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(Journal of Leukocyte Biology. 2000;68:373-382.)
© 2000 by Society for Leukocyte Biology

The emerging role of CD40 ligand in HIV infection

Richard S. Kornbluth

Department of Medicine, University of California San Diego and the VA San Diego Healthcare System, La Jolla, California

Correspondence: Richard S. Kornbluth, Department of Medicine - 0679, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093.


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ABSTRACT
 
CD40 ligand (also called CD40L, CD154, or TNFSF5) is a membrane protein expressed mainly by activated CD4+ T cells, which interacts with its receptor, CD40, on a variety of cells. The crucial importance of the CD40L-CD40 system for many immune responses has been extensively described. This review focuses on the multiple roles that this system may play in HIV infection. In early HIV infection, CD40L expression contributes to the immunological control of viral replication by inducing HIV-suppressive chemokines and supporting the production of anti-HIV antibodies and cytotoxic T cells. However, by activating antigen-presenting cells, such as dendritic cells and macrophages, CD40L can also lead to increased CD4+ T cell activation, which promotes the replication of HIV in these lymphocytes. Later, with the development of AIDS, CD40L-expressing CD4+ T cells become selectively depleted, perhaps as a result of a gp120-induced signal through CD4 that down-regulates CD40L expression. This acquired CD40L deficiency may explain the similarity between the types of opportunistic infections that occur in AIDS and in congenital CD40L deficiency. Vaccines or other strategies that promote the growth of CD4+ T cells capable of expressing CD40L may help to sustain host immunity against HIV and prevent AIDS-defining opportunistic infections.

Key Words: CD40 ligand • HIV • CD4+ T cells


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INTRODUCTION
 
CD40 ligand (CD40L) and its receptor, CD40, have become the subject of intense investigation by immunologists because of their fundamental roles in the development of cellular and humoral responses. The inappropriate expression of CD40L has been causally related to diseases as diverse as autoimmunity, transplant rejection, atherosclerosis, and Alzheimer’s disease. Conversely, a deficiency of CD40L stimulation has been linked to certain infections and cancers. Further details about these associations can be found in comprehensive reviews that portray the range and power of this ligand-receptor pair [1 2 3 4 ].


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THE CD40L/CD40 SYSTEM IS A CENTRAL REGULATOR OF ADAPTIVE IMMUNITY
 
Numerous studies have indicated that CD40L is a major regulator of immune responses. Briefly, CD40L affects the immune system in the following four ways (5): (1) CD40L "activates" or "matures" antigen-presenting cells (APCs, mainly macrophages and dendritic cells) to express co-stimulatory molecules including B7 (CD80 and CD86, both ligands for CD28), ICAM-1 (CD54), and CD44. These co-stimulatory signals are needed for T cells to become fully activated, rather than anergic, after T cell receptor (TCR)-stimulation. (2) CD40L induces macrophages and dendritic cells to make interleukin-12 (IL-12), IL-18, and other cytokines. In an immunological response, CD40L is the primary stimulus for IL-12 production (in the absence of microbial invasion). IL-12 and IL-18 stimulate NK cells for interferon-{gamma} (IFN-{gamma}) production. IL-12 causes CD4+ T cells to differentiate into type 1 helper T cells (Th1) that mediate delayed-type hypersensitivity responses. (3) CD40L-expressing CD4+ T cells are generally required for the generation of cytotoxic T lymphocytes (CTLs) against tumors and virus-infected cells. As in CD4+ T cell activation, CD40L activates APCs to express the co-stimulatory molecules needed to fully activate ("cross-prime") CTLs already responding to antigen/MHC class I complexes [6 ]. (4) CD40L promotes the differentiation of activated B cells and, with few exceptions, is required for the "class switch" from IgM to IgG production.


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CD40 SIGNAL TRANSDUCTION BY TRAF6
 
CD40 (also called TNFRSF5) is a type 1 membrane protein in the TNF receptor superfamily (TNFRSF) that is found on B cells, macrophages, dendritic cells, endothelial cells, and even T cells (see Table 1 ). The study of signal transduction by CD40 is an important area of research that has been reviewed elsewhere [7 ] and will only be touched upon in this review. Most notably, CD40 signaling leads to the activation of NF-{kappa}B, which in turn has anti-apoptotic effects [8 ] leading to the enhanced survival of macrophages [9 ] and dendritic cells [10 ].


