NIH conference. Immunopathogenic mechanisms in human immunodeficiency virus (HIV) infection.

NIH

CONFERENCE

Immunopathogenic Mechanisms in Human Immunodeficiency Virus (HIV) Infection Moderator: Anthony S. Fauci, MD; Discussants: Steven M. Schnittman, MD; Guido Poli, MD; Scott Koenig, MD; and Giuseppe Pantaleo, MD

An understanding of the immunopathogenic mechanisms of infection with human immunodeficiency virus (HIV) is fundamental in developing successful approaches to designing effective therapeutic and vaccine strategies. In this regard, we have investigated the mechanisms by which HIV inserts itself into the human immune system and uses the elaborate cytokine network to its own replicative advantage. We have also shown that the burden of HIV in CD4+ T cells is directly associated with a decline in this cell population in vivo and a progression to disease. Mononuclear phagocytes may play a role in the pathogenesis of HIV infection by serving as reservoirs of the virus. Of note is the fact that monocytes in the peripheral blood of HIV-infected individuals are rarely infected in vivo, whereas infected-tissue macrophages may play a role in organ-specific HIV-related pathogenesis. The role of HIVspecific humoral and cell-mediated immunity in HIV infection is not well understood. However, fine specificity of responses against HIV have been delineated in some in-vitro systems. It is unclear why these responses, particularly HIV-specific cytolytic T-cell responses, diminish over the course of infection and are unable to contain progression of infection. Annals of Internal Medicine. 1991;114:678-693.

Anthony S. Fauci, MD (Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland): The acquired immunodeficiency syndrome (AIDS) results from infection of certain components of the human immune system by the human immunodeficiency virus (HIV). From the initial pathogenic event of the binding of HIV to a functionally important receptor (the CD4 molecule) on a subset of T cells and monocytemacrophages, the virus establishes an extraordinary interaction with the human immune system (Table 1). The ultimate consequence of this interaction is a profound immunosuppression resulting from quantitative depleAn edited summary of a Clinical Staff Conference held 27 June 1990 at the Amphitheater, Building 10, Bethesda, Maryland. The conference was sponsored by the National Institutes of Health, U.S. Department nen of Health and Human Services. Authors who wish to cite a section of the conference and specifically indicate its author can use this example for the form of reference: Schnittman SM. Viral burden in human immunodeficiency virus type 1 (HIV-1) infection, pp 680-683. In: Fauci AS, moderator. Immunopathogenic mechanisms in human immunodeficiency virus (HIV) infection. Ann Intern Med. 1991;114:678-693. 678

tion and functional abnormalities of the CD4+ T-cell subset (the helper/inducer subset, reviewed in [1]). Of particular interest is the fact that HIV can remain latent or chronically expressed at a low level for extended periods. Despite the fact that there is always some degree of virus replication even early in the course of HIV infection (2, 3), this "microbiologic" latency certainly contributes to the fact that the interval of time from initial infection to clinical disease in HIVinfected individuals averages about 10 years (Figure 1). Nonetheless, even during this prolonged disease-free period, there is a gradual and progressive diminution of CD4+ T-cell numbers, which occurs in almost all HIVinfected persons. This ultimately leads to serious immunosuppression and opportunistic disease (Figure 1). The Relation between HIV and the Human Immune System It has been clearly shown that the expression of HIV in infected cells depends on the state of activation of the cell (4, 5). In this regard, mitogenic and antigenic stimuli as well as transfected heterologous virus genes have been shown to induce efficiently virus expression (reviewed in [6]). Over the last several years, we have been interested in the induction of HIV expression by naturally occurring endogenous cytokines whose fundamental purpose is the homeostatic regulation of the human immune response (7, 8). This cytokine network is pleotropic and redundant and operates in an autocrine and paracrine manner (9). Because induction of virus expression can occur in vivo in the absence of such recognizable stimuli as opportunistic infections, it was important to determine whether normal endogenous cytokines could actually induce virus expression. Thus, we established a panel of T-cell and monocytoid cell lines that were infected in vitro with HIV (reviewed in [7]). A chronic infection was established, and the chron-

Table 1. Multifaceted Mechanisms by which HIV Interacts with and Affects the Human Immune System 1. HIV (gp 120) binds to a functionally important cell surface marker (CD4) on an immune competent cell (T4 lymphocyte) 2. HIV products (gpl20, tat protein, gp41) can suppress T-cell function 3. HIV potentially induces autoimmune mechanisms that can kill innocent bystander cells 4. Macrophages can be infected with HIV and can serve as a reservoir of infection as well as pass the virus to susceptible T4 cells during normal immune interactions 5. HIV intercalates itself into the normal immunoregulatory network and uses cytokines that modulate immune cell function to regulate its own expression

15 April 1991 • Annals of Internal Medicine • Volume 114 • Number 8

Downloaded From: http://annals.org/ by a Brown University User on 09/30/2013

Figure 1. Typical course of HIV infection. After infection with HIV, there is a modest decrease in circulating CD4+ T cells (•-—•) and an initial burst of virus replication as shown by p24 antigenemia (O O). Patients then usually have a prolonged asymptomatic course measured in years. Over this period, the number of CD4+ T cells gradually decreases. Many patients have an abrupt drop in CD4+ T cells later in the course of disease. At this time there may be sustained p24 antigenemia. Once CD4+ T cells decrease below a critical level (usually 200 cells/mm3), the risk for such opportunistic infections as Pneumocystis carinii pneumonia (PCP) is high.

ically infected cells were cloned. These lines constitutively expressed variable levels of virus and were induced to virtually 100% expression by phorbol myristate acetate. We first established that crude supernatants from lipopolysaccharide-stimulated monocytes or phytohemagglutinin-stimulated mononuclear cells (which contain a wide variety of cytokines) could potently induce HIV expression in the T-cell and monocyte lines (10). We then examined a panel of recombinant DNA-derived cytokines for their ability to induce HIV expression in this system. We found that tumor necrosis factor-alpha (TNF-a) alone among the cytokines examined could induce HIV expression in the chronically infected T-cell line (ACH-2), whereas TNF-a as well as granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin-6 could induce HIV expression in the chronically infected monocyte line (Ul) (10-12) (Figure 2). In a manner similar to that in which they regulate the immune system itself, these cytokines, particularly TNF-a, induced HIV expression in an autocrine and paracrine manner and synergized with other cytokines in this process (11). In fact, in the case of TNF-a, constitutive expression of HIV in certain cell lines could be blocked by antibody to TNF, and the induction of HIV expression by phorbol myristate acetate involved TNF-dependent mechanisms because antiTNF-a antibody could partially block induction (11). These studies indicated that endogenous TNF can play an important role in the constitutive regulation of HIV expression in vivo. This is of particular relevance because plasma levels of TNF-a have been shown to be elevated in HIV-infected persons (13) and TNF-a is present in high levels in the plasma of humans and animals during infection with several of the same organisms that commonly infect HIV-infected persons during

their stage of profound immunosuppression (14). Furthermore, increased levels of interleukin-6 have been reported both in the serum and in the cerebrospinal fluids of HIV-infected persons (15, 16). The molecular mechanisms by which these cytokines induce HIV expression also parallel the molecular mechanisms by which certain cytokines regulate the immune system. In particular, TNF-a induces expression in these cell lines by transcriptional mechanisms involving the activation of N F - K B (17), which is a pleotropic mediator of inducible and tissue-specific gene control (18). N F - K B is also involved in T-cell activation and cytokine regulation. With regard to HIV, oligonucleotide consensus sequences that bind N F - K B are located in the core enhancer region of the promoter in the HIV long terminal repeat sequence of the proviral DNA. In contrast to the transcriptional mechanisms involved in the TNF induction of HIV expression, GM-CSF and interleukin-6 either alone or in combination induce HIV expression by post-transcriptional mechanisms ([12], Poli G. and colleagues. Unpublished observations). In contrast to the induction of HIV expression, cer-

Figure 2. Cytokine induction of HIV expression. Tumor necrosis factor-a (TNF-a) induces the expression of HIV in both chronically infected promonocytic (Ul) and T-cell (ACH-2) lines. Interleukin-6 (IL-6) and GM-CSF induce the expression of HIV in the Ul cell line only. RT = reverse transcriptase; GM-CSF = granulocyte-macrophage-colony stimulating factor.

15 April 1991 • Annals of Internal Medicine • Volume 114 • Number 8 Downloaded From: http://annals.org/ by a Brown University User on 09/30/2013

679

tain cytokines can actually downregulate the expression of HIV, and this, of course, has potentially important therapeutic implications. One cytokine, interferon-a, which has been demonstrated in vitro to decrease virus expression and which we have shown blocks the budding of HIV from our chronically infected cell lines (19), has already been shown in clinical trials to be an effective antiretroviral agent (20-22). Another cytokine of particular interest is transforming growth factor-0, which is a 25-kD molecule that was originally described in its capacity to stimulate reversible transformation of non-neoplastic murine fibroblasts (23). It has subsequently been shown to have pleotropic physiologic effects. Of note with regard to the immunoregulatory network, transforming growth factor-/3 has been shown to have either growth-enhancing or growthstimulating properties depending on the particular cell type and other growth factors present. In general, however, it is most commonly thought of as an immunosuppressive molecule. We have recently shown that transforming growth factor-/? suppresses the phorbol myristate acetate-induced, but not the TNF-induced, expression of HIV in the chronically infected Ul cell line (Poli G. and Fauci AS. Unpublished observations). This observation confirms, as mentioned above, that there is a TNF-dependent and a TNF-independent component of the phorbol myristate acetate-induced expression of HIV. In this regard, transforming growth factor-/3 blocks the TNF-independent component, but not the TNF-dependent component (Figure 3). Furthermore, transforming growth factor-/? blocks the induction of HIV expression in Ul cells by interleukin-6 and GM-CSF when these cytokines are present alone or in synergistic combination (Poli G. and Fauci AS. Unpublished observations). Of particular note is the fact that

Figure 3. Pathways of phorbol myristate acetate induction of HIV. Tumor necrosis factor (TNF)-dependent and TNF-independent pathways of phorbol myristate acetate (PMA) induction of HIV expression in chronically infected promonocytic (Ul) cells. Transforming growth factor-/? (TGF-p) blocks the TNF-independent pathway but not the TNF-dependent pathway of HIV induction, whereas antibody to TNF (anti-TNF) blocks the TNF-dependent pathway. 680

