune system and continuous dissemination of the virus to other organs. Opportunistic infections or tumors can then develop. Many questions remain to be answered concerning HIV infection. This partly hypothetical model could contribute to a better understanding of some aspects of the immune reaction and will enable the delineation of new therapeutical strategies.
We thank Prof. L. Montagnier for fruitful discussionsand Dr J.P. Van Eendenburg for critical review of the manuscript. Supported in part by grants from CNAMTS, ARC and University Paris VI. References 1 Barr~-Sinoussi,F., Chermann, J.C., Rey,F. eta/. (1983) Science 220, 868 2 Popovic, M.N., Sarngadharan, MG., Read, E. eta/. (1984) Science 224, 497 3 Levy,J., Hoffman, A.D., Kramer, S.M. eta/. (1984) Science 225, 840 4 Coffin, J., Haase,A., Levy,J.A. eta/. (1986) Nature (London) 321, 10 5 Coates, R.A., Soskolne, Cl., Read, Se. etaL (1986)Abstract, International Conference on AIDS, Paris 60smond, D., Moss, A., Kelly,T. eta/. (1986) Abstract, international Conference on AIDS, Paris 7 Hardy, W.J., Jr (1980) FelineLeukemia Virus33, Elsevier/ North-Holland, New York 8 Seligman,M., Chess,L., Fahey,J.L. etal. (1984) N. Engl. J. Med. 311, 1286 9 Cavaill~-Coll,M., Messiah,A., Klatzmann, D. etal. (1984) Clin. Exp. Immunol. 57, 511 10 Gluckman, J.C., Klatzmann, D., CavaillE-Coll,M. eta/. (1985) Clin. Exp. Immunol. 60, 8 11 Klatzmann, D., Barr~-Sinoussi,F., Nugeyre, M.T. eta/. (1984) Science 225, 59
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eds), p. 139, NewYork, Masson Publishing USA 65 Chassagne, J., Verrelle, P., Dionet, C. etaL (1986) J. ImmunoL 136, 1442
the veto cell-mediated inactivation of anti-H-43 a CTLp. This explanation is supported by the fact4 that the (anti-H-43 a) CTLp of H-43 b responder mice are primed by immunization with H-2 compatible H-43 a donor spleen cells carrying additional minor-H-alloantigens (e.g. H-Y antigen in the case of female H-43 b responder mice)4. This priming probably occurs via the process of linked recognition. If CTLp in the thymus are much more vulnerable to the veto mechanism than those in the spleen, due either to the unique thymic environment or to their cellu-
rostrum lar immaturity, the above rescue from the CTLp tolerance would not take place in the thymus. Consequently, the developing anti-self CTLp would be continuously eliminated in the thymus, and this, in conjunction with the concept of 'positive selection '6, may account in part for the massive cell death occurring in the thymus.
Hiromichi Ishikawa, Tomoo Hino, Hidehito Kato, Hiroko Suzuki and Kazuhisa Saito
Departmentof Microbiology,KeioUniversity Schoolof Medicine,Shinjuku-ku,Tokyo160, Japan
References 1 Muraoka, S., Ehman, D.L and Miller, R.G. (1984) Eur. J. Immunol. 14, 1010-1016 2 Ishikawa, H., Kubota, E., Suzuki, H. et a/. (1985)J. Immunol. 134, 2953-2959 3 Ishikawa, H., Suzuki, H., Hino, T. etaL (1985)2. Immunol. 135, 3681-3685 4 Ishikawa, H., Hino, T., Kato, H. etaL (1986) J. Immunol. 137 (in press) 5 Schwarz, R.H. (1986)Adv. ImmunoL 38, 31-201 6 Jerne, N.K. (1971 ) Eur. J. Immunol. 1, 1-9
HIV infection:facts and hypotheses The etiological agent of the acquired immunodeficiency syndrome (AIDS) was first isolated in 1983 and called lymphadenopathy associated virus (LAY)I. Other isolates of similar viruses have been named HTLV-III or ARV2'3. Numerous studies of their biological and molecular characteristics have confirmed that they are all different isolates of the same virus for which the name human immunodeficiency virus (HIV) has recently been proposed by an intemational committee 4. The current understanding of HIV's biological properties, supported by epidemiological and clinical