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  		Table 1. Nomenclature of Selected TNF Superfamily Ligands and Receptors

A recent area of emphasis has been the interactions of CD40 with TNF receptor- associated factors (TRAFs). TRAF6 is particularly important because it is an essential adapter protein in both Toll-like receptor (Tlr) and IL-1 receptor pathways [11 ] and in certain TNFRSF pathways, particularly CD40, RANK [12 , 13 ], and the neurotrophin receptor p75 protein [14 , 15 ]. Whereas the Toll receptors and the IL-1 receptor bind indirectly to TRAF6 through two cytoplasmic proteins, MyD88 and IL-1 receptor-associated kinase (IRAK) (Fig. 1) , the cytoplasmic domains of ligated CD40, RANK [13 , 16 , 17 ], or neurotropin receptor p75 [14 ] bind directly to TRAF6. For CD40, a high density of CD40L molecules is needed to induce the degree of CD40 multimerization required for TRAF6 to bind to the CD40 cytoplasmic domain [18 ]. For macrophages [19 ] and resting B cells [20 ], CD40 stimulation is most efficiently provided by CD40L expressed on the membranes of activated T cells, rather than by soluble forms of the ligand [21 ].



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  		Figure 1. CD40L recreates aspects of the "danger" stimulus for antigen-presenting cells (APCs). A) In innate immune responses, receptors that recognize "danger" (such as Tlr4, which recognizes microbial products such as LPS or the IL-1 receptor) convey signals that stimulate dendritic cells, macrophages, and B cells through a MyD88/IRAK pathway that engages TRAF6 indirectly. This APC stimulation leads to B7 expression (signal 2) and fully activates naive T cells recognizing antigen presented on MHC (signal 1). These activated T cells proliferate, express their effector functions, and some become memory T cells. B) In adaptive immune responses, memory T cells encounter antigen in the absence of danger, so that B7 molecules are absent from the APCs. However, T cell receptor (TCR) stimulation (signal 1) induces memory T cells to express CD40L, which then interacts with CD40 on the APC to engage TRAF6 directly. This TRAF6-mediated APC stimulation again leads to B7 expression (signal 2) and fully activates the memory T cell. In addition, chemokines produced by CD40L-stimulated APC attract additional naive T cells that can become activated by these APCs if they recognize the MHC-presented antigen.

TRAF6 knock-out mice have been constructed. Although homozygous TRAF6-deficient mice survive for <2 weeks, macrophage cultures can be derived from their bone marrow cells for study. As expected, the production of nitric oxide after lipopolysaccharide (LPS) (a Tlr4 ligand) or IFN-{gamma} plus IL-1ß stimulation does not occur in mice lacking TRAF6. In addition, B cells derived from these mice fail to activate NF-{kappa}B in response to LPS, IL-1ß, or CD40 ligation [22 ]. Because these TRAF6-deficient mice develop osteopetrosis as a result of impaired functioning of osteoclasts (a monocyte-derived cell lineage) [22 , 23 ], it is likely that signaling induced by RANKL is also impaired because RANK is needed for osteoclast activation [24 ].

These studies show that a single adapter protein, TRAF6, is required for both the innate responses to LPS and injury, with its attendant release of IL-1 [25 ], and for CD40 signaling. Janeway [26 ] and Matzinger [27 ] first proposed that some type of noxious stimulus is necessary to trigger an immune response. LPS (a Tlr4 ligand) serves as the prototypic molecular embodiment of "danger" (Matzinger’s term), which induces B7 molecules (CD80 and CD86) and other APC factors needed to stimulate naive T cells. Once stimulated, a fraction of these T cells become memory cells. As recently shown by Lanzavecchia’s group, memory T cells are distinguished by the acquisition of the CCR7 chemokine receptor, which directs their migration out of the periphery and toward lymph nodes and by a marked enhancement in CD40L expression after TCR stimulation. As a consequence of this enhanced CD40L expression, activated memory T cells are much more effective at stimulating dendritic cells to produce IL-12 than are activated naive T cells [28 ]. Therefore, the TRAF6 pathway provides a common pathway for APC stimulation both at the site of "danger" (leading to the initial immune response) and at the site of a cognate interaction between a specific memory T cell and an antigen-bearing APC (leading to an expansion of antigen-specific responsive T cells).


