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94 Fanta, C. et al. (1999) Systemic immunological changes induced by administration of grass pollen allergens via the oral mucosa during sublingual immunotherapy. Int. Arch. Allergy Immunol. 120, 218–224 95 Passalacqua, G. et al. (1998) Randomised controlled trial of local allergoid immunotherapy on allergic inflammation in mite-induced rhinoconjunctivitis. Lancet 351, 629–632 96 Oshiba, A. et al. (1996) Pretreatment with allergen prevents immediate hypersensitivity and airway hyperresponsiveness. Am. J. Respir. Crit. Care Med. 153, 102–109 97 Norman, P. et al. (1996) Treatment of cat allergy with T-cell reactive peptides. Am. J. Respir. Crit. Care Med. 154, 1623–1628

98 Boutin, Y. et al. (1997) Distinct biochemical signals characterize agonist- and altered-peptideligand-induced differentiation of naive CD4+ T cells into Th1 and Th2 subsets. J. Immunol. 159, 5802–5809 99 Janssen, E. et al. (2000) Modulation of Th2 responses by peptide analogues in a murine model of allergic asthma: amelioration or deterioration of the disease process depends on the Th1 or Th2 skewing characteristics of the therapeutic peptide. J. Immunol. 164, 580–588 100 Arkwright, P. and David, T. (2001) Intradermal administration of a killed Mycobacterium vaccae suspension (SRL 172) is associated with improvement in atopic dermatitis in children with moderate-to-severe disease. J. Allergy Clin. Immunol. 107, 531–534

The mucosal immune system: primary target for HIV infection and AIDS Ronald S. Veazey, Preston A. Marx and Andrew A. Lackner Despite intensive research, several questions remain regarding the pathogenesis of infection with HIV-1. Recently, it has been shown that simian immunodeficiency virus (SIV) selectively targets and destroys specific subsets of CD4+ T cells that are abundant in mucosal tissues but rare in peripheral lymphoid tissues. This finding could be highly relevant in explaining a major paradox in the infection and elimination of CD4+ T cells during HIV infection: the progressive decline in the number of CD4+ T cells in the blood, despite the paucity of HIV-infected cells in this tissue. This article discusses the hypothesis that infection with HIV and SIV, and the resulting disease, is governed by the state of cellular activation and the expression of chemokine receptors by specific subsets of CD4+ T cells residing in mucosal lymphoid tissues, rather than those found in the peripheral blood or lymph nodes.

Ronald S. Veazey* Preston A. Marx Andrew A. Lackner Tulane Regional Primate Research Center, Tulane University Health Sciences Center, 18703 Three Rivers Rd, Covington, LA 70433, USA. *e-mail: veazey@ tpc.tulane.edu Preston A. Marx Aaron Diamond AIDS Research Center, 455 1st Avenue, New York, NY 10016, USA.

The discovery that specific chemokine receptors act as co-receptors for both human and simian immunodeficiency viruses (HIV and SIV) has shed light upon the mechanisms underlying viral entry and tropism1–6. It is now known that, in addition to the CD4 molecule, most strains of HIV and SIV use the CC-chemokine receptor CCR5 for viral attachment and entry into host cells1,6–9. Furthermore, most, if not all, of the strains responsible for the transmission of HIV use CCR5, whereas viral strains that emerge later in the disease process might use the CXC-chemokine receptor CXCR4 instead of, or in addition to, CCR5 (Refs 2,10,11). It has even been hypothesized that progression from the asymptomatic stage of infection with HIV to the development of full-blown AIDS (and depletion of CD4+ T cells in peripheral blood) might be triggered by a switch in the viral usage of chemokine receptors from CCR5 to CXCR4 (Refs 10,11). If correct, this could, at least partially, explain the http://immunology.trends.com

mechanism behind the apparent delay in disease progression, as well as the profound drop in the number of peripheral CD4+ T cells observed in the later stages of infection with HIV. However, morerecent studies have demonstrated that CCR5utilizing strains predominate in cells from chronically infected patients12. Moreover, studies in the SIV rhesus macaque model indicate that such a switch in the expression of chemokine receptors is not necessary for peripheral CD4+ T-cell depletion and the development of AIDS in macaques. Recent studies indicate that all pathogenic SIV strains tested to date use CCR5 primarily1,9. Furthermore, CCR5 tropism persists in SIV-infected macaques sacrificed in advanced stages of SIV infection or AIDS (Ref. 13). Therefore, we hypothesize that it is the state of cellular activation and CD4+ T-cell turnover within mucosal tissues that determines the progression of disease and pathogenesis of AIDS, rather than a switch in the use of chemokine receptors. The current controversy over CD4+ T-cell turnover in HIV infection