Table 2. Effect of Cytokines on the Induction Expression in Chronically Infected Cell Lines

1. Various cytokines (TNF-a, GM-CSF, interleukin-6) induce the expression of HIV in chronically infected T-cell and promonocyte lines 2. These cytokines can synergize in the induction of HIV expression 3. Tumor necrosis factor-a induces HIV expression in an autocrine and paracrine manner 4. Tumor necrosis factor-a induces HIV expression in these models by transcriptional mechanisms involving activation of NF-KB

5. Interleukin-6 and GM-CSF induce HIV expression predominantly by post-transcriptional mechanisms

transforming growth factor-/? blocks HIV expression by interleukin-6 and GM-CSF at the post-transcriptional level whereas it blocks HIV expression induced by phorbol myristate acetate at the transcriptional level. The regulation of HIV expression by cytokines is summarized in Table 2. An in-vivo physiologic correlate in HIV-infected persons of the in-vitro induction of HIV expression is readily apparent in the case of the state of activation of B cells. We showed several years ago that peripheralblood B cells of HIV-infected persons are polyclonally activated (24). The same is true of lymph-node B cells that are in close contact with HIV-infected T cells. We have recently demonstrated that supernatants from invitro-activated B cells of HIV-seronegative persons can induce expression of HIV in the chronically infected Ul and ACH-2 cell lines (25). In addition, the HIV-inducing capacity of these supernatants can be neutralized by antibodies to TNF-a and interleukin-6. Furthermore, these activated B cells can induce HIV expression in coculture with these cell lines. Of particular interest is the fact that B cells from HIV-infected individuals with hypergammaglobulinemia constitutively secrete high levels of TNF-a and interleukin-6 in the absence of exogenous stimuli, whereas B cells from seronegative persons require in-vitro stimulation with such activators as Staphylococcus aureus Cowan strain I and interleukin-2 in order to secrete TNF-a and interleukin-6. Furthermore, unstimulated B cells from HIV-infected persons, but not from seronegative persons, can induce the expression of HIV in coculture with chronically infected cell lines. These studies indicate that B cells from HIV-infected persons may be capable of inducing HIV expression in vivo by virtue of secretion of cytokines such as TNF-a and interleukin-6 (Figure 4). Thus, it is remarkable that HIV has intercalated itself into the normal immunoregulatory network of the human immune system and uses for its self-propagation the very cellular and molecular mechanisms that the immune system uses for its homeostatic regulation. Viral Burden in Human Immunodeficiency Virus Type 1 (HIV-1) Infection Steven M. Schnittman, MD (Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland): The focal point for the immunopathogenesis



of HIV

of infection with human immunodeficiency virus type 1 (HIV-1) is the depletion of the helper/inducer subset of T lymphocytes that express the CD4 surface molecule, T4 cells (reviewed in [26]). Because the T4 lymphocyte is the central cell involved directly or indirectly in the induction of most immunologic functions (26), its loss results in the profound immunosuppression that characterizes infection with HIV-1. After infection with HIV-1, substantial quantitative and qualitative immunologic defects develop that may lead to opportunistic infections and neoplasms (26, 27). A strong body of evidence demonstrates that HIV-1 selectively infects cells expressing the CD4 molecule, particularly lymphocytes and cells of the monocytemacrophage lineage (28-31). After infecting cells bearing the CD4 receptor, HIV-1 may undergo active viral replication leading to cytopathicity and death of the host cell. Alternatively, an infected cell could enter a latent or chronic low-level state of infection that could be triggered into active viral replication. Previous studies using in-situ hybridization techniques to detect cells expressing viral mRNA and immunofluorescence to detect cells expressing viral proteins have shown a low frequency of circulating lymphocytes expressing viral RNA or protein in HIVinfected individuals, ranging from 1 in 10 000 to 1 in 100 000 (32, 33). Consequently, there has been considerable discussion concerning whether or not infection of CD4+ T cells plays a direct role in causing the profound depletion of CD4+ T lymphocytes and, consequently, the immunosuppression associated with HIV infection (reviewed in [1]). Because of the possibility that a larger proportion of cells may be latently infected, that is, containing pro viral DNA but not expressing viral mRNA or protein, it was necessary to use methods sensitive enough to detect HIV pro viral DNA. Until the gene amplification (polymerase chain reaction, PCR) method was developed, HIV-1-infected cells that did not express virus were not readily detectable (34, 35). To address the question of viral latency and to determine the reservoir for HIV-1 in human peripheral blood mononuclear cells (PBMC), we used flow cytometry to sort highly purified subpopulations of these cells from HIV-1 infected persons. Polymerase chain reaction was then done to determine the presence of HIV-1 DNA in the various enriched cell subpopulations that included CD4+ T lymphocytes, CD8+ T lymphocytes, CD 14+ monocytes, CD 16+ natural killer lymphocytes, and CD 19+ B lymphocytes. In. every patient examined by PCR, HIV-1 DNA was detected in the enriched CD4+ T-lymphocyte population of cells (33, 36). In fewer than 10% of persons did PCR detect HIV-1 DNA in the enriched peripheral monocyte fraction. In no patient was HIV-1 DNA detected in the CD8+ T-cell, CD 19+ B-cell, or CD 16+ natural killer-cell populations. To determine the frequency of HIV-1 DNA in CD4+ T cells, quantitative PCR was done on serial dilutions of the purified CD4+ T-cell population from HIV-infected individuals and compared with the PCR amplifications done on serial dilutions of the ACH-2 cell line, a chronically infected T-cell line containing one DNA copy of HIV per cell (37). We determined that the frequency of

CD4+ T cells that contain HIV-1 DNA is at least 1/100 cells in patients with AIDS (33). In contrast to patients with AIDS, asymptomatic HIV-1-seropositive persons have a frequency of HIV-1 DNA in CD4+ T cells that varies greatly, between 1/100 000 and 1/100 cells (36). These studies suggested that using PCR to quantitatively analyze HIV-1 DNA from infected individuals is feasible. We next addressed the relation between viral burden and immunologic status in HIV-infected individuals. We used quantitative PCR to examine viral burden over time in a group of HIV-seropositive persons, half of whom remained stable and half of whom had a rapidly progressive immunologic and clinical decline. In those HIV-1-infected persons who developed progressive disease (onset of opportunistic infections, Kaposi sarcoma, or significant symptoms), the CD4 cell count and percent CD4+ T cells declined significantly (P < 0.01), whereas CD4 cell counts and percent CD4+ T cells were unchanged in those individuals who remained asymptomatic over a comparable follow-up period. Quantitative PCR was done to determine the HIV-1 viral burden in sorted, purified CD4+ T cells derived from patients at various time points. In those persons who remained asymptomatic, frequencies of HIV-infected CD4+ T cells were low (< 1/10 000 to 1/1000) at study entry and increased only minimally (none higher than 1/1000) over 14 months of follow-up. In contrast, among those individuals who developed HIV-related symptoms (including AIDS), despite having similar CD4 counts at study entry, frequencies of HIV-infected CD4+ T cells were higher at entry (> 1/1000) and increased substantially (> 1/100) in most within 3 months of developing progressive disease (38) (Figure 5). This clear-cut increase in viral burden per constant number of CD4+ T cells as persons progress to clinical disease, as well as the association of increased viral burden with quantitative depletion of CD4+ T cells in persons who are clinically stable, strongly suggests a direct and causal relationship between viral burden and immunosuppression and disease. To further support the association between HIV-1 viral burden and clinical disease, recent reports have found levels of HIV-1 in plasma and PBMC to have been much higher than were previously estimated (2) and demonstrated that the degree of

Figure 4. Effect of activated B cells on HIV expression. Exposure to various stimuli such as viral antigens (from cytomegalovirus [CMV], Epstein-Barr virus [EBV], or HIV) results in the activation of a resting B cell and induction of secretion of interleukin-6 (IL-6) and TNF-a. These cytokines, in turn, can induce the expression of HIV from latently or chronically infected T cells or macrophages.


681

Figure 5. Relation between viral burden and disease progression. Changes in the frequency of HIV-1 DNA in purified CD4+ T cells in two healthy seropositive persons over time. Polymerase chain reaction was done at two time points on tenfold serial dilutions of highly enriched CD4+ T cells obtained from seropositive patients who developed clinically progressive disease {left, representative Patient A, Pneumocystis carinii pneumonia) or who remained asymptomatic {right, representative Patient B). These signals were compared with those obtained from the polymerase chain reaction of serial dilutions of the ACH-2 cell-line standard. The amplifications (SK68/69 env primer, SK70 probe) show a significant increase in the frequency of HIV-1 infected CD4+ T cells between time 1 and time 2 in Patient A compared with Patient B (Adapted with permission from [38]). plasma viremia is a sensitive marker of the clinical stage of HIV infection and viral replication (3). These findings suggest that quantitative PCR may be a useful surrogate marker to measure the effects of antiretroviral agents on viral burden in clinical trials. In recently reported preliminary studies, the amount of HIV-1 proviral DNA was determined by quantitative PCR in the blood of patients before and during antiviral therapy with dideoxyadenosine and zidovudine (39). HIV-1 proviral DNA decreased in six out of ten patients during treatment, a finding that is rarely seen in untreated patients. CD4+ T cells of individuals with HIV infection, particularly early in the course of infection, exhibit a qualitative defect in the ability to respond to soluble antigen, whereas responses to mitogens remain normal (40). CD4+ T cells can be broadly divided phenotypically into "naive" (CD45RA+) and "memory" (CD29+, CD45RO+) cell subpopulations, which represent dis682

tinct maturation stages (reviewed in [41]). These CD4+ T-cell subsets demonstrate differential functional capabilities. In particular, CD45RA+ (naive) cells respond well to stimulation by mitogen and in autologous mixed lymphocyte reactions; however, these cells do not respond to recall antigens, but they do induce suppression of B-cell immunoglobulin production. In contrast, CD29+/CD45RO+ (memory) cells respond well to stimulation by recall antigens and to monoclonal antibodies to CD2 and CD3 antigens, and they provide help to B cells for immunoglobulin production. The role of these CD4 subsets in the pathogenesis of HIV infection has not been investigated extensively. To determine the in-vivo preference of HIV-1 infection in these CD4+ lymphocyte subsets, we obtained PBMC from HIV-infected persons, sorted them into naive and memory CD4+ T-cell subpopulations, and studied them by quantitative PCR. We determined that the memory cells formed the principal reservoir for