observations, enables David Klatzmann and John Gluckman to propose a general model for its pathogenicity: a complex pathway of interaction between host and virus properties controls the stepwise evolution from primary infection to disease. Very low amounts of HIV infectious ~articles can be found in the serum, sperm, saliva, tears, or any other body fluid of an infected individual. This is probably why HIV is a poorly contagious virus and is almost exclusively transmitted via sexual contact or blood products. Indeed, except for massive injection of contaminated fluids (e.g. transfusion of plasma and factor VIII), we think that infection probably occurs more efficiently when infected lymphocytes are transmitted, each of them capable of producing thousands of viral particles. This also implies that allospecific T lymphocytes will be the first victims. Such a mode of infection has already been documented for the ovine lentivirus visna whose experimental infection is more easily obtained by injection of infected cells (G. Querat, unpublished). Of particular interest is the recent observation that the number of lymphocytes in the sperm is elevated in individuals with a history of repeated sexually transmitted diseasess. After exposure to HIV some individuals may resist infection, since 30% of the regular sex partners of infected patients do not show any evidence of infection 6. Animals models offer two possible explanations for such resistance:
Laboratoired'lmmunologieN~phrologiqueet Transplantation H(~pital, La Pitie-Salp~triere,75634ParisCedex13, France ~) 1986, Elsevier Science Publishers B.V., Amsterdam 0167 4919/86/$02 O0
D. Klatzmannand J.C. Gluckman (1) Sustained experimental bovine leukemia virus infection can be achieved by transmission of infected blood, but clearly depends on the inoculum size (A. Burny, unpublished). (2) After challenge with high doses of feline leukemia virus, approximately 30% of the cats resist infection without any evidence of specific cellular immune response, another 40% demonstrate a specific cellular and humoral response leading to immune protection, leaving only 30% to develop signs of chronic infection 7. With respect to these findings, it is possible that either an immune response or a classical non-specific clearance mechanism may protect against primary challenge with HIV.
Tropismof HIV Like most viruses HIV has preferential target cells in which it replicates after entering the organism. It was indeed the remarkable selective CD4 ÷ lymphocyte defect noted in AiDS patients8-1° that led us to investigate whether HIV displayed a selective tropism for CD4 ÷ lymphocytes: in vivo, HIV was only recovered from CD4 ÷ lymphocytes of infected patients, and only normal CD4 ÷ and not CD8 ÷ cells could be successfully infected in vitro 11. While HTLV-I, the first human retrovirus to be discovered, has also been claimed to display a CD4 + lymphotropism on the basis that HTLV-I infected cells are often CD4 +, recent experiments have demonstrated that CD8 ÷ lymphocytes are also infected and transformed by the virus 12. However, it has subsequently been noted that HIV can be present, or even replicate in various other cell types. Viral particles have thus been observed in normal B lymphocytes and cells of the monocytemacrophage lineage 13 , and HIV has been shown to actually replicate in some EBV-transformed B lymphoblastoid cell lines14 and cell lines of monocytic origin 1 2 1' 5 . Similarly, viral antigens or particles have been found in follicular dendritic cells of the lymphnode reticulum 16-18 and in cells of the brain 19, that are either passenger lymphocytes and monocytes, or microglial cells (Ref. 20, and R. Vazeux, unpublished)(Table 1).