Early in the HIV epidemic, there seemed to be little controversy over the theory that HIV preferentially infected and then destroyed CD4+ T cells as a result of lytic viral replication. This assumption was supported by the profound loss of CD4+ T cells in the blood of patients with AIDS, as well as the discovery that the CD4 molecule is the major receptor for the attachment and entry of HIV into cells14. However, this relatively simple explanation was complicated by data showing that the number of infected cells in the

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blood is too small to account for the profound CD4+ T-cell depletion observed15,16. Furthermore, several functional deficits were described in CD4+ T cells in the early stages of infection with HIV, long before the onset of CD4+ T-cell depletion was observed15. These data indicated that a selective loss of CD4+ T-cell memory function occurs in early infection15. Together, these findings suggested that the direct viral lysis of infected CD4+ T cells alone could not account for the T-cell depletion observed in AIDS patients, and that deficits might be occurring in CD4+ T cells that are not infected directly. As a result, additional mechanisms were proposed to explain the loss of CD4+ T cells, including bystander apoptosis triggered by soluble HIV antigens (Ags) or cytokine production15,17–19, cytotoxic T lymphocyte (CTL)-mediated destruction of CD4+ T cells20 and down-regulation of expression of the CD4 Ag induced by HIV nef gene products21–23. In addition, the concept of ‘viral latency’ was proposed to explain the prolonged period between infection with HIV and the development of AIDS. In 1995, Ho et al. introduced further evidence suggesting that direct viral lysis is the major mechanism responsible for the loss of CD4+ T cells24. Using highly active anti-retroviral therapy (HAART) as a tool to measure rates of CD4+ T-cell replenishment, they demonstrated that CD4+ T cells repopulated the blood of HIV-infected patients rapidly once viral replication was suppressed effectively. These, and other studies, challenged the concept of viral latency, proposing instead that viral replication and the destruction of CD4+ T cells were occurring at a very high rate, and that the rate of CD4+ T-cell destruction was nearly balanced by an equally high rate of production of CD4+ T cells24–27. In this ‘tap and drain’ hypothesis, CD4+ T cells are being produced continually at a high rate by the thymus and other sites of T-cell generation (the ‘tap’) in an attempt to maintain T-cell homeostasis in the face of massive, continuous, viral-induced destruction of CD4+ T cells (the ‘drain’). However, the ‘tap and drain’ hypothesis of HIV infection remains highly controversial, as data continue to emerge that both support28,29 and refute30–33 this theory. The latter data argue that the major proportion of the increase in the number of CD4+ T cells observed in patients undergoing HAART is the result of redistribution of existing CD4+ T cells from sequestered tissues to the peripheral blood, rather than de novo production33. Moreover, others suggest that suppression or impairment of de novo CD4+ T-cell production, rather than the destruction of CD4+ T cells, is responsible primarily for the gradual decline in the number of CD4+ T cells during infection with HIV (Refs 30,31). The major limitation of most studies of CD4+ T-cell proliferation during infection with HIV and SIV is that they have all been performed using lymphocytes derived from peripheral blood or lymph nodes. These studies might have assumed incorrectly that http://immunology.trends.com

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peripheral-blood lymphocytes provide an accurate reflection of what is occurring in the rest of the lymphoid tissues, and CD4+ T cells in the blood are representative of CD4+ T cells found in other tissues. By limiting the analysis to peripheral blood, important issues regarding lymphocyte trafficking, immunological compartmentalization and lymphocyte expansion in tissues are largely ignored. Although blood is easy to sample, the SIV macaque model of AIDS has provided an opportunity to examine simultaneously the proliferation of CD4+ T cells and their turnover in various immunological compartments and tissues, something that cannot be examined in humans. Recent studies in this model indicate that the major cellular targets for viral infection and replication are abundant in the mucosal lymphoid tissues, and the dynamics of the destruction and turnover of CD4+ T cells in these tissues differ drastically from those observed in the peripheral blood or lymph nodes. The role of differential chemokine-receptor expression between tissues and blood