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MEASUREMENT OF CD40L EXPRESSION BY ACTIVATED CD4+ T CELLS: CD40L EXPRESSION DECLINES WITH THE DEVELOPMENT OF AIDS
 
CD40L (also called CD154 or TNFSF5) is a type II transmembrane trimeric protein that is primarily expressed on the surface of a subset of CD4+ T cells upon cellular activation. The time course of CD40L expression is critically dependent upon the stimulus used to activate the T cells. Using phorbol myristate acetate (PMA) plus calcium ionophore, CD40L appears on a majority of T cells beginning at 2 h, peaking at 6 h, and lasting for >20 h. Anti-CD3 antibody, which acts through the TCR, is a more physiological stimulus that induces CD40L on a small subset of T cells beginning in 5 min and lasting for only 2–6 h [29 ]. A secondary effect of T cell stimulation is the up-regulation of CD40L transcription, and concurrent CD28 stimulation stabilizes the CD40L mRNA.

The very early appearance of CD40L on the surface of activated T cells after TCR stimulation is unusual for a T cell activation marker. It is now recognized that CD40L pre-exists within cytoplasmic granules in a subset of CD4+ and CD8+ T cells and that TCR stimulation leads to the rapid exteriorization of this storage depot [29 ]. However, this phenomenon cannot be appreciated when T cells are stimulated in mixed cultures such as PBMCs, because contact with CD40 receptor-bearing cells transmits a reverse signal that causes CD40L to become rapidly re-internalized through a pathway that leads to its degradation in lysosomes [30 ]. Even using purified CD4+ T cells, immobilized anti-CD3 stimulation of CD4+ T cells from healthy individuals results in <1% of cells staining positively for CD40L at 6 h [31 ]. An additional new technique is flow cytometric analysis of permeabilized cells that have been intracellularly stained for CD40L, but this method has so far only been applied to murine cells [32 , 33 ].

The importance of the inducing stimulus on the dynamics of CD40L expression complicates measurements of the expression of CD40L on T cells from HIV-infected individuals. Using PMA and calcium ionophore to maximally stimulate T cells, a reduction in CD40L-expressing CD4+ T cells was found in one study [34 ] but not another [35 ] (Table 2) . In contrast, Vanham et al. [36 ] used the more natural TCR stimulation provided by immobilized anti-CD3 antibody and found a severe defect in CD40L in CD4+ T cells in AIDS but not in earlier stages of HIV infection. These authors used Fc receptor-bearing murine P815 mastocytoma cells as a surface for the immobilization of anti-CD3 antibody, which was added to the media to gradually assemble on the P815 cells during a convenient overnight co-culture with T cells. At the same time this culture was set up, a fluorochrome-conjugated anti-CD40L antibody was added to the culture media to capture CD40L as it surfaced for subsequent detection by flow cytometry. Using uninfected subjects, 0.1% of unstimulated CD4+ T cells were positive for CD40L and ~50% were positive after anti-CD3 stimulation. HIV-infected subjects with >200 CD4+ T cells/µL behaved similarly. However, for subjects with AIDS (<200 CD4+T cells/µL), only ~18% of CD4+ T cells expressed CD40L after anti-CD3 stimulation, a significant reduction [36 ]. With the development of AIDS, not only are total CD4+ T cells depleted, but also the percentage capable of expressing CD40L is reduced. Because CD40L is a limiting factor for immune responses in a mouse model in vivo [37 ], it is likely that the reduced CD40L expression on CD4+ T cells in AIDS plays a role in the immunodeficiency of HIV infection.


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  		Table 2. Studies on CD40L Expression by T Cells in HIV Infection

It is not yet clear if the basis for a reduction in CD40L-expressing CD4+ T cells in HIV infection is a result of a defect in the signalling pathways needed for CD40L expression or whether there is a depletion of the differentiated CD4+ T cell subset capable of expressing CD40L. In this regard, CD27L (also called CD70 or TNFSF7) has been reported to promote the development of CD40L-expressing CD4+ T cells in T cell-dendritic cell co-cultures [38 ]. Because CD27L expression is greatly reduced on B cells from HIV-infected individuals [34 ], it is possible that deficient CD27L expression leads to a cell production defect that contributes to the reduced numbers of CD40L-expressing CD4+ T cells in AIDS.