HIV-1 (by fourfold to tenfold) within the CD4+ T cells of infected individuals (42). We also determined that the functional abnormalities attributable to CD4+ T cells in HIV-infected persons (for example, failure to respond to soluble antigen) reside primarily within these memory cells. We have concluded that the selective functional defects present in the memory CD4+ T-cell subset may be a direct result of the preferential infection and consequential greater viral burden within these cells. Any explanation for the increased susceptibility to HIV-1 of memory and naive cells remains speculative. However, the recent finding that both CD4 and CD45R molecules possess enzymatic activity located on intracellular domains that can regulate levels of activation of T cells may prove relevant (reviewed in [43]). The presence of HIV-1 DNA in CD4+ T cells in the peripheral blood is not the only correlate of HIV-1 viral burden directly affecting function. It has been recently shown that PBMC in infected individuals contain minimal quantities of unintegrated, compared with integrated, HIV-1 DNA. In contrast, brain tissue from patients with HIV encephalitis has a considerably higher proportion (> 10:1) of unintegrated viral DNA (44). It will be important to determine whether high levels of unintegrated viral DNA correlate with the onset of pathogenic effects of HIV-1 in the brains of patients with HIV encephalitis. In addition, the presence of HIV-1 RNA in the lungs of pediatric patients with AIDS may also correlate with dysfunction. In children with AIDS who develop the chronic respiratory disorder known as lymphocytic interstitial pneumonitis, a relatively high frequency of cells (0.1%) was found to be expressing HIV-1 RNA by in-situ hybridization, whereas the lungs of patients with AIDS without lymphocytic interstitial pneumonitis were nearly devoid of HIV-1 RNA (0% to 0.002%) (45). Thus, HIV-1 may play a direct causal role in the development of lymphocytic interstitial pneumonitis in infected patients. Thus, the HIV-1 viral burden is directly associated with the progressive decline in CD4+ T cells and the deteriorating course of immunodeficiency in HIV-infected persons. Moreover, our present understanding of the pathogenesis of HIV infection strongly supports a direct role for HIV in the development of the clinical manifestations of AIDS. The Role of Mononuclear Phagocytes in the Pathogenesis of HIV Infection Guido Poli, MD (Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland): Although several functions of mononuclear phagocytes, including secretion of cytokines and production of superoxide anion, have been found to be essentially intact throughout the different stages of HIV infection (reviewed in [46]), we have reported a severe impairment of monocyte chemotactic response even before the clinical onset of full-blown AIDS (47, 48). Although direct infection of monocytes with HIV seems unlikely to be the sole cause because of the low frequency of infected PBMC (1/1000 to 1/100 000), no conclusive explanation has been provided for this dysfunction of the phagocytic

system (33). More likely, the functional defects of mononuclear phagocytes observed in HIV-infected individuals are the consequence of a direct or indirect immunosuppressive effect of HIV envelope proteins (49, 50), as previously suggested for the immunodeficiencies caused by murine and feline retroviruses (51). Shortly after mononuclear phagocyte dysfunctions were described, several laboratories demonstrated unequivocally that mononuclear phagocytes, similar to the CD4+ subset of T lymphocytes, were a target of HIV infection both in vitro (52, 53) and in vivo (54, 55). Furthermore, certain characteristics of mononuclear phagocyte infection suggested that these cells could function as an important reservoir of HIV in vivo. First, in-vitro-infected mononuclear phagocytes showed a reduced or insignificant cytopathicity in sharp contrast with the rapid cell killing observed with CD4+ T cells (52). Second, budding and accumulation of viral particles occurred both at the plasma membrane level (as with T cells) and in intracellular compartments (52, 53), suggesting that infected mononuclear phagocytes may be responsible for spreading HIV to different organs and tissues. The most striking feature of mononuclear phagocyte infection, however, was the ability to isolate strains of HIV that could replicate with up to 10 000fold increased efficiency in these cells as opposed to T lymphocytes (52). These HIV isolates, operationally defined as ''macrophage tropic strains," had their counterparts in viral strains that could preferentially replicate in T cells, suggesting that the immunologic, and ultimately clinical, aspects of HIV infection may result from diverse strains of HIV that target different cell types. This possibility has potentially important implications for the design of effective therapeutic interventions as well as for the development of vaccines. However, Weiss and colleagues (56) reported that in the unique case of an individual infected with a laboratory-adapted T-lymphocytotropic strain of HIV (IIIB), isolation was accomplished exclusively by cocultivation of the individual's PBMC with normal monocyte-derived macrophages, but not with phytohemagglutininstimulated T-cell blasts. Furthermore, the molecular comparison of the newly isolated macrophage tropic variant with the original T-cell-adapted strain failed to reveal significant structural changes. In addition, infection of circulating monocytes has not yet been formally demonstrated. Therefore, we decided to investigate whether the ability to isolate in-vitro macrophage tropic HIV strains was correlated with infection of circulating monocytes in a group of 23 asymptomatic, HIV-infected individuals with high (above 400/mm3) CD4+ T-cell counts. In these experiments, PBMC from HIV-infected individuals were cocultivated with allogeneic phytohemagglutinin-stimulated PBMC from HIV-negative individuals, according to a standard protocol. Simultaneously, PBMC from HIVinfected persons were cocultivated with allogeneic monocyte-derived macrophages obtained from normal seronegative persons. These monocyte-derived macrophages were allowed to undergo terminal maturation in vitro 10 to 14 days before cocultivation. The culture media contained pooled human serum supplemented with either recombinant M-CSF (52) or crude superna-


683

Figure 6. Replication of a macrophage-tropic strain of HIV. Typical profile of virus production during isolation of a macrophage-tropic strain of HIV. Significant levels of HIV replication, as determined by p24 gag antigen production, were observed exclusively when patients' peripheral blood monocytes (PBMC) were cocultivated with allogeneic monocytederived macrophages (MDM), but not with allogeneic phytohemagglutinin-stimulated PBMC. Saturating levels of p24 antigen (which saturate the optical density [O.D.] capacity of the ELISA reader) were produced 60 days after cocultivation. The coculture was artificially terminated after 80 days. (Adapted with permission from [58]). tant from the GCT histiocytic cell line, known to contain several cytokines and colony-stimulating factors (57). This approach allowed us to isolate several macrophage tropic strains of HIV. Production of HIV p24 antigen in the culture supernatants typically began between days 15 and 21 after cocultivation, in contrast to T-cell blast cocultures in which virus replication occurred between days 5 and 14. Replication of HIV in monocyte-derived macrophages was also characterized by a prolonged plateau phase (up to 75 days after cocultivation) in association with the absence of significant cytopathicity (Figure 6). Having established that a significant number (approximately 50%) of these asymptomatic individuals harbored strains of HIV that showed in-vitro tropism for

mononuclear phagocytes, we next investigated the invivo cell origin of these viruses by the combined use of fluorescence-activated cell sorting and PCR. We first verified that our PCR set of primer pairs and probes (chosen according to highly conserved sequences in the virus long terminal repeat, gag, and env genes [33]) could efficiently amplify HIV DNA obtained from monocytederived macrophages infected with a previously established macrophage tropic HIV strain (AD-'87) (53). Amplification was accomplished efficiently, indicating that no gross structural differences were present, at least for the sequences tested, between AD-'87 and a prototypic T lymphocytotropic clone of HIV-1. A sufficient number of PBMC was obtained from 5 out of the 9 patients harboring macrophage tropic strains of HIV. A portion of these PBMC was first depleted of T lymphocytes by sheep red blood cells rosetting and then stained with the anti-monocyte specific (anti-CD 14) monoclonal antibody (mAb) LeuM3. A second aliquot of patients' PBMC was stained directly with the anti-T lymphocyte specific mAb Leu3a (anti-CD4) and Leu2a (anti-CD8). Cells were then sorted with an EPICS C fluorescence activated cell sorting apparatus (Coulter Corporation, Hyalea, Florida) into LeuM3+, Leu3a+, Leu2a+ cells, and their negatively selected counterparts (Figure 7). The purity of these populations was verified by fluorescence-activated cell-sorting analysis, and was consistently found to be > 98% to 99%. Polymerase chain reaction analysis of the sorted PBMC subsets revealed that in all the patients tested, HIV DNA was present both in the unfractionated PBMC as well as in the CD4+ T-lymphocyte fraction (Figure 7). In a single case, monocytes were also found to be PCR positive, whereas CD8+ T cells were always negative. Analysis of the negatively sorted cells confirmed these findings. Strong positivity was associated with the C D 8 - (predominantly CD4+ T cells), but not with the C D 4 (predominantly CD8+ T-cells) fraction, whereas C D 1 4 sorted cells frequently showed a weak reactivity, reflecting the presence of residual contaminating T cells (Figure 7) (58). These observations were supported by our inability to isolate HIV by cocultivation of the sorted monocytes with normal monocyte-derived macrophages (58). Furthermore, we extended these obserFigure 7. The in-vivo cell origin of macrophage-tropic strains of HIV. Flow chart of purification of peripheral blood mononuclear cell {PBMC) subsets from HIV-infected patients and their respective states of infection {bottom). Purity of each PBMC subset was always above 95% as assessed by fluorescence-activated cell-sorting analysis. Polymerase chain reaction {PCR) for HIV sequence was done using SK68/69, SK38/39, and SK29/30 primer pairs for highly conserved sequences present in the env, gag, and long terminal repeat regions, respectively. Results were comparable using each set of primer pairs and related probes. (+) = weak reactivity.