In humans and macaques, CXCR4 is expressed primarily on naive (CD45RA+CD62L+) lymphocytes, whereas the expression of CCR5 is limited primarily to memory (CD45R0+CD62L−) lymphocytes13,34,35. Furthermore, it is increasingly apparent that CCR5expressing CD4+ T cells are activated, terminally differentiated effector cells that induce T helper 1 (Th1)-type (cell-mediated) cytokine responses preferentially35. Therefore, the expression of specific chemokine receptors appears to correlate with the state of cellular activation and prior exposure to Ag. Moreover, it appears that CD4+ T cells responsible for directing cell-mediated immune responses express CCR5 selectively, whereas those that express CXCR4 are mostly resting, naive cells35. It is now known that the majority of CD4+ T cells in the peripheral blood lacks expression of CCR5 and therefore, is not susceptible to infection with most (CCR5-utilizing) strains of HIV. In fact, peripheral blood mononuclear cells (PBMCs) usually require several days of mitogenic stimulation before they will support high levels of replication of HIV or SIV. Similarly, most CD4+ T cells in the lymph nodes and spleen also lack expression of CCR5, although levels are slightly higher in the latter13. By contrast, CD4+ T-cell populations in the mucosal surfaces are rich in CCR5-expressing CD4+ T cells. Indeed, the vast majority of CD4+ T cells residing in the intestinal tracts of humans and monkeys express high levels of CCR5 (Refs 13,34, 36,37) (Fig. 1). Similarly, CD4+ T cells in the female reproductive tract (vagina) express high levels of CCR5 (Fig. 1). Furthermore, this expression of CCR5 correlates with the predominance of activated, memory CD4+ T cells residing in these tissues13. Because the mucosal surfaces of the intestinal and reproductive tracts are exposed frequently to

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Fig. 1. Comparison of the coexpression of CC-chemokine receptor 5 (CCR5) and CD45RA on CD4+ T cells in various tissues of macaques. Note that the majority of viral target cells (CCR5+CD45RA−), shown in red, resides in mucosal tissues (e.g. intestinal and reproductive tracts). Plots were generated by gating on CD4+ lymphocytes.

environmental Ags, lymphocytes in these tissues are predominantly activated, terminally differentiated, effector cells. Together, the constitutively high level of cellular activation and CCR5 expression of CD4+ T cells in mucosal tissues explains why these cells are the optimal targets for viral infection and replication during primary HIV infection. Furthermore, the sheer abundance of CD4+CCR5+ T cells in the vagina and rectum could explain why CCR5-utilizing strains of HIV are transmitted selectively through mucosal exposures. Additional factors within the mucosal surfaces might contribute to the selective transmission of viruses that use CCR5. Although CXCR4 is also expressed on mucosal T cells (and is often coexpressed with CCR5 on intestinal CD4+ T cells)13,36, recently, it has been shown that both reproductive and intestinal mucosal epithelial cells produce and secrete large quantities of the CXC-chemokine stromal-cellderived factor 1α (SDF-1α)38. SDF-1α is the natural ligand for CXCR4, and it has been shown to inhibit the infection of T cells with X4 (CXCR4-dependent) Before infection 6%

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Fig. 2. Dot plots demonstrating typical changes that occur in CD4+ T-cell subsets during infection with simian immunodeficiency virus (SIV). Marked and rapid elimination of viral target cells (CCR5+CD45RA−), shown in red, occurs in all tissues within 11 days of infection with SIV, but this loss is most prominent in the intestinal tract, owing to the predominance of CCR5+ memory CD4+ T cells at this site. Also, note that attempts to replenish the number of CD4+ T cells in the intestinal tract are evident in both early (11 days) and later (three months) stages of infection, as shown by the appearance of naive (CD45RA+), CCR5+CD4+ T cells (upper right quadrants). However, the numbers of optimal target cells for viral infection remains depleted throughout the course of infection, presumably due to ongoing, lytic replication in this specific CD4+ T-cell subset. Abbreviation: CCR5, CC-chemokine receptor 5.