Similar studies have examined the effects of SIV infection upon CD40L expression in nonhuman primates. Using PMA and calcium ionophore, Brice et al. [39 ] found no differences in CD40L expression on CD4+ T cells from rhesus macaques at any stage of SIV infection, consistent with a similarly performed study in humans with HIV infection [35 ]. Interestingly, however, there were significant differences in the normal physiology of CD40L expression between rhesus macaques, sooty mangabees (SMs), and African green monkeys (AGMs). In SMs, CD40L is expressed on activated CD4+ T cells at almost twice the density measured on activated rhesus CD4+ T cells. In AGMs, CD40L is expressed mainly on a subset of activated CD8+ T cells rather than on CD4+ T cells. Only the rhesus macaque expresses CD40L in a manner similar to humans. Because SIV-induced AIDS develops only in rhesus macaques and not in SMs or AGMs, it is tempting to speculate that a relative preservation of CD40L expression contributes to the disease resistance of SIV-infected SMs and AGMs [39 ].


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CD40L EFFECTS THAT INCREASE HIV REPLICATION
 
One of the most established concepts in HIV retrovirology is that CD4+ T cell activation is required for HIV to replicate in these cells [40 ]. Consequently, it is not surprising that the first observations on the effects of CD40L on HIV replication by Clark’s group showed that CD40L expression by anti-CD3 activated CD4+ T cells was needed to induce B7 molecule expression on dendritic cells, resulting in full CD4+ T cell activation and the enhanced growth of HIV-1 IIIB (LAI), a CXCR4-utilizing (X4) syncytium-inducing strain of HIV-1 [41 ]. Similar results were reported by Tsunetsugu-Yokota et al. [42 ]. Because CD40L induces macrophages and dendritic cells to produce MIP-1{alpha}, MIP-1ß, and RANTES, it is notable that these same ß-chemokines enhance the replication of X4 strains of HIV in CD4+ T cells [43 ]. These factors could explain why the peak of viremia that occurs in SIV-infected macaques 2 weeks post-infection correlates in time with a transient surge in CD40L expression by peripheral blood CD4+ T cells [44 ]. Additionally, CD40L expression is needed for retroviral replication in MAIDS, a murine model of AIDS [45 ].

CD40L also stimulates HIV production by infected dendritic cells. These cells can become infected by CCR5-utilizing (R5) nonsyncytium-inducing strains when they are immature, but viral reverse transcription does not complete in these resting cells [46 ]. After CD40L stimulation, however, dendritic cells become resistant to de novo infection yet complete reverse transcription of the viral genomes already within them, leading to the production of infectious virions [47 ]. As an additional factor, CD40L stimulation activates NF-{kappa}B, which in turn promotes transcription from the HIV LTR [48 ].

CD40L has other, less important, effects in promoting HIV replication and disease. For a small subset of B cells, CD40L induced the expression of both CD4 and CXCR4, making these cells susceptible to HIV infection [49 , 50 ]. Microvascular endothelial cells expressing abnormally high levels of CD40 have been found to stimulate CD40L-expressing B cells, favoring the development of lymphoma [51 ]. Perhaps because CD40 is present on normal endothelial cells, a proportion of Kaposi’s sarcoma tumor cells express CD40 [52 ].


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CD40L EFFECTS THAT DECREASE HIV REPLICATION: CHEMOKINES
 
CD40L can limit HIV replication by inducing the production of HIV-suppressive ß-chemokines. Our studies found that contact with CD40L-expressing cells induced monocyte-derived macrophages (MDM) to produce copious amounts of MIP-1{alpha}, MIP-1ß, and RANTES. For MIP-1{alpha} and MIP-1ß, the amount produced per cell per day exceeded that of any nontransformed CD8+ T cell, CD4+ T cell, or NK cell yet described. The ß-chemokine-containing supernatants from these CD40L-stimulated macrophages protected purified CD4+ T cells from infection by the R5 strain, HIV-1-SF162, and this protection was abrogated using a cocktail of antibodies which neutralized these three ß-chemokines [53 ]. Analogous results were obtained using CD40L-stimulated monocyte-derived dendritic cells (DC) [54 55 56 ]. CD40L also caused the down-regulation of CCR5 from the surface of dendritic cells [46 , 55 , 56 ] and macrophages [57 ], protecting these cells from infection by R5 viruses.