684



vations to a larger number (35) of asymptomatic infected individuals. Human immunodeficiency virus DNA was found to be present in the monocytes of only two additional patients out of 35 individuals studied (< 10%), whereas CD4+ T cells were found to be positive in 100% of the cases (Schnittman S. Unpublished data). These findings confirm and extend our previous observation that the cell harboring HIV DNA in the peripheral blood is predominantly the CD4+ T lymphocyte (33). Furthermore, the present study provides the first direct demonstration that infection of circulating monocytes, although rare, may occur in vivo. In summary, our findings suggest that HIV transmission between T lymphocytes and mononuclear phagocytes may not be restricted in vivo by cell tropism barriers, which seem to be more representative of the phenomena of in-vitro adaptation in tissue culture or selection of HIV variants. Another implication of these studies is that terminally differentiated macrophages may be more susceptible to HIV infection than peripheral blood monocytes (46, 54, 55). This hypothesis is supported by the observation that infection of monocytes is more efficient when these cells are allowed to mature in vitro (52, 53, 58). Whether every mononuclear phagocyte represents a potential target for HIV infection or whether only a susceptible monocyte subset exists that is selectively susceptible during this culture period remains to be established. Recent observations by Perno and colleagues (59) indicate that HIV infection of mononuclear phagocytes occurs predominantly, if not exclusively, via interaction with the CD4 molecule, as previously demonstrated for T lymphocytes (46). In this regard, we and others (60) have observed increased surface expression of CD4 concurrent with the in-vitro maturation of monocytes to macrophages. Increased surface expression of CD4 may represent a potentially important factor correlated with the higher susceptibility to HIV infection shown by monocyte-derived macrophages compared with freshly isolated monocytes. Finally, because HIV infection of mononuclear phagocytes can be upregulated, at least in vitro, by multiple colonystimulating factors (such as M-CSF [53] and GM-CSF [12, 61]) and cytokines such as interleukin-3 (61), TNF-a, and interleukin-6 (7, 12), it is possible that the production of these potent immunoregulatory molecules in the tissues of HIV-infected individuals may influence in situ the infectibility or the levels of virus expression of mononuclear phagocytes. HIV-Specific Immunity and the Pathogenesis of AIDS Scott Koenig, MD (Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland): The pathogenesis of AIDS is intimately related to the success of the immune system in curbing the replication and spread of HIV. Failure of this function results in the inexorable decline of immune competency and renders the infected individual susceptible to myriad opportunistic pathogens. Although a better understanding has evolved of the specific components of the immune system that are induced by infection with HIV and their

potential in controlling viral replication, the conditions for sustaining persistent immunity and the essential elements for maintaining viral suppression are only now being elucidated. Although protective immune responses against lentiviral-induced diseases had been considered feasible, it was only recently that experimental proof emerged to support this concept in a lentiviral model simulating AIDS. This was established by two research groups demonstrating that immunization of inactivated forms of the simian immunodeficiency virus could protect monkeys from being infected after challenge with homologous strains of that virus (62, 63). Although the specific components of the immune system that successfully fended off simian immunodeficiency virus replication were not defined, these observations provided clear experimental justification for pursuing the development of a vaccine to HIV. In addition to attempting to achieve protection for uninfected, at-risk groups, some investigators have tried to bolster specific immunity to HIV through immunization of HIV-seropositive persons with inactivated HIV or recombinant viral products (64, 65). This is an inventive approach whose efficacy needs to be established, particularly in infected individuals with relatively intact immune functions. This approach presumes that protective immunity is inducible, but that adequate levels are not achieved by natural infection. Preliminary work in animals has suggested that immune function to specific viral components is enhanced and associated with some antiviral effects. Whether these efforts will translate into an arrest of disease progression in patients, however, remains to be determined. These observations, however, raise some basic questions regarding the tenets of immunity to HIV in particular and to viruses in general. Clearly, HIV induces an immune response in infected persons. The production of antibodies to the various viral components serves as the primary means by which we determine if a person has been infected with this virus. In addition, HIV-specific, cell-mediated responses, as characterized either by T-cell proliferation or cytotoxicity, can be detected in a large segment of the infected population (64). Presuming that the results established in the simian immunodeficiency virus system regarding protection against infection can be applied to HIV infection of humans, it raises the question of why specific immunity to HIV fails to prevent disseminated infection and disease in most persons. Possibilities include the following: inadequate levels of the specific humoral and cell-mediated responses at the sites of primary infection that fail to eliminate virions before dissemination or viral latency occurs; induction of immune responses directed against immunodominant regions that are not protective or induction of competing responses that may facilitate viral spread; high rates of viral mutation and recombination that result in the repeated evasion of immune surveillance until compromise of immune function no longer allows for specific recognition of escape mutants; induction of initially protective responses that create, because of cross-reactivity with other cell components, autoimmune effects that result in the loss of protective immune function; compromise of potent effector functions through altered pat-


685

Figure 8. Cytotoxic T-cell recognition. Schematic representation of cytotoxic T-cell recognition by major histocompatibility complex (MHC) class I and class II restricted cytotoxic T cells.

terns of differentiation, or the production of normal cytokines (for example, transforming growth factor-f$) and the generation of regulatory mechanisms that contend with the hyperactivity (for example, hypergammaglobulinemia) of the immune system in viremic patients and secondarily impede protective responses. These possibilities should be assessed in the context of our present understanding of the immune response to HIV. It had been observed in other viral infections in humans and in nonhuman species that antibodies and cellular responses, including cytotoxicity and cytokine production, can contribute to antiviral immunity. Putative protective mechanisms include the following: antibodies that are neutralizing for virions or cytolytic for infected cells; natural killer cells that are lytic for virally infected cells; natural killer cells or macrophages that bind antibodies with specificity for HIV and lyse infected target cells (antibody-dependent cellular cytotoxicity, or ADCC); cytotoxic T cells (CTL) that kill HIV-infected cells by recognizing a viral peptide product in association with a major histocompatibility complex (MHC) protein (Figure 8); activated T cells that secrete antiviral cytokines (for example, interferon-a) or lyse infected cells in an MHC-unrestricted manner. These mechanisms may work independently, competitively, or in concert with each other. One of the major antiviral effects of antibodies has been ascribed to their "neutralizing" properties, an invitro measure of the ability of such antibodies to inhibit acute viral infections. Sera obtained from many HIVinfected persons from all clinical stages appear to have antibodies with these effects (66). There seems to be a tendency for neutralizing activity to decline as clinical disease progresses, although neutralizing activity in a given patient's serum is not predictive of clinical status. Antibodies can neutralize free virions or membranebound particles before entry in the cell (67). Neutralizing antibodies may act by altering or masking critical sites on the virion membrane to impede viral entry. Yet, antibodies directed against internal structural proteins have been reported to be neutralizing (68); thus, several mechanisms may be responsible for this property. The principal neutralizing determinant is located within a hypervariable region of the envelope protein (amino acids 296 to 331) (69). Antibodies to this region are type specific. Additional sites have been described 686

in gpl20 and gp41 that are targets for neutralization and, because of recognition of conserved envelope segments, they may have broader activity against different strains of HIV-1 (70-72). Despite the variation seen in the principal neutralizing determinant, a sequence common to over 60% of 245 field isolates was observed that could be recognized by a neutralizing antibody with a single specificity (73). This suggests that if protection is best conferred by neutralizing antibodies to this region, immunization against a limited number of variants would be required to achieve broad protection. Although it has not been demonstrated that such antibodies are truly protective, a recent study has shown that immunization of two chimpanzees with recombinant gpl20, but not gpl60, could protect these animals from infection with a challenge of a homologous strain of HIV-1. This immunity appeared to be associated with high levels of neutralizing antibody to the principal neutralizing determinant (74). Further, it has been shown that a pregnant woman is less likely to give birth to an HIV-infected child if she has antibodies to the principal neutralizing determinant, although the manner in which protection is conferred is not clear (75-77). Antibodies directed to other portions of the envelope protein may have some protective properties by virtue of their ability to mediate ADCC (78). Sera from patients from all clinical stages can mediate ADCC responses (79). Antibodies responsible for such responses are produced soon after infection with HIV and can even be detected in sera from high-risk donors before full seroconversion can be demonstrated in conventional enzyme-linked immunosorbent assays (ELISA) or radioimmunoprecipitation assays ([80], Koenig S. Unpublished observations). The observation that antibodies with ADCC activity can be found in most patients with AIDS would seem to argue against a significant role for this mechanism in providing protection. However, the failure may be associated with the impaired function of the cells mediating this process in vivo in patients with AIDS and not with the antibodies produced to facilitate such responses. In fact, it has been shown that CD16+ cells, armed with antibody in vivo, can mediate ADCC-like responses in vitro, and activity of these cells does appear to dissipate with clinical progression ([80], Koenig S. Unpublished observations). Therefore, an initially effective protective mechanism may be rendered ineffectual. ADCC responses are di-



rected primarily against portions of the envelope protein and recently, we have been able to map some of the B-cell epitopes of the envelope protein within gpl20 and gp41 to which these responses are directed. If ADCC mechanisms show promise in regulating viral replication, the use in mapping such specific sites would be to direct vaccination efforts toward optimizing the responses to these particular envelope sequences. Further efforts will be needed to establish the true role of this mechanism in vivo, and if found to be important, to devise ways of enhancing the function of the effector cells (natural killer cells) in immunocompromised patients with AIDS. In this regard, CD 16+ natural killer cells (not bearing antibody) from healthy seronegative donors have been shown to lyse HIV-infected cells in vitro (81). In general, natural killer cell activity against tumor cell targets appears to wane with clinical progression (82). This does not seem to result from a quantitative decrease of these cells in patients with AIDS, although recent studies have shown that CD 16+ cells are composed of phenotypically distinct subpopulations, and alterations may therefore occur within functionally active subsets (83). Natural killer cell activity from HIV-infected donors can be enhanced with interleukin-2 in vitro and in vivo ([84-86], Weinhold K. Personal communication). The importance of these cells in preventing the spread of HIV is unknown. Major histocompatibility class-restricted CTL directed against HIV may play a critical role in limiting viral spread. These cells have been isolated from the peripheral blood, cerebrospinal fluid, and bronchoalveolar lavage fluid of infected individuals (87-93). Although occasional CD4+, class II-restricted CTL can be isolated from infected donors, most such HIV-specific CTL is CD8+ and class I-restricted. These cells appear in many HIV-infected persons in such high frequencies that their activity can be measured directly in fresh PBMC (90, 94). This is the first chronic viral disease associated with such prominent CTL activity. The frequency and activity of HIV-specific CTL appear to decline with clinical progression, although there are insufficient data to implicate the loss of this function as causally related to clinical status (94). Cytotoxic T-cell responses are directed to both structural and regulatory proteins, although the former predominate (87-93, 95, 96). Activity is most frequently directed against the env and gag gene products, with significant responses also seen against reverse transcriptase, nef, and vif. Cytotoxic T-cell responses can be detected in HIV-infected persons in about 25% to 40% of individuals who have not progressed to AIDS (Koenig S. Unpublished observations). The specific epitopes recognized by CTL can be mapped by the use of synthetic peptides that bind to MHC class I proteins (97). The usual methods used for epitope mapping have consisted of developing antigenspecific lines or clones to serve as effectors and MHCmatched cells pulsed with overlapping synthetic peptides, which function as targets. As the magnitude of specific CTL responses to some HIV proteins is often sufficient to detect in whole PBMC, we have shown that mapping can be done using freshly separated cells, thus