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-rostrum Such tropism can well account for the clinical signs of HIV infection: predominantly CD4 ÷ immune deficiency, lymphadenopathy, brain lesions, and lymphoma of B cell origin. It remains to be determined whether Kaposi's sarcoma as well as some intestinal and lung lesions are due to direct viral infection or are merely signs of opportunistic infections or tumors. Cell susceptibility to viruses can be controlled either at the genomic level by regulatory sequences, as described for HTLV-121, or at the membrane level through the interaction between a specific receptor and viral proteins, or at both levels. In the case of HIV, transfection experiments in various cell types, including animal cells in which HIV cannot replicate, have not shown any cell specificity for its regulatory sequences 22'23. On the contrary, there is now strong evidence that the CD4 molecule is (part of) an HIV-specific receptor. Indeed, monoclonal antibodies directed against different epitopes of the CD4 molecule can prevent HIV infection of CD4 + lymphocytes24. Similarly, they block penetration of, and fusion induced by, pseudotypes constructed with vesicular somatitis virus inserted in an HIV envelope2s. Recently, co-precipitation experiments have shown that HIV binding to CD4 + lymphocytes involves a close association between the viral envelope glycoprotein (gp 1 10) and the CD4 molecule 26. However, the exact nature of the HIV receptor may be more complex. Recent experiments have shown that transfection of the CD4 gene leads to virus binding in CD4 + transfected human cell lines (Hela, Raji) or murine fibroblasts27. However, the latter27 as well as human/ murine CD4 ÷ T-lymphocytes hybrids28 cannot sustain subsequent virus replication. Furthermore, there is some evidence that, at a given time, only a fraction of normal CD4 + lymphocytes can be infected by HIV, since no more than 5-10% of these lymphocytes express cytoplasmic viral antigens or are affected by HIV's cytopathic effect at the peak of virus replication in vitro 24. Table 1. Specificity of the cellular tropism of HIV Cell types and organs
Presence of Replication b in antigen or normal transformed viral cells cells particles a
Human: T lymphocytes: CD4 + + + + CD8 + _ _ B lymphocytes + + monocyte/macrophages + + + follicular dendritic cells + ? microglial brain cells + + ? Animal: chimpanzee, baboon or + + ? Rhesus lymphocytes other primate ? lymphocytes rodent lymphocytes or fibroblasts CD4-transfected cells: human + murine CD4+ murine/human T cell hybrids aHIV particles and/or antigens have been respectivelydetected by electron microscopy or immunohistological methods. bVirus replication has been determined by reversetranscriptase assaysin cell cultures, or by viral RNA detection by in situ hybridization.
To explain these findings, we propose that HIV cell infection is controlled by two different mechanisms: binding, which clearly depends on virus interaction with CD4; and penetration, which is controlled either by another molecule (not specific for CD4 ÷ lymphocytes) or by the availability (i.e. number, configuration, and mobility) of the CD4 molecule at the cell membrane. The results29 we obtained using a binding assay with fluorescein-isothiocyonate labelled HIV (HIV-FITC) are in line with this hypothesis. Indeed, while others have observed the binding of purified HIV to approximately 100% of CD4 ÷ lymphocytes using an indirect staining with purified human anti-HIV immunoglobulins 3°, HIVFITC stains only 10% of CD4 + lymphocytes. Since this assay has been shown to be specific, and because preincubation of CD4 ÷ lymphocytes with heatinactivated HIV-FITC before HIV infection blocks further virus replication 29, we think that this test detects the HIV high affinity binding, which is the only binding to result in virus penetration into the cell. Using this technique we also showed that 'HIV receptors' could be functionally regulated at the cell membrane. We used various agonist or antagonist stimuli of cell activation, including mitogens, monoclonal antibodies and drugs, to analyse the modifications of HIV binding independently of any change in the expression of the CD4 molecule. Functional interaction with early activation events, such as preincubation with cyclosporin A, a drug known to inhibit T-lymphocyte activation, can modify HIV receptor expression so as to render the cells unable to bind HIV (Table 2)31,32. As alternative models for HIV receptors emerge from all these findings (Fig. 1). First, CD4 is the HIV receptor on CD4 expressing cells. The virus penetration is controlled by the availability (number, mobility, configuration) of CD4 at the cell membrane. However, cells normally negative for CD4 expression can occasionally be infected provided they express a CD4-1ike molecule or a phagocitic capability facilitated by opsonization through Fc receptors. Secondly, CD4 mediates HIV binding to CD4 + cells, but another molecule, which does not necessarily interact with the viral proteins, is part Of the 'functional receptor' which is required for