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strains of HIV (Ref. 38). Therefore, the abundance of activated, CCR5-expressing CD4+ T cells in the mucosa, combined with the abundant production of SDF-1α in the mucosal tissues, could explain why CCR5-utilizing strains of HIV and SIV are transmitted selectively across mucosal tissues. Furthermore, the fact that optimal viral replication occurs specifically within activated CD4+ T cells (which predominantly coexpress CCR5) could explain why dominance of X4 strains is seldom observed in humans, even in cases where this is the dominant transmitted strain. In this case, there will still be selective pressure favoring the virus that replicates most efficiently within CCR5+ (activated) cells, whereas those strains that use CXCR4 exclusively are likely to remain in proviral form within naive CD4+ T cells, until sufficient antigenic stimulation of the T cell triggers their replication. Selective and rapid intestinal CD4+ T-cell depletion occurs in primary SIV infection

The predominance of activated, CCR5-expressing CD4+ T cells in the intestine also explains the rapid and selective nature of the loss of CD4+ T cells that occurs in the intestine during primary SIV infection, long before significant changes in CD4+ T-cell counts are detected in the peripheral blood or lymph nodes39–41. A profound and relatively rapid depletion of the number of intestinal CD4+ T cells has been demonstrated also in HIV-infected humans42–45 (although studies in primary HIV infection have yet to be performed). More recently, it has been shown that this profound and rapid CD4+ T-cell loss is selective for specific subsets of activated, memory CD4+ T cells that coexpress CCR5 (Ref. 13) (Fig. 2). Moreover, it was demonstrated that these subsets are lost not only in the intestinal tract, but also, in the lymph nodes, spleen and blood during acute infection13. However, because effector CCR5-expressing CD4+ T cells are rare in the latter tissues, their loss does not impact significantly on the total number of CD4+ T cells in these tissue sites. Clearly, the kinetics of this selective CD4+ T-cell depletion are far too rapid to be explained by attrition owing to impaired production of CD4+ T cells (i.e. suppression of the bone marrow or thymus). Equally improbable is the possibility that these CD4+ T cells are being eliminated through ‘bystander’ apoptosis associated with chronic stimulation of the immune system, which has also been postulated as a major mechanism for the loss of CD4+ T cells during HIV infection20. The rapid kinetics of this CD4+ T-cell depletion (i.e. 11–14 days after infection), as well as the selective loss of CCR5+CD4+ (but not CCR5+CD8+) T cells during primary infection, argue against a theory of chronic immune stimulation. Instead, the data support the hypothesis that specific subsets of CD4+ T cells are being eliminated by direct, lytic viral replication. Intriguingly, the rate of this depletion

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corresponds also with the incubation period and pathogenesis associated with several other nonlentiviral infections46. In further support of this lytic hypothesis, experiments using SIV–HIV chimeric viruses (SHIVs) also indicate that the expression of chemokine receptors on cells is the primary factor in determining the subsets of CD4+ T cells that are depleted47. SHIVs that strictly use CXCR4 as their coreceptor deplete the population of CD4+ T cells in peripheral lymphoid tissues (which are mostly CXCR4+) rapidly, but spare intestinal CD4+ T cells. Conversely, SHIVs that utilize CCR5 exclusively deplete the population of intestinal CD4+ T cells (which are mostly CCR5+) rapidly, but spare peripheral blood CD4+ T cells47. Supporting results have also been demonstrated with human cells, at least under in vitro conditions10. PBMCs and tissueculture explants from humans are markedly depleted of CD4+ T cells when cultured with CXCR4-utilizing strains of HIV, whereas CCR5-utilizing strains only slightly reduce the number of CD4+ T cells in these tissues10,48. In combination, these data suggest strongly that the expression of chemokine receptors and the state of cellular activation determine whether CD4+ T cells are targeted for infection and destruction during infection with HIV. Here, we hypothesize that most, if not all, of this selective CD4+ T-cell loss can be attributed to direct, lytic viral replication. If ‘bystander apoptosis’ of uninfected CD4+ T cells were involved, the expression of chemokine receptors would be irrelevant in determining which CD4+ T cells are lost. Because chemokine receptors are involved in the attachment and binding of the virus to CD4+ T cells, these results argue that only those cells that are infected, or at least have attached viral proteins, are being targeted for elimination during infection with SIV and HIV. Viral-load dynamics also support, at least in part, the hypothesis that the number of effector CCR5expressing CD4+ T cells is a limiting factor in viral replication. Peak plasma viral loads occur 11–14 days after infection with SIV, followed (in most cases) by a marked decrease in viral loads and the establishment of a viral ‘set point’ that persists until the onset of AIDS (Refs 49,50). Most researchers attribute the early decline in viral loads to the development of specific antiviral immune responses50–53. Indeed, studies in CD8+ T-cell-depleted macaques demonstrate sustained viral loads following peak viremia, although the timing and magnitude of peak viral loads do not seem to be affected51. However, it is also probable that some of the rapid decline in viral loads during primary infection could be attributed to the rapid loss of optimal viral target cells necessary to sustain high levels of viral replication. A similar hypothesis, based upon mathematical modeling of CD4+ T-cell kinetics and viral loads during infection with HIV, was proposed originally by Phillips et al.54 http://immunology.trends.com