obviating the need to generate antigen-specific lines or clones (95). CD8+ cells from HIV-seropositive donors have been shown to inhibit HIV replication in vitro (98, 99). In one study, bulk CD8+ cells appeared to inhibit viral replication in an MHC-unrestricted manner, suggesting that more than one CD8+ subpopulation may participate in the regulation of HIV replication (98). To this end, we have cloned CTL to several HIV-1 proteins and have shown that not only can these CD8+ cells lyse targets acutely infected with HIV, but they can inhibit HIV replication in vitro (Koenig S. Unpublished observations). Preliminary studies suggest that inhibition may occur both by lytic and nonlytic mechanisms. It will be critical to test the CTL clones of different HIV proteins in animal models, isolated from other forms of immunity to HIV, to determine their true role in regulating HIV replication. In conclusion, given that animal studies seem to indicate that protection against lentiviral infection is possible, and the magnitude of both cellular- and humoralspecific immune responses to HIV in many infected individuals is truly unparalleled when compared to other viral infections, it raises the important question of why the immune system does not prevail, clearing this virus sufficiently to prevent AIDS. Obviously, there is no clear-cut answer, but the data do support the hypothesis that, in fact, HIV is controlled for long periods due to effective immunity. The virus may not be totally cleared initially because the primary immune response to HIV is inadequate at the sites of entry (for example, mucosal surfaces) to prevent viral dissemination. Rapid changes in the primary structure of individual isolates of HIV undoubtedly occur in vivo (100). Unpublished observations have shown that as much as 11% of the amino acid sequence within the envelope region of a molecularly cloned isolate of simian immunodeficiency virus changes after 20 weeks' infection in macaques (Johnson P. Personal communications), and one would not expect the circumstances to be different for HIV. With time and progressive decline in the quantity and function of the antigen-specific CD4+ population and the function of CD8+ and CD 16+ cells, immune responses cannot be generated to these new variants and the infection ultimately progresses. While these events are occurring, immunity cannot be generated against the opportunistic pathogens. Therefore, the greatest chance of success in preventing AIDS from the vantage point of specific immunity seems to be by stimulating protective responses in high-risk individuals before exposure to HIV and by augmenting and maintaining a potent immune response to HIV during the earliest periods after infection. Mechanisms of CD8+ Cell Dysfunction in HIV Infection Giuseppe Pantaleo, MD (Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland): The most striking phenomenon of HIV infection is the depletion in CD4+ lymphocytes (31). However, other cell lineages (such as B cells [24], natural killer cells [84], and CD8+ lymphocytes [101, 102]), which


687

688



are not known to be infected with HIV in vivo, show profound functional defects in persons infected with HIV. Although the mechanisms responsible for the dysfunction of these other cell lineages have not been determined, the quantitative deficiency of CD4+ lymphocytes may explain, in part, the functional abnormalities of these other cells (1). Because of their antiviral effector function, CD8+ T lymphocytes may play a particularly important role in the pathogenesis of AIDS. In fact, HIV-specific cytotoxic CD8+ T cells have been found in the peripheral blood of HIV-infected patients (87, 89, 90), and CD8+ T lymphocytes can suppress HIV replication in vitro (103). These results suggest that a dysfunction of the CD8+ T-cell subset, the cell type that plays a potentially crucial role in the immune response against HIV, may contribute to clinical progression in HIV-infected persons. The functional abnormalities of CD8+ T lymphocytes in patients with AIDS include reduced capacity to undergo clonal expansion (104) and loss of HIV-specific cytolytic activity (94). We have investigated the mechanisms responsible for the defective clonogenic potential and the loss of HIVspecific cytolytic activity (105, 106). By use of one-color cytofluorometric analysis of resting peripheral blood lymphocytes of patients with AIDS, we consistently observed an increase in the number of human leukocyte antigen (HLA)-DR+ cells that was not caused by an increased percentage of B cells or monocytes. By use of two-color fluorescence cytofluorometry with a phycoerythrin (PE)-conjugated anti-HLA-DR monoclonal antibody (mAb) and a fluorescein isothiocyanate-conjugated anti-CD3 mAb, we demonstrated that a large proportion of T lymphocytes expressed HLA-DR antigen. As shown in Figure 9 (left), approximately one third of the CD3 + cells co-expressed HLA-DR (panel A). Two-color analysis with anti-CD8 or anti-CD4 mAb demonstrated that most CD3+DR+ cells belonged to the CD8+ subset (approximately 30%, panel B), and a significant proportion were CD4+ cells (approximately 15%, panel C). In addition, as previously reported (107), we have found that expression of DR antigen on T lymphocytes in patients with AIDS was not associated with the expression of CD25 antigen (the receptor for interleukin-2, Figure 9, left; panel D), a surface marker generally expressed on normal activated T lymphocytes. These results suggest that T lymphocytes are atypically activated in patients with AIDS and the expansion of the C D 8 + D R + C D 2 5 - cell subset could be

responsible for the decreased proliferative potential of CD8+ cells in these patients. In order to examine this hypothesis, sorted CD8+ DR+ and C D 8 + D R - cell subsets obtained from the peripheral blood of patients with AIDS were stimulated with anti-CD3 mAb (104). As shown in Figure 9 (right; panel A), CD8+DR+ cells showed a severe proliferative defect. The decreased proliferative potential of CD8+DR+ cells could not be ascribed to a deficiency of interleukin-2 in the cultures, since exogenous interleukin-2 was added 24 hours after initiating the cultures. The inability of CD8+DR+ cells to respond to anti-CD3 mAb resulted from the failure to induce expression of CD25 antigen after anti-CD3 mAb exposure in CD8 + DR+ cells compared with C D 8 + D R - cells (Figure 9, right; panels C and D). However, CD8+DR+ cells failed to proliferate even when interleukin-4, instead of interleukin-2, was added to the cell microcultures 24 hours after the addition of anti-CD3 mAb (Figure 9, right; panel B). In this regard, it should be stressed that interleukin-4 enhances the proliferative responses of preactivated T cells in a manner that is independent of interleukin-2 (108). This indicates that the decreased potential of the CD8+DR+ subset to proliferate was not exclusively related to the reduced expression of interleukin-2R, and that a more general mechanism or mechanisms could be responsible for the defective function of this subset. In addition, CD8+DR+ cells showed similar proliferative defects in response to such stimuli as anti-CD2 mAb, alone or in combination with anti-CD28 mAb, and such mitogens as phytohemagglutinin, alone or in combination with phorbol myristate acetate (105). The reduced proliferative potential of CD8+DR+ cells was further confirmed in experiments at the clonal level. In fact, the analysis of the frequency of the proliferating T-lymphocyte precursors in sorted CD8+DR+ and C D 8 + D R - cell subsets showed that only 5% of CD8+DR+ were clonogenic compared with the 25% to 30% of C D 8 + D R - cells (105). These results indicate that the proliferative defect of the CD8+ cells in patients with AIDS may be in large part explained by the in vivo expansion of the CD8+DR+ subset, which is apparently refractory to further in-vitro signals for proliferation. We then extended our analyses to the characterization of the cytolytic properties of the CD8+DR+ and C D 8 + D R - T-cell subsets (106). To this end, we selected three asymptomatic HIV-1 seropositive individu-

Figure 9. HLA-DR antigen expression and proliferative capacity of peripheral blood lymphocytes in HIV infection. Left. Cytofluorometric analysis of CD3, CD8, CD4, CD25, compared with HLA-DR antigen expression of peripheral blood lymphocytes from a patient with AIDS. Peripheral blood lymphocytes were stained with (A)fluoresceinisothiocyanate-conjugated anti-CD3 mAb and PE anti-HLA-DR mAb; (B) fluorescein isothiocyanate-conjugated anti-CD8 mAb and PE anti-HLA-DR mAb; (C) fluorescein isothiocyanate-conjugated anti-CD4 and PE anti-HLA-DR mAb; and (D)fluoresceinisothiocyanate-conjugated CD25 and PE anti-HLA-DR mAb. A control sample is shown in (E). In this patient, the percentages of CD3+, CD8+, and CD4+ cells co-expressing HLA-DR were 26%, 24%, and 2%, respectively. Cells co-expressing HLA-DR and CD25 (0.4%) were virtually absent. Right. In panels A and B, sorted CD8+DR+ and CD8+DR- cell populations from a patient with AIDS were stimulated with anti-CD3 mAb in soluble form (1/50 000 of ascitic fluid) in the presence of irradiated (4000 rad) allogeneic exogenous monocytes (104 per well), purified interleukin (IL)-2 (10% final concentration) or recombinant interleukin-4 (rIL-4) (200 U/well). After 72 hours, cell proliferation was evaluated by [3H] TdR uptake (16-h pulse). Panel A. bar 1: CD8+ cells alone; bar 2: + monocytes; bar 3: + IL-2; bar 4: + monocytes + IL-2; bar 5: + anti-CD3; bar 6: + anti-CD3 + monocytes; bar 7: + anti-CD3 + monocytes + IL-2. Panel B. bar 1: CD8+ cells alone; bar 2: + monocytes; bar 3: + IL-4; bar 4: + monocytes + IL-4; bar 5: + anti-CD3; bar 6: + anti-CD3 + monocytes; bar 7: + anti-CD3 + monocytes + IL-4. Panels C and D. The distribution of CD25 antigen in the CD8+ subsets stimulated for 72 hours with anti-CD3 mAb and cultured in the presence of IL-2 was measured. In this patient, 10% of CD8+DR+ cells expressed CD25 antigen compared with 50% of CD8+DR- cells. (Adapted with permission from [105].) 15 April 1991 • Annals of Internal Medicine • Volume 114 • Number 8 Downloaded From: http://annals.org/ by a Brown University User on 09/30/2013