virus penetration. Such a two-step infection (binding and penetration) has already been described for the endocytosis of West Nile virus in macrophages 33. In any case, signals which interfere with cellular activation might either 'freeze' the membrane or modify the relationship of the receptor components. The infected cell HIV is a lentivirus34. Retroviruses of this subgroup cause slowly developing non-tumorigenic diseases and replicate through complementary DNA (cDNA) intermediates that can be found either as unintegrated circular or linear double strands, or as integrated cDNA in the host's genome (provirus form). One of the main characteristics of lentiviruses is that their replication is restricted, which results in latent cell infection. For example, monocytes, the preferential target of visna, are latently infected without expressing viral proteins but their activation and differentiation to macrophages leads to virus replication 3s. As a consequence, the latently infected cells remain invisible to the immune system and persist. Similarly, we have already
ImmunologyToday,vol. 7, No. 10, 1986
rostr.m
-
Table 2. Inhibition of HIV binding on normal CD4 +
T-lymphocytes and subsequent replication after culture Cell pretreatment a HIV-FITCbinding b HIV replication c -
-I-
+
CD4 antibodies a
-
-
- (transient) + +
- (transient) Not tested Not tested
PHA PMA Con A CD3 antibody CD2 antibodies
+ -
Not tested -
Cyclosporin A
-
-
aBefore HIV-FITC incubation, cells were preincubated for 30 min. to 1 h with: saturating concentration of CD4 monoclonal antibodies optimal mitogenic concentration of PHA, PMA or Con A mitogenic concentration of anti-CD3 saturating concentration of anti-CD2. Only antibodies directed to CD2 epitope 1 induce the inhibition of binding and replication 500 ng/ml of Cyclosporin A for 1 h blsothiocyanate-labelled, heat-inactivated, virus (HIV-FITC)was incubated for 1 h with the target ceils, before the percentage of positively labelled virus was determined by direct immunofluorescence29. cVirus replication was determined as described1.
demonstrated that activation of infected lymphocytes is necessary for HIV replication 36. So far, all lymphocyte stimuli tested - mitogens (PHA, ConA, WGA), soluble antigens extracted from bacteria, viruses, fungi, or allogeneic cells - result in virus replication37: When initiated, HIV replication is very efficient because of the powerful viral cis- and trans-acting regulatory elements 38~o. These two properties, latency and powerful activation, may be differently expressed according to the infected cell types. For example, comparison of virus replication in CD4 + lymphocytes and CD4 + transformed cell lines has shown that, with respect to the percentage of infected cells, replication is more efficient in normal lymphocytes 41. Such a heterogeneity can also be noted among the cells of continuous lines, in most of which the virus is replicating without any additional stimulation while a small percentage latently harbors the virus47.
CD4 is the only I spec f c receptor I
I
O t h e r molecules p a r t i c i p o t e in the receptor
Virus
Virus
CD4 + cell
CO4- cell
]
Virus
Virus
CD 4 ~"cell
Penetration in o CD4* Penetration in cell requires adequate other cells con binding between viral use either CD4proteins and CD 4 like epitopes or other non specific receptors like FC
Other cell
Penetration of H I V in CD4" lymphocytes requires both molecules
Penetration in other cells con use eitherone of these two specific binding proteins with or without other non specific molecules
Fig.1. Alternative hypotheses for HIV receptor. bodies can protect non-infected CD4 lymphocytes from such fusion process ~. The question remains: why does the cell die? Activation of a viral or cellular cytotoxic protein has been postulated 4s,46. Moreover, it has recently been proposed that this cell damage could be the result of the interaction between viral and CD4 protein on the membrane of the same cell47. Alternatively, it could be due to intracytoplasmic interaction of viral protein and CD447.48 which also explains why CD4 is no more detectable at the cell membrane during virus replication, while specific CD4 mRNA is present and even up-regulated 48. However, all these mechanisms are still hypothetical and the only evidence of a cytopathic effect in vivo is the description of giant cells in the brain of infected individuals2°.
Soluble proteins The follow-up of CD4+-enriched cultured cells after infection shows the progressive disappearance of CD4 from the membrane of most cells, although only 5-10% of them express cytoplasmic viral antigens 1~. This rather indirect effect of viral replication can be explained by the masking of the CD4 molecule by free soluble viral proteins. These proteins are difficult to detect, probably because the epitopes that are usually recognized are those involved in the interaction. Free viral proteins are produced in larger amounts than the viral RNA available for encapsidation, due to the fact that HIV activating elements (trans-activating factors) act, at least partly, at the translational level 49.