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and has been substantiated recently (at least in part) by models based on data from untreated HIV-1infected patients55. However, not until the discovery of the role of chemokine receptors in infection with HIV, and the subsequent characterization of the highly variable distribution of these receptors on various CD4+ T-cell subsets, was it possible to develop a hypothesis to explain the pathogenesis of infection with HIV. A unifying hypothesis for the pathogenesis of HIV infection and AIDS

On the basis of the above observations, it is hypothesized that primary HIV and/or SIV infection results in widespread lytic destruction of the majority of the susceptible (CD4+CCR5+) host cells that support viral replication (activated, memory cells) (Fig. 3). Because most of these cells reside in mucosal surfaces, little change is observed in the peripheral blood or lymph nodes at this stage. After these mucosal ‘target’ cells have been depleted, viral loads diminish and viral reservoirs are established in susceptible cells that can be infected but do not support high levels of viral replication, such as dendritic cells. Furthermore, viral reservoirs are established within resting, memory CD4+ T cells that are infectable, yet not actively transcribing DNA and, therefore, not supportive of lytic replication. Such resting, memory CD4+ T cells are increasingly being recognized as the major viral reservoir in HIVinfected patients on HAART, and are a major target of anti-HIV therapeutic strategies aimed at viral eradication12. Because the effector CD4+ T-cell population is greatly decreased in primary infection, ‘help’ for initiating new immune responses is diminished, but cell-mediated and humoral immune responses to previously encountered Ags might be maintained for extended periods by memory CD8+ T and B cells, respectively. Furthermore, several structural and functional alterations occur in the intestine during HIV infection, including intestinal villous atrophy56, epithelial-cell vacuolation and apoptosis57,58, mucosal inflammation59,60, and a variety of functional and/or absorptive deficits57,61–63. Although it is conceivable that these changes might be attributed directly to HIV infection, it is more probable that they are a consequence of immunological alterations of the mucosal environment. It is well established that immunological perturbations in the intestinal mucosa result in marked changes in intestinal architecture and function64–67. Such alterations could lead to an increased exposure to lumenal Ags, resulting in increased turnover of both CD4+ and CD8+ T cells. Eventually, however, the number of memory CD8+ T cells could be depleted through simple attrition (over years), and opportunistic infections (OIs) eventually take advantage of steadily diminishing immune responses. Once established, the OIs induce additional mucosal damage, and