689

Figure 10. HTV-specific cytolytic properties of CD8+ T-cell subsets in HIV infection. Panel A. Cytolytic activity against target cells expressing HIV-HUB env, gag, pol, and nef proteins by freshly sorted CD8+ DR+ and CD8+DR- cells from a HIV-1 seropositive healthy homosexual man. Panel B. Cytolytic activity against P815 target cells in the presence of anti-CD3 mAb (1:50 000 dilution of ascitic fluid) by the same CD8+ subset. Panels C and D. HIV-1-specific cytolytic activity and broad cytolytic potential, respectively, in CD8+DR+ T-cell subsets in a patient with AIDS. Panels E and F. Comparative analysis over time of the HIV-1 specific cytolytic activity and cytolytic potential during the progression of HIV-1 infection (patient was healthy and seropositive in 1986 and developed AIDS in 1989). (Adapted with permission from [106].)

als with significant levels of CD8+DR+ cells (30% to 60% of the total number of CD8+ cells) and HIVspecific cytolytic activity in the peripheral blood. As previously shown in patients with AIDS (105), the CD8+DR+ cells in these healthy HIV-1 seropositive individuals showed a substantial defect (as compared with C D 8 + D R - T cells) in their ability to proliferate in response to different stimuli and to undergo clonal expansion (106). In order to assess the HIV-specific cytolytic properties of the CD8+ T-cell subsets, autologous EpsteinBarr-virus-transformed lymphoblastoid cell lines were infected with HIV vaccinia recombinant viruses and used as target cells in a chromium release assay. As shown in Figure 10 (panel A), in one representative asymptomatic HIV-seropositive individual, the HIVspecific cytolytic activity was restricted predominantly 690

to the CD8+DR+ subset. The inability of C D 8 + D R cells to lyse target cells expressing HIV proteins was not caused by a defective function of the cytolytic machinery in this cell subset. In fact, the levels of broad cytolytic potential (as measured by a redirected killing assay) mediated by C D 8 + D R - cells were comparable to those of CD8+DR+ cells (Figure 10, panel B). This assay uses FcR+ target cells (we have used the murine mastocytoma cell line P815) and mAbs of the appropriate immunoglobulin G isotype. The anti-CD3 mAb used in this study mediates cross-linking between target and effector cells and triggers the cytolytic machinery of CTL by signaling the CD3-TCR complex, leading to the lysis of target cells if the effector cells have intact cytolytic potential. Freshly sorted CD8+DR+ cells obtained from one representative patient with AIDS did not display HIV-specific cytolytic potential (Figure 10,



panel Q. However, they retained their cytolytic potential as measured by the redirected killing assay (Figure 10, panel D). By doing a comparative analysis over time of both the HIV-1-specific cytolytic activity and the broad cytolytic potential, we have confirmed these results. As shown in Figure 10 (panel E), consistent cytolytic activity was mediated by CD8+DR+ cells in the early asymptomatic stages of HIV-1 infection, whereas in the same patient CD8+DR+ cells were not cytolytic in the advanced stages of AIDS (Figure 10, panel E). On the contrary, the levels of broad cytolytic potential remained unchanged regardless of the stage of HIV infection. In summary, we have provided evidence that the loss of HIV-specific cytolytic activity cannot be explained by a defective function of the cytolytic machinery and, as we have previously reported (106), it is unlikely that a cell-mediated suppression of HIV-1-specific CTL occurs in vivo. In addition, we have shown that HIVspecific cytolytic T lymphocytes belong predominantly to the CD8+DR+ subset whose ability to respond to further stimulation in vitro has been impaired. Therefore, our results, together with the recent demonstration that a progressive decrease in the frequency of HIV-1specific CTL occurs during the progression of HIV infection (94), suggest that the loss of HIV-specific cytolytic activity results in part from a reduced ability of the HIV-specific CTL population to expand in vivo. Other mechanisms, however, that may contribute to the loss of HIV-specific cytolytic activity are the in-vivo selection of HIV mutants, the loss of CD4+ helperdependent HIV-specific effector cell responses owing to the depletion in CD4+ lymphocytes, as well as the possibility that under certain circumstances CD8+ CTL may become infected with HIV on contact with HIVinfected CD4+ cells. These mechanisms are being investigated in our laboratory. Of particular interest, we have shown that a dissociation exists in the functional behaviors of CD8+DR+ T lymphocytes after triggering via the CD3-TCR complex. As a consequence of the signaling via the CD3-TCR complex, CD8+DR+ cells displayed a normal broad cytolytic potential, whereas the proliferative response to anti-CD3 mAb was defective. These findings indicate that the CD3-TCR complex is functional in these cells and that the reduced ability to proliferate in vitro may be caused by more general mechanisms. Further, it is not possible to exclude the possibility that the decreased proliferative potential of CD8+DR+ cells is a characteristic of cells in a specific functional and maturational stage in which the cells mediate cytolytic activity and proliferate poorly. In this regard, it should be stressed that expression of HLA-DR antigen, even in other cell lineages, is always associated with cell differentiation. Finally, the appearance in the circulation of this CD8+ subset does not seem to be specific for HIV infection, but it is a feature of an ongoing chronic stimulation of the immune system. In fact, in-vivo expansion of CD8+DR+ cells is a finding common to such viral infections as Epstein-Barr virus and cytomegalovirus (109, 110) as well as in bone marrow transplantation (111) and such chronic inflammatory diseases as sys-

temic lupus erythematosus (112) and rheumatoid arthritis (113). Requests for Reprints: Anthony S. Fauci, MD, National Institute of Allergy and Infectious Diseases, Building 31, Room 7A03, 9000 Rockville Pike, Bethesda, MD 20892. Current Author Addresses: Dr. Fauci: National Institute of Allergy and Infectious Diseases, Building 31, Room 7A03, 9000 Rockville Pike, Bethesda, MD 20892. Drs. Schnittman, Poli, Koenig, and Pantaleo: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Building 10, Room 11B-13, 9000 Rockville Pike, Bethesda, MD 20892.

References 1. Fauci AS. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science. 1988;239:617-22. 2. Ho DD, Moudgil T, Alam M. Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. N Engl J Med. 1989;321:1621-5. 3. Coombs RW, Collier AC, Allain JP, et al. Plasma viremia in human immunodeficiency virus infection. N Engl J Med. 1989;321:162631. 4. Zagury D, Bernard J, Leonard R, et al. Long-term cultures of HTLV-III—infected T cells: a model of cytopathology of T-cell depletion in AIDS. Science. 1986;231:850-3. 5. Folks T, Kelly J, Benn S, et al. Susceptibility of normal human lymphocytes to infection with HTLV-III/LAV. J Immunol. 1986; 136:4049-53. 6. Rosenberg ZF, Fauci AS. Induction of expression of HIV in latently or chronically infected cells. AIDS Res Hum Retroviruses. 1989;5:1-4. 7. Rosenberg ZF, Fauci AS. Immunopathogenic mechanisms of HIV infection: cytokine induction of HIV expression. Immunol Today. 1990;11:176-80. 8. Rosenberg ZF, Fauci AS. Activation of latent HIV infection. J NIH Res. 1990;2:41-5. 9. Balkwill FR, Burke F. The cytokine network. Immunol Today. 1989;10:299-304. 10. Folks TM, Justement J, Kinter A, Dinarello CA, Fauci AS. Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line. Science. 1987;238:800-2. 11. Poli G, Kinter A, Justement JS, et al. Tumor necrosis factor alpha functions in an autocrine manner in the induction of human immunodeficiency virus expression. Proc Natl Acad Sci USA. 1990; 87:782-5. 12. Poli G, Bressler P, Kinter A, et al. Interleukin-6 induces HIV expression in infected monocytic cells alone and in synergy with tumor necrosis factor alpha by transcriptional and post-transcriptional mechanisms. J Exp Med. 1990;172:151-8. 13. Lahdevirta J, Maury CP, Teppo AM, Repo H. Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome. Am J Med. 1988;85:289-91. 14. Cerami A, Beutler B. The role of cachectin/TNF in endotoxic shock and cachexia. Immunol Today. 1988;9:28-31. 15. Breen EC, Rezai AR, Nakajima K, et al. Infection with HIV is associated with elevated IL-6 levels and production. J Immunol. 1990;144:480-4. 16. Gallo P, Frei K, Rordorf C, Lazdins J, Tavolato B, Fontana A. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system: an evaluation of cytokines in cerebrospinal fluid. J Neuroimmunol. 1989;23:109-16. 17. Duh EJ, Maury WJ, Folks TM, Fauci AS, Rabson AB. Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat. Proc Natl Acad Sci USA. 1989; 86:5974-8. 18. Lenardo MJ, Baltimore D. NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell. 1989;58:227-9. 19. Poli G, Orenstein JM, Kinter A, Folks TM, Fauci AS. Interferonalpha but not AZT suppresses HIV expression in chronically infected cell lines. Science. 1989;244:575-7. 20. Lane HC, Kovacs JA, Feinberg J, et al. Anti-retroviral effects of interferon-alpha in AIDS-associated Kaposi's sarcoma. Lancet. 1988;2:1218-22. 21. Lane HC, Davey V, Kovacs JA, et al. Interferon-alpha in patients with asymptomatic human immunodeficiency virus (HIV) infection. A randomized, placebo-controlled trial. Ann Intern Med. 1990;112:805-11. 22. Krown SE, Gold JW, Niedzwiecki D, et al. Interferon-alpha with zidovudine: safety, tolerance, and clinical and virologic effects in patients with Kaposi sarcoma associated with the acquired immunodeficiency syndrome (AIDS). Ann Intern Med. 1990;112:812-21. 23. Roberts AB, Sporn MB. The transforming growth factor-betas. In:

15 April 1991 • Annals


of Internal

Medicine

• V o l u m e 114 • N u m b e r 8

691

24.

25.

26.

27. 28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45. 46. 47.