The effects of viral replication
These are shown in Fig. 2. Cytopathogenicity The first biological property of HIV which distinguished it from other previously described human retroviruses was its strong cytopathic effect 36. At the time, or soon before reverse transcriptase activity becomes detectable in culture supernatants, giant multinucleated cells can be observed 11. This cell fusion process probably depends on the interaction between the viral proteins (gp 110) expressed at the membrane of virus producing cells and the CD4 protein expressed by other - not necessarily infected - cells. Indeed we have shown that the addition of normal CD4 + MOLT-4 cells to infected CEM cells results in rapid appearance of giant cells42, and others have recently observed that anti-CD4 monoclonal anti-
Normal uninfecfed CD4 + cells Activation
®/.~ "~ ®
/~
J f~.~..,~:..~.~-,.."~
o [nfected cells
~'~ celhl
particles ' -
\'-.-J )
j
® "~J.,
®
'~,J®
""
"~'~Virot soluble
®
__~CO)
Cytopothic e f f e c t plus possible fusion to non-infected cells
Inhibition of T lymphocytes
~...
proteins
"
\\\
"~" ( ~
celIP°lycl°nalactlvationB
-%
Destruction of normal cells coated with
virol proteinl
Fig.2. The effects of viral replication.
293
Immunology Today, vol. 7, No. 10, 1986
-ros/rum
will help to delineate the properties of each viral component. It is obvious that the ultimate result of viral replication, which seems to occur in 10 -3 to 10 -5 lymphocytes s~, will be the release of numerous viable viral particles, infecting additional cells, expanding the virus reservoir (Fig. 2). The immune responseto HIV Humoral response
Antibody responses observed in HIV-infected individuals indicate that all of HIV's gene products are immunogenic, but the predictive value of each pattern (antibodies directed either at the gag and/or the env products) has not yet been determined. Apparently conflicting results have been reported regarding neutralizing antibodies 42,52,s3. But, if the conclusions of the authors are different, their results are similar, showing that when they exist, neutralizing antibodies are present in very low concentrations. Genetic variability
Fig.3. Relationship between destruction of the follicular dendritic network and the presence of CD8+ infiltrates of germinative centres in lymph nodes of HIV-infected patients. (a) Disrupted follicular dendritic cells stained by a CVK anti-HIV antibody 66. (b) Double-labelling technique showing infected FDC (in red) and CD8+ cells (in blue) at their contact. (c) An activated CD8+ cell presenting a cytotoxic granule, in close contact with a destroyed FDC, nevertheless characterized by its desmosome. (Pictures kindly provided by C. Parravicini; see Ref 18 and 62.)
294
This large release of free soluble proteins after cell lysis might also be responsible for various effects on the immune system, such as a decrease in lymphocyte proliferation capabilities 1ts0, a polyclonal immunoglobulin secretion by B lymphocytes, or - on,the contrary - a depressed responsiveness to polyclonal B-cell activators such as PWM, SAC, and EBVs°. In this respect, the availability of recombinant proteins or synthetic peptides
One of the most striking properties of HIV is its important genetic variability, especially in the env and F proteins 54's5. Such variability might indeed represent a potential selective advantage of mutations in these regions and could represent an "escape' mechanism from the immune response as described for other lentiviruses (EIAV) 56. Follow-up of the virus in a single patient using restriction mapping and sequencing has not shown any important genomic shift 57. In contrast, comparing the restriction map patterns of two isolates, one from an infected blood donor, the other from his recipient, showed an individual adaptation of the virus; although both isolates resembled each other more than two non-related isolates would, they were still clearly different s8. It is also important to note that, in spite of the fact that some individuals are potentially contaminated by hundreds of different viruses, only one can be isolated from their lymphocytes. To explain these findings, we propose thatthis genetic variability enables the virus to express - early after initial infection - those epitopes to which the host is a low responder, making clear that once an optimally adapted virus emerges, little change in its 'antigenic make-up' is expected to be observed. Such a 'low-high responder' immune mechanism has been documented for the transplantation antigens, and it is interesting to note here that HIV env and/or F proteins possibly share some homology with MHC class II proteins 59 '60 . Molecular analysis of different viral isolates has clearly demonstrated that some conserved regions are shared by all isolates s4'55, which might be crucial for the conservation of some of the basic viral properties, i.e. the CD4 tropism. Again, the availability of synthetic peptides or recombinant proteins expressed in a different immunogenic context may help to develop a response not observed during natural infection. Cellular response
The early augmentation of the number of CD8 ÷ lymphocytes is suggestive of a specific proliferative response against HIV infected cells. Recently HIV-specific HLA-class I-restricted cytotoxic T cells and lymphokineactivated killer cells have been described 61. However,