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Fig. 3. Hypothetical model of the pathogenesis of HIV, focusing on events that occur in the intestinal mucosa. (a) In the normal intestinal tract, large numbers of activated, memory, terminally differentiated effector T cells (both CD4+ and CD8+) reside throughout the lamina propria. Resting, naive T cells are usually restricted to the inductive lymphoid tissues [i.e. Peyer’s patches (PP)]. Immunity to microbes and antigens (Ags) is maintained by a complex interplay of exposure to Ag in the inductive lymphoid tissue, followed by activation of Ag-specific CD4+ and CD8+ T cells. These cells recirculate briefly in the blood, and then disseminate to the effector sites, where CD4+ and CD8+ T-cell cooperation prevents the establishment of active infections in the tissues. (b) During primary infection with HIV, effector CD4+ T cells are eliminated rapidly owing to their high expression of CCR5 and their highly activated state, which supports lytic viral replication. Although CD4+ T cells are essentially depleted from the effector lymphoid tissues, most of the CD4+ T cells residing in the inductive lymphoid tissues (PP) are spared owing to their lack of CCR5 expression. However, low-level antigenic exposure to microbes in the lumen results in continuous priming, recirculation and homing of Agspecific cells to the effector lymphoid tissues. Naive CD4+ T cells, initially primed in inductive lymphoid tissues, soon become optimal target cells for HIV infection and replication, and might be destroyed in the inductive sites or shortly after trafficking to effector lymphoid tissues. (c) During the asymptomatic period of HIV infection, numbers of effector CD4+ T cells remain depleted, but immunity to previously encountered Ags might be maintained by the persistence and expansion of effector CD8+ T cells and memory B cells (not shown). However, CD4+ T-cell help for initiating new responses remains unavailable owing to the continuous elimination of Ag-primed CD4+ T cells as they begin to coexpress CCR5 and become fully activated, making them optimal targets for HIV infection and viral replication, respectively. (d) At the onset of AIDS, mucosal immunity diminishes, owing to the continued attrition of Ag-specific effector CD8+ T cells, and the continued absence of new CD4+ T-cell help. Opportunistic infections become established in tissues, resulting in a marked escalation in exposure to Ags, both to the offending Ag, as well as to other Ags entering through the compromised mucosal tissue. This results in a marked increase in the priming and activation of CD4+ T cells, leading to increased numbers of viral target cells, increased viral replication and rapid exhaustion of the naive CD4+ T-cell pool.

together, the OIs and increased exposure to lumenal Ags could prime the remaining naive CD4+ T cells in the inductive tissues (e.g. lymph nodes and Peyer’s patches). This would result in increased CD4+ T-cell activation, up-regulation of expression of CCR5, and recirculation and homing of these newly formed viral target cells throughout the intestinal tract (Fig. 3), as http://immunology.trends.com

well as in the systemic lymphoid tissues of chronically infected patients. An up-regulation of expression of CCR5 on CD4+ T cells has been described recently in the blood of untreated patients chronically infected with HIV (Ref. 68). This sustained increase in the number of viral target cells could explain the eventual global lymphoid depletion (of both inductive and effector sites), as well as the increased viral replication and higher viral loads, typical of patients in the advanced stages of AIDS. However, if the establishment of an intestinal or pulmonary infection (triggered by depletion of mucosal immunity) was responsible for the proliferation of local cells and expansion of CCR5-expressing CD4+ T-cell populations in mucosal tissues, this turnover might not be readily detectable in the peripheral lymphoid tissues. Because different trafficking patterns exist for peripheral and mucosal lymphoid compartments, it is improbable that such a regional, proliferative immune response would be detected in the peripheral circulation. Finally, this hypothesis could explain why certain pathogens result in OIs, whereas other agents do not. Perhaps, it is no coincidence that most of the OIs that develop in AIDS patients (e.g. Mycobacterium sp., cryptosporidiosis, candidiasis, cytomegalovirus, herpesviruses and pneumocystis), primarily, are diseases of the mucosal surfaces. These results could also shed light on recent observations of CD4+ T-cell replenishment and/or

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Acknowledgements This work was supported in part by NIH grants HD36310, DK50550 and AI49080, and Elizabeth Glaser Pediatric AIDS Foundation Grant No. PG-50861. A.A.L. is a recipient of the Elizabeth Glaser Scientist Award. We wish to thank L. Martin, C. Lanclos and J. Bruhn for technical support, and R. Rodriguez for assistance in preparing the figures.