692

Sporn MB, Roberts AB, eds. Peptide Growth Factors and Their Receptors. New York: Springer-Verlag; 1990:419-72. Lane HC, Masur H, Edgar LC, Whalen G, Rook AH, Fauci AS. Abnormalities of B-cell activation and immunoregulation in patients with the acquired immunodeficiency syndrome. N Engl J Med. 1983;309:453-8. Rieckmann P, Poli G, Kehrl JH, Fauci AS. Activated B-lymphocytes from human immunodeficiency virus infected individuals induce virus expression in infected T cells and monocytes. J Exp Med. 1991;173:1-5. Bowen DL, Lane HC, Fauci AS. Immunopathogenesis of the acquired immunodeficiency syndrome. Ann Intern Med. 1985;103: 704-9. Lane HC, Fauci AS. Immunologic abnormalities in the acquired immunodeficiency syndrome. Annu Rev Immunol. 1985;3:477-500. Klatzmann D, Barre-Sinoussi F, Nugeyre MT, et al. Selective tropism of lymphadenopathy associated virus (LAV) for helper-inducer T lymphocytes. Science. 1984;225:59-63. Folks T, Benn S, Rabson A, et al. Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus. Proc Natl Acad Sci USA. 1985;82:4539-43. McDougal JS, Mawle A, Cort SP, et al. Cellular tropism of the human retrovirus HTLV-III/LAV. I. Role of T cell activation and expression of the T4 antigen. J Immunol. 1985;135:3151-62. Popovic M, Read-Connole E, Gallo RC. T4 positive human neoplastic cell lines susceptible to and permissive for HTLV-III [Letter]. Lancet. 1984;1:1472-3. Harper ME, Marselle LM, Gallo RC, Wong-Staal F. Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc Natl Acad Sci USA. 1986;83:772-6. Schnittman SM, Psallidopoulos MC, Lane HC, et al. The reservoir for HIV-1 in human peripheral blood is a T cell that maintains expression of CD4. Science. 1989;245:305-8. Kwok S, Mack DH, Mullis KB, et al. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J Virol. 1987;61: 1690-4. Ou CY, Kwok S, Mitchell SW, et al. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science. 1988;239:295-7. Psallidopoulos MC, Schnittman SM, Thompson LM 3d, et al. Integrated proviral human immunodeficiency virus type 1 is present in CD4+ peripheral blood lymphocytes in healthy seropositive individuals. J Virol. 1989;63:4626-31. Clouse KA, Powell D, Washington I, et al. Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol. 1989;142:431-8. Schnittman SM, Greenhouse J J , Psallidopoulos MC, et al. Increasing viral burden in CD4+ T cells from patients with human immunodeficiency virus (HIV) infection reflects rapidly progressive immunosuppression and clinical disease. Ann Intern Med. 1990; 113:438-43. Aoki S, Yarchoan R, Pludor JM, et al. Quantitative analysis of human immunodeficiency virus-1 (HIV-1) pro virus in peripheral blood mononuclear cells (PBMC) from patients with HIV-1 infection [Abstract]. Interscience Conference on Antimicrobials and Chemotherapy, 1989. Lane HC, Depper JM, Greene WC, Whalen G, Waldmann TA, Fauci AS. Qualitative analysis of immune function in patients with the acquired immunodeficiency syndrome. Evidence for a selective defect in soluble antigen recognition. N Engl J Med. 1985,313:7984. Sanders ME, Makgoba MW, Shaw S. Human naive and memory T cells: reinterpretation of helper-inducer and suppressor-inducer subsets. Immunol Today. 1988;9:195-9. Schnittman SM, Lane HC, Greenhouse J, Justement JS, Baseler M, Fauci AS. Preferential infection of CD4+ memory T cells by human immunodeficiency virus type 1: evidence for a role in the selective T-cell functional defects observed in infected individuals. Proc Natl Acad Sci USA. 1990;87:6058-62. Clark EA, Ledbetter JA. Leukocyte cell surface enzymology: CD45 (LCA, T200) is a protein tyrosine phosphatase. Immunol Today. 1989;10:225-8. Pang S, Koyanagi Y, Miles S, Wiley C, Vinters HV, Chen IS. High levels of unintegrated HIV-1 DNA in brain tissue of AIDS dementia patients. Nature. 1990;343:85-9. Chayt KJ, Harper ME, Marselle LM, et al. Detection of HTLV-III RNA in lungs of patients with AIDS and pulmonary involvement. JAMA. 1986;256:2356-9. Rosenberg ZF, Fauci AS. The immunopathogenesis of HIV infection. Adv Immunol. 1989;47:377-431. Smith PD, Ohura K, Masur H, Lane HC, Fauci AS, Wahl SM. Monocyte function in the acquired immune deficiency syndrome. Defective chemotaxis. J Clin Invest. 1984;74:2121-8. 15 April 1991 • Annals

of Internal

Medicine

48. Poli G, Bottazzi B, Acero R, et al. Monocyte function in intravenous drug abusers with lymphadenopathy syndrome and in patients with acquired immunodeficiency syndrome: selective impairment of chemotaxis. Clin Exp Immunol. 1985;62:136-42. 49. Wahl SM, Allen JB, Gartner S, et al. HIV-1 and its envelope glycoprotein down-regulate chemotactic ligand receptors and chemotactic function of peripheral blood monocytes. J Immunol. 1989;142:3553-9. 50. Tas M, Drexhage HA, Goudsmit J. A monocyte chemotaxis inhibiting factor in serum of HIV infected men shares epitopes with the HIV transmembrane protein gp41. Clin Exp Immunol. 1988;71: 13-8. 51. Snyderman R, Cianciolo GJ. Immunosuppressive activity of the retroviral envelope protein pl5E and its possible relationship to neoplasia. Immunol Today. 1984;5:240-4. 52. Gartner S, Markovits P, Markovitz DM, Kaplan MH, Gallo RC, Popovic M. The role of mononuclear phagocytes in HTLV-III/LAV infection. Science. 1986;233:215-9. 53. Gendelman HE, Orenstein JM, Martin MA, et al. Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. J Exp Med. 1988; 167:1428-41. 54. Koenig S, Gendelman HE, Orenstein JM, et al. Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science. 1986;233:1089-93. 55. Wiley CA, Schrier RD, Nelson JA, Lampert PW, Oldstone MB. Cellular localization of human immunodeficiency virus infection within the brains of acquired immune deficiency syndrome patients. Proc Natl Acad Sci USA. 1986;83:7089-93. 56. Weiss SH, Goedert JJ, Gartner S, et al. Risk of human immunodeficiency virus (HIV-1) infection among laboratory workers. Science. 1988;239:68-71. 57. Erickson-Miller CL, Abboud CN, Brennan JK. Purification of GCT cell-derived human colony stimulating factors. Exp Hematol. 1988; 16:184-9. 58. Massari FE, Poli G, Schnittman SM, Psallidopoulos MC, Davey V, Fauci AS. In vivo T lymphocyte origin of macrophage-tropic strains of HIV. Role of monocytes during in vitro isolation and in vivo infection. J Immunol. 1990;144:4628-32. 59. Perno CF, Baseler MW, Broder S, Yarchoan R. Infection of monocytes by human immunodeficiency virus type 1 blocked by inhibitors of CD4-gpl20 binding, even in the presence of enhancing antibodies. J Exp Med. 1990;171:1043-56. 60. Potts BJ, Maury W, Martin MA. Replication of HIV-1 in primary monocyte cultures. Virology. 1990;175:465-76. 61. Koyanagi Y, O'Brien WA, Zhao J Q , Golde DW, Gasson J C , Chen IS. Cytokines alter production of HIV-1 from primary mononuclear phagocytes. Science. 1988;241:1673-5. 62. Desrosiers RC, Wyand MS, Kodama T, et al. Vaccine protection against simian immunodeficiency virus infection. Proc Natl Acad Sci USA. 1989;86:6353-7. 63. Murphey-Corb M, Martin LN, Davison-Fairburn B, et al. A formalin-inactivated whole SIV vaccine confers protection in macaques. Science. 1989;246:1293-7. 64. Salk J. The whole virus approach to immunization, pp 378-381. In: Fauci AS, moderator. Development and evaluation of a vaccine for human immunodeficiency virus (HIV) infection. Ann Intern Med. 1989;110:373-85. 65. Redfield R, Davis C, Birx D, et al. Active immunization of recombinant produced gpl60 in patients with early HIV infection: phase 1 trial immunogenicity and toxicity [Abstract]. Proceedings of the Sixth International Conference on AIDS. 1990;3:209. 66. Robert-Gurofif M, Goedert J J , Naugle CJ, Jennings AM, Blattner WA, Gallo RC. Spectrum of HIV-1 neutralizing antibodies in a cohort of homosexual men: results of a 6 year prospective study. AIDS Res Hum Retroviruses. 1988;4:343-50. 67. Nara P. HIV neutralization: evidence for rapid binding and postbinding neutralization from infected human, chimpanzee, and gpl20 vaccinated animals. In: Lerner RA, Ginsburg H, Chanock R, Brown F, eds. Vaccines '89: Modern Approaches to New Vaccines Including Prevention of AIDS. Cold Spring Harbor, New York: Cold Spring Harbor Press; 1989:137-44. 68. Sarin PS, Sun DK, Thornton AH, Naylor PH, Goldstein AL. Neutralization of HTLV-III/LAV replication by antiserum to thymosin alpha 1. Science. 1986;232:1135-7. 69. Javaherian K, Langlois AJ, McDanal C, et al. Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc Natl Acad Sci USA. 1989;86:6768-72. 70. Ho DD, Kaplan JC, Rackauskas IE, Gurney ME. Second conserved domain of gpl20 is important for HIV infectivity and antibody neutralization. Science. 1988;239:1021-3. 71. Warren RQ, Wolf H, Shuler KR, et al. Synthetic peptides define the fine specificity of the human immunodeficiency virus (HIV) gpl60 humoral immune response in HIV type 1-infected chimpanzees. J Virol. 1990;64:486-92. 72. Ho DD, Li XL, Daar ES, et al. A neutralizing human monoclonal

• V o l u m e 114 • N u m b e r 8


73.

74.

75.

76.

77.

78.

79.

80. 81.

82.

83.

84.

85.

86.

87. 88.

89. 90.

91.

92.