For technical reasons we are unable to reproduce this figure in colour. See the October issue of Immunology Today for full colour illustration,
Immunology Today, voL 7, No. 10, 1986
rostru since latently infected cells do not express viral antigens, and because expression of these antigens is associated with the cell death, cytotoxic cells may not play a significant role in the control of HIV infection. On the contrary, they may participate in an 'autoimmune' reaction. Circumstantial evidence indicates that cytotoxic CD8 ÷ cells might be involved in the destruction of HIV-presenting follicular dendritic cells (FDC) of the lymph node reticulum 18. Sequential follow-up of lymph node biopsies from lymphadenopathy patients shows that at first the germinal center reticulum is enlarged and many viral antigens are present on the FDC, while later evolution of the disease is accompanied by penetration of CD8 ÷ cells into the germinal centers. Such cells are found in close contact with FDC, and their presence is correlated with destruction of the latter62. It can be hypothesized that the same mechanism leads to an autoimmune destruction of non-infected CD4 ÷ lymphocytes coated with soluble proteins specifically bound to the HIV receptor (Fig. 3)63. Immune status of the host Immune status of the infected host might be crucial for controlling immune response to HIV. The pre-existing mild immune defects that can be observed in healthy homosexuals before HIV infection 36, could tip the balance in favor of establishment of infection rather than virus control. The multiple viral, bacterial, or fungal infections observed in these subjects are probably the cause of such pre-existing immune abnormalities. In addition, they will contribute, after primary infection, to the permanent activation of the immune system, which enhances HIV dissemination. In this respect, activation of allospecific T cells might be crucial. The allogeneic sperm lymphocytes which can occasionally enter the organism during sexual intercourse may be responsible for a graft-versus-host-like reaction leading to immunodeficiency 64. We think that they also play a major role because they could activate already infected alloantigen-specific T-cells, leading to a more rapid viral dissemination. Genetic factors As in other viral infections, genetic factors could control the level of the immune response against HIV. A strong response might result in the elimination of a few contaminating viral particles, but also in an accelerated evolution towards immune deficiency by enhancing the autoimmune destruction of CD4 + cells. Except for the case of Kaposi's sarcoma, which is significantly associated with HLA-DR 5 (Ref. 65), there is currently no evidence of major histocompatibility gene association with HIV-related diseases. 'Suicide" of HIV specific cells The local recruitment of HIV specific CD4 + lymphocytes at the site of the immune reaction might result in their 'suicide' since they will be exposed to viral infection. Such a mechanism may explain the disappearance of antibodies specific for viral core protein observed during the progression of the disease. The persistence of antibodies specific for the envelope will then represent the stigma of early infection, before the variation of the envelope.
-
A model for the pathology of HIV infection We propose that progressive infection is a step-wise process, each step involving more cells and more extensive virus replication. The first step in infection represents the group of healthy carriers. Few CD4 + lymphocytes (being the target cells of primary infection) are infected. Virus replication occurs only after their activation, an event unlikely to happen since T-lymphocytes need to be specifically stimulated through its antigen receptor. A balance exists between the occasional stimulation of infected cells and their 'natural clearance', but each viral replication event increases the pool of infected cells and augments the probability that other cells than CD4 ÷ lymphocytes can be infected. Later, in some individuals, multiple infections or allostimulation will finally enhance viral replication. When macrophages are involved, the chance of virus replication probably increases because these cells are more often (not specifically) stimulated than lymphocytes. Progressive increase of viral replication will enhance the impairment of the immune system. B lymphocytes will be affected through their regulation by CD4 + cells but also by the effect of soluble viral proteins. A new balance is established, with virus replicating and attacking new cells on one side, an autoimmune reaction on the other, and immunodeficiency in progress. This fragile status quo might last until replication is suddenly enhanced when, for example, EBV-transformed cells become infected. They will be a reservoir for permanent and active replication, which will contribute to the final active destruction of the immune system and continuous dissemination of the virus to other organs. Opportunistic infections or tumors can then develop. Many questions remain to be answered concerning HIV infection. This partly hypothetical model could contribute to a better understanding of some aspects of the immune reaction and will enable the delineation of new therapeutical strategies.
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