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turnover in the peripheral blood. The relatively low rate of production of new CD4+ T cells detected in the peripheral blood does not support the ‘tap and drain’ hypothesis fully. Thus, perhaps we should examine the rates of conversion from resting, CXCR4expressing CD4+ T cells to activated, CCR5expressing CD4+ T cells in patients infected with HIV to help explain the pathogenesis of infection. Although it has been shown recently that chronically infected patients have higher expression of CCR5 on CD4+ T-cell subsets in the blood68, prospective studies examining the rate of conversion and production of these specific cell subsets might provide additional insight into the pathogenesis of chronic infection with HIV. Moreover, turnover rates of cells that home specifically to mucosal tissues could be measured by examining the expression of mucosal homing markers (i.e. α4β7, αEβ7 and/or β7 integrins) on well-defined T-cell subsets simultaneously. Finally, these discoveries have important implications for understanding the pathogenesis of infection with HIV and SIV and how these viruses are viewed. Although the need for a new classification of HIV terminology has been recognized previously7, the misleading terms of ‘macrophage tropic’ and ‘T-cell tropic’ remain widely used. These terms should be reconsidered, because all HIV strains are ‘T-cell tropic’, particularly in activated T-cell cultures. The differences in viral tropism have more to do with the differential expression of chemokine receptors on T cells obtained from different areas of the body. Intestinal CD4+ T cells are primarily CCR5+ and, thus, appear to be the major targets for R5 viruses (previously termed ‘macrophage tropic’), whereas peripheral blood CD4+ T cells primarily express CXCR4 and, thus, are the targets for X4 viruses (hence the term ‘T-cell tropic’). Given that the vast majority of experiments are performed on peripheral blood lymphocytes, it is easy to see why this terminology evolved and how our views of HIV pathogenesis have become so ‘hemocentric’. Furthermore, this differential expression could also explain some of the mysteries involved in transmission of the virus. For example, needlestick injuries result in a relatively low incidence of transmission (approximately 0.3%), whereas rectal exposures during anal intercourse and oral exposures in infants appear to have a much higher frequency of transmission. In light of this recent data, one possible explanation for these differences lies in the differential expression of chemokine receptors by the cells in these tissues.

References 1 Chen, Z. et al. (1997) Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry. J. Virol. 71, 2705–2714 2 Feng, Y. et al. (1996) HIV-1 entry cofactor: functional cDNA cloning of a seventransmembrane, G-protein-coupled receptor. Science 272, 872–877 http://immunology.trends.com

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Recent findings in the SIV macaque model of HIV infection indicate that the vast majority of the cellular targets for viral infection, replication and persistence lies within the intestinal and reproductive mucosal tissues. In this regard, primary SIV infection seems to be a disease of the mucosal immune system predominantly. If applicable to humans, these findings should impact profoundly on our understanding of the pathogenesis of infection with HIV, as well as offer logical explanations for many of the mysteries of infection. On the basis of the current data, we hypothesize that it could be the rate of conversion of resting, naive CD4+ T cells to activated, memory CD4+ T cells that coexpress CCR5 that determines the severity and rate of progression to AIDS in HIV-infected patients. Unlike blood, the mucosal tissue environment appears to match the requirements of naturally transmitted (CCR5-utilizing) immunodeficiency viruses, primarily owing to the high numbers of activated, CCR5-expressing cells in these tissues. The long ‘asymptomatic’ period of infection that follows acute infection could simply reflect the turnover rate of CCR5-expressing cells specifically within mucosal tissues, which could be highly variable depending upon the level of cellular activation in the mucosal immune system. Both prospective and retrospective studies of mucosal immune activation could be performed in humans, such as examining the effects of intestinal and reproductive diseases as co-factors in HIV infection and progression to disease. For example, diseases that induce cellular activation specifically in mucosal tissues, such as inflammatory bowel diseases, other sexually transmitted diseases and perhaps even dietary allergens, could lead to increased susceptibility to infection with HIV, and might accelerate disease progression and/or severity in HIV-infected patients. If correct, these hypotheses could explain much of the pathogenesis of HIV infection, as well as affect our strategies for both vaccine development and therapy regimes profoundly. Future vaccination and treatment strategies should be directed primarily towards stimulating effector cells residing within mucosal tissue and eliminating virus that persists in the mucosal tissues. Moreover, studies of HIV pathogenesis should consider approaching infection with HIV principally as a disease of the mucosal immune system.