93.

antibody (HMab) identifies an epitope within the putative CD4binding domain (CD4-BD) of HIV-1 gpl20 [Abstract]. Proceedings of the Sixth International Conference on AIDS. 1990;1:152. LaRosa GJ, Davide JT, Weinhold K, et al. Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science. 1990;249:932-5. Berman PW, Gregory TJ, Riddle L, et al. Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gpl20 but not gpl60. Nature. 1990;345:622-5. Goedert J J, Mendez H, Drummond JE, et al. Mother-to-infant transmission of human immunodeficiency virus type 1: association with prematurity or low anti-gpl20. Lancet. 1989;2:1351-4. Rossi P, Moschese V, Broliden PA, et al. Presence of maternal antibodies to human immunodeficiency virus 1 envelope glycoprotein gpl20 epitopes correlates with the uninfected status of children born to seropositive mothers. Proc Natl Acad Sci USA. 1989;86:8055-8. Devash Y, Calvelli TA, Wood DG, Reagan KJ, Rubinstein A. Vertical transmission of human immunodeficiency virus is correlated with the absence of high-affinity/avidity maternal antibodies to the gpl20 principal neutralizing domain. Proc Natl Acad Sci USA. 1990;87:3445-9. Rook AH, Lane HC, Folks T, McCoy S, Alter H, Fauci AS. Sera from HTLV-III/LAV antibody-positive individuals mediate antibody-dependent cellular cytotoxicity against HTLV-III/LAV-infected T cells. J Immunol. 1987;138:1064-7. Lyerly HK, Reed DL, Matthews TJ, et al. Anti-GP 120 antibodies from HIV seropositive individuals mediate broadly reactive antiHIV ADCC. AIDS Res Hum Retroviruses. 1987;3:409-22. Tyler DS, Lyerly HK, Weinhold KJ. Anti-HIV-1 ADCC. AIDS Res Hum Retroviruses. 1989;5:557-63. Ruscetti FW, Mikovits JA, Kalyanaraman VS, et al. Analysis of effector mechanisms against HTLV-I- and HTLV-III/LAV-infected lymphoid cells. J Immunol. 1986;136:3619-24. Bonavida B, Katz J, Gottlieb M. Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. I. Defective trigger on NK cells for NKCF production by target cells, and partial restoration by IL 2. J Immunol. 1986;137:1157-63. Moretta A, Tambussi G, Bottino C, et al. A novel surface antigen expressed by a subset of human C D 3 - CD16+ natural killer cells. Role in cell activation and regulation of cytolytic function. J Exp Med. 1990;171:695-714. Rook AH, Masur H, Lane HC, et al. Interleukin-2 enhances the depressed natural killer and cytomegalovirus-specific cytotoxic activities of lymphocytes from patients with the acquired immune deficiency syndrome. J Clin Invest. 1983;72:398-403. Rook AH, Hooks J J , Quinnan GV, et al. Interleukin 2 enhances the natural killer cell activity of acquired immunodeficiency syndrome patients through a gamma-interferon-independent mechanism. J Immunol. 1985;134:1503-7. Weinhold KJ, Lyerly HK, Matthews TJ, et al. Cellular anti-GP120 cytolytic reactivities in HIV-1 seropositive individuals. Lancet. 1988;1:902-5. Walker BD, Chakrabarti S, Moss B, et al. HIV-specific cytotoxic T lymphocytes in seropositive individuals. Nature. 1987;328:345-8. Sethi KK, Naher H, Stroehmann I. Phenotypic heterogeneity of cerebrospinal fluid-derived HIV-specific and HLA-restricted cytotoxic T-cell clones. Nature. 1988;335:178-81. Plata F, Autran B, Martins LP, et al. AIDS virus-specific cytotoxic T lymphocytes in lung disorders. Nature. 1987;328:348-51. Koenig S, Earl P, Powell D, et al. Group-specific, major histocompatibility complex class I-restricted cytotoxic responses to human immunodeficiency virus 1 (HIV-1) envelope proteins by cloned peripheral blood T cells from an HIV-1-infected individual. Proc Natl Acad Sci USA. 1988;85:8638-42. Koup RA, Sullivan JL, Levine PH, et al. Detection of major histocompatibility complex class I-restricted, HIV-specific cytotoxic T lymphocytes in the blood of infected hemophiliacs. Blood. 1989; 73:1909-14. Riviere Y, Tanneau-Salvadori F, Regnault A, et al. Human immunodeficiency virus-specific cytotoxic responses of seropositive individuals: distinct types of effector cells mediate killing of targets expressing gag and env proteins. J Virol. 1989;63:2270-7. Nixon DF, Townsend AR, Elvin JG, Rizza CR, Gallwey J, McMichael AJ. HIV-1 gag-specific cytotoxic T lymphocytes defined

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108. 109.

110.

111. 112.

113.

15 April 1991 • Annals


with recombinant vaccinia virus and synthetic peptides. Nature. 1988;336:484-7. Hoffenbach A, Langlade-Demoyen P, Dadaglio G, et al. Unusually high frequencies of HIV-specific cytotoxic T lymphocytes in humans. J Immunol. 1989;142:452-62. Koenig S, Fuerst TR, Wood LV, et al. Mapping the fine specificity of a cytolytic T cell response to HIV-1 nef protein. J Immunol. 1990;145:127-35. Walker BD, Flexner C, Paradis TJ, et al. HIV-1 reverse transcriptase is a target for cytotoxic T lymphocytes in infected individuals. Science. 1988;240:64-6. Townsend AR, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael J. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell. 1986;44:959-68. Walker CM, Moody DJ, Stites DP, Levy JA. CD8+ T lymphocyte control of HIV replication in cultured CD4+ cells varies among infected individuals. Cell Immunol. 1989;119:470-5. Tsubota H, Lord CI, Watkins DI, Morimoto C, Letvin NL. A cytotoxic T lymphocyte inhibits acquired immunodeficiency syndrome virus replication in peripheral blood lymphocytes. J Exp Med. 1989;169:1421-34. Tersmette M, Gruters RA, de Wolf F, et al. Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential HIV isolates. J Virol. 1989;63:2118-25. Rook AH, Manischewitz J F , Frederick WR, et al. Deficient, HLArestricted, cytomegalovirus-specific cytotoxic T cells and natural killer cells in patients with the acquired immunodeficiency syndrome. J Infect Dis. 1985;152:627-30. Shearer GM, Salahuddin SZ, Markham PD, et al. Prospective study of cytotoxic T lymphocyte responses to influenza and antibodies to human T lymphotropic virus-Ill in homosexual men. Selective loss of an influenza-specific, human leukocyte antigenrestricted cytotoxic T lymphocyte response in human T lymphotropic virus-Ill positive individuals with symptoms of acquired immunodeficiency syndrome and in a patient with acquired immunodeficiency syndrome. J Clin Invest. 1985;76:1699-704. Walker CM, Moody DJ, Stites DP, Levy J A. CD8+ lymphocytes can control HIV infection in vitro by suppressing virus replication. Science. 1986;234:1563-6. Margolick JB, Volkman DJ, Lane HC, Fauci AS. Clonal analysis of T lymphocytes in the acquired immunodeficiency syndrome. Evidence for an abnormality affecting individual helper and suppressor T cells. J Clin Invest. 1985;76:709-15. Pantaleo G, Koenig S, Baseler M, Lane HC, Fauci AS. Defective clonogenic potential of CD8+ T lymphocytes in patients with AIDS. Expansion in vivo of a nonclonogenic CD3+CD8+ D R + C D 2 5 - T cell population. J Immunol. 1990;144:1696-704. Pantaleo G, De Maria A, Koenig S, et al. CD8 + T lymphocytes of patients with AIDS maintain broad cytolytic function despite the loss of human immunodeficiency virus-specific cytotoxicity. Proc Natl Acad Sci USA. 1990;87:4818-22. Salazar-Gonzales J F , Moody DJ, Giorgi JV, Martinez-Maza O, Mitsuyasu RT, Fahey JL. Reduced ecto-5'-nucleotidase activity and enhanced OKT10 and HLA-DR expression on CD8 (T suppressor/ cytotoxic) lymphocytes in the acquired immune deficiency syndrome: evidence of CD8 cell immaturity. J Immunol. 1985; 135: 1778-85. Mitchell LC, Davis LS, Lipsky PE. Promotion of human T lymphocyte proliferation by IL-4. J Immunol. 1989;142:1148-57. Tomkinson BE, Wagner DK, Nelson DL, Sullivan JL. Activated lymphocytes during acute Epstein-Barr virus infection. J Immunol. 1987;139:3802-7. Carney WP, Iacoviello V, Hirsch HS. Functional properties of T lymphocytes and their subsets in cytomegalovirus mononucleosis. J Immunol. 1983;130:390-3. Lum LG. The kinetics of immune reconstitution after bone marrow transplantation. Blood. 1987;69:369-80. Linker-Israeli M, Gray JD, Quismorio FP Jr, Horwitz DA. Characterization of lymphocytes that suppress IL-2 production in systemic lupus erythematosus. Clin Exp Immunol. 1988;73:236-41. Pitzalis C, Kingsley G, Lanchbury JS, Murphy J, Panayi GS. Expression of HLA-DR, DQ and DP antigens and interleukin-2 receptor on synovial fluid T lymphocyte subsets in rheumatoid arthritis: evidence for "frustrated" activation. J Rheumatol. 1987; 14:662-6.

of Internal

Medicine

• V o l u m e 114 • N u m b e r 8

693

THERAPY AND PROPHYLAXIS IN HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION.

Frailty in people aging with human immunodeficiency virus (HIV) infection.

Human Immunodeficiency Virus (HIV).

[Extensive bilateral chorioretinitis revealing human immunodeficiency virus (HIV) infection].

Serum IgE and human immunodeficiency virus (HIV) infection.

Allergic manifestations of human immunodeficiency virus (HIV) infection.

Transforming growth factor beta and noncytopathic mechanisms of immunodeficiency in human immunodeficiency virus infection.

Increased production of human immunodeficiency virus (HIV) in HIV-induced syncytia formation: an efficient infection process.

Serum enhancement of human immunodeficiency virus (HIV) infection correlates with disease in HIV-infected individuals.

Human immunodeficiency virus (HIV) infection, sexually transmitted diseases and HIV-antibody testing practices in Belgian prostitutes.

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infection in Spanish leprosy patients.

Microbiome in human immunodeficiency virus infection.

Lymphoreticular Involvement in Human Immunodeficiency Virus Infection.

Pancreatitis in pediatric human immunodeficiency virus infection.

Human immunodeficiency virus infection in children.

Pancreatic involvement in human immunodeficiency virus infection.

Human immunodeficiency virus infection acquired in Thailand.

Brain imaging in human immunodeficiency virus infection.

Acquired immunodeficiency syndrome and human immunodeficiency virus infection in Nevada.

Human immunodeficiency virus (HIV)-indeterminate western blots and latent HIV infection.

Human immunodeficiency virus (HIV) associated nephropathy.

[Microbiological diagnosis of human immunodeficiency virus infection].

Human immunodeficiency virus infection and pneumothorax.

Malaria and Human Immunodeficiency Virus Infection.