3 Choe, H. et al. (1996) The β-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 86, 1135–1148 4 Doranz, B.J. et al. (1996) A dual-tropic primary HIV-1 isolate that uses fusin and the β-chemokine receptors CKR-5, CKR-2b as fusion cofactors. Cell 86, 1149–1159 5 Alkhatib, G. et al. (1996) CC CKR5: a RANTES, MIP-1α, MIP-1β receptor as a fusion cofactor for

macrophage-tropic HIV-1. Science 272, 1955–1958 6 Mellado, M. et al. (1999) Chemokine control of HIV-1 infection. Nature 400, 723–724 7 Berger, E.A. et al. (1998) A new classification for HIV-1. Nature 391, 240 8 Marx, P.A. and Chen, Z. (1998) The function of simian chemokine receptors in the replication of SIV. Semin. Immunol. 10, 215–223

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The biology of human natural killer-cell subsets Megan A. Cooper, Todd A. Fehniger and Michael A. Caligiuri

Megan A. Cooper Dept of Veterinary Biosciences and the Comprehensive Cancer Center, Todd A. Fehniger Michael A. Caligiuri* Dept of Medicine and the Comprehensive Cancer Center, The Ohio State University, 458A Starling-Loving Hall, 320 West 10th Ave, Columbus, OH 43210, USA. *e-mail: caligiuri-1@ medctr.osu.edu

Natural killer (NK) cells are one component of the innate immune system and have the ability to both lyse target cells and provide an early source of immunoregulatory cytokines1. Human NK cells comprise ≈15% of all lymphocytes and are defined phenotypically by their expression of CD56 and lack of expression of CD3 (Ref. 1). Almost 15 years ago, with the advent of monoclonal antibodies (Abs) for specific NK-cell markers, it was recognized that two distinct populations of human NK cells could be identified, based upon their cell-surface density of CD56 (Ref. 2). The majority (≈90%) of human NK cells have low-density expression of CD56 (CD56dim) and express high levels of Fcγ receptor III (FcγRIII, CD16), whereas ≈10% of NK cells are CD56brightCD16dim or CD56brightCD16− (Fig. 1). Early functional studies of these subsets by Lanier and colleagues revealed that resting CD56dim cells are the more cytotoxic subset2; however, many questions remained regarding the developmental state and functional relevance of these subsets in vivo. Over the past decade, a number of phenotypic and functional properties of human NK cells have been characterized, and there is now good evidence to suggest that distinct immunoregulatory http://immunology.trends.com

CD56bright CD56–PE

Human natural killer (NK) cells comprise ≈15% of all circulating lymphocytes. Owing to their early production of cytokines and chemokines, and ability to lyse target cells without prior sensitization, NK cells are crucial components of the innate immune system. Human NK cells can be divided into two subsets based on their cell-surface density of CD56 – CD56bright and CD56dim – each with distinct phenotypic properties. Now, there is ample evidence to suggest that these NK-cell subsets have unique functional attributes and, therefore, distinct roles in the human immune response. The CD56dim NK-cell subset is more naturally cytotoxic and expresses higher levels of Ig-like NK receptors and FCγγ receptor III (CD16) than the CD56bright NK-cell subset. By contrast, the CD56bright subset has the capacity to produce abundant cytokines following activation of monocytes, but has low natural cytotoxicity and is CD16dim or CD16−. In addition, we will discuss other cell-surface receptors expressed differentially by human NK-cell subsets and the distinct functional properties of these subsets.

CD56dim

CD16–FITC TRENDS in Immunology

Fig. 1. Flow cytometric analysis of CD56bright and CD56dim natural killer (NK) cells. CD56bright NK cells (red box) comprise ≈10% of all human NK cells and are CD16− or CD16dim. The majority (≈90%) of NK cells is CD56dim (blue box) and has high-density expression of CD16 (CD16bright). Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin.

roles can be assigned to the CD56bright and CD56dim NK-cell subsets. This review summarizes our current knowledge of the unique functions of these NK-cell subsets in the immune response and discusses relevant questions regarding the development of human NK-cell subsets. It is important to note that although murine NK cells resemble their human counterparts in many regards, including their ability to lyse tumor cells and produce cytokines, and their expression of inhibitory receptors for MHC class I molecules, they do not express a murine homolog of CD56. Consequently, it is not known yet whether mice have NK-cell subsets analogous to CD56bright and CD56dim cells. Thus, studies of the cell biology of CD56bright and CD56dim

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