In addition to inducing the CCR5-binding ß-chemokines, CD40L also stimulates the production of other chemokines. Monocyte-derived chemokine (MDC) is strongly induced by CD40L in DC [58 , 59 ]. This may be significant because some [60 ] but not all studies have found that a processed form of MDC has anti-HIV activity. More recently, CD40L has been shown to stimulate fractalkine expression by dendritic cells and B cells [61 , 62 ]. Fractalkine may also have anti-HIV activity against isolates that use the fractalkine receptor (CX3CR1) as a co-receptor [63 ]. Finally, in macaque PBMCs, CD40L induced the production of IL-16, which suppresses SIV replication by reducing the transcription of viral mRNA [64 ]. Taken together, CD40L provides a molecular link between antigen-specific T cell stimulation and the production of anti-HIV chemokines by macrophages and dendritic cells.


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CD40L EFFECTS THAT DECREASE HIV REPLICATION: IL-12 AND IFN-{gamma} INDUCTION
 
CD40L induces macrophages and dendritic cells to produce IL-12. IL-12 promotes the production of IFN-{gamma}, Th1 T cell differentiation, and CTL activity. CD40L also induces IL-18 mRNA in dendritic cells [10 ] and MDM (unpublished results). Although IL-18 is molecularly unrelated to IL-12, IL-18’s biological activities overlap with IL-12 and the two molecules synergize to stimulate IFN-{gamma} production [65 ].

Staphylococcus aureus Cowan strain (SAC) has been used as a stimulant to uncover the severe IL-12 production defect that occurs in AIDS (1% of normal levels) [66 , 67 ]. Chougnet et al. [68 ] confirmed this result and used anti-CD40L antibodies to determine that ~60% of the IL-12 response to SAC in semi-purified monocytes is a result of SAC-induced CD40L expression. The addition of soluble CD40L and IFN-{gamma} to monocyte cultures from HIV-infected subjects restored SAC-induced IL-12 production to normal [68 ]. Therefore, as the crucial T cell molecule needed for IL-12 production by macrophages and dendritic cells, CD40L is important for the immunological production of IFN-{gamma}, which in turn strongly protects macrophages from HIV infection [69 70 71 ].


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EVIDENCE THAT HIV ACTIVELY SUPPRESSES CD40L EXPRESSION
 
Given the CD40L-dependent anti-HIV activities described above, it is not surprising that HIV-1 has evolved a means to suppress CD40L expression. In a pioneering study, Chirmule et al. [72 ] found that purified gp120 suppressed the expression of CD40L on anti-CD3-stimulated CD4+ T cells, in turn preventing the induction of B7-1 expression on B cell APCs [72 ]. More recently, DNA microarray technology (Affymetrix GeneChip) was used to detect the mRNAs present in PBMCs from HIV-infected individuals during successful antiretroviral therapy and after discontinuing this therapy. On treatment when virus was undetectable, CD40L mRNA was present in PBMCs. However, after treatment was stopped, CD40L mRNA was 1 of only 20 out of 6,800 mRNAs that was down-regulated as the virus levels rebounded [73 ].

The mechanism whereby HIV suppresses CD40L expression likely involves the binding of gp120 to CD4 because certain anti-CD4 antibodies also suppress CD40L expression [72 ]. This effect of anti-CD4 antibody is relatively selective, however, and does not extend to certain other aspects of T cell activation such as the expression of the CD69 activation marker [74 ]. Of great interest, as originally shown in 1984 in Chess’s laboratory, only certain anti-CD4 antibodies suppress CD40L expression (measured then as the ability of anti-CD3 stimulated T cells to provide help for antibody production by B cells) [75 ]. The anti-CD4 antibodies that most strongly inhibit T cell help (i.e., CD40L) are the same clones as those that interfere with HIV infection [76 ]. This suggests that HIV interacts with the surface of CD4 that delivers a signal to suppress CD40L expression (Fig. 2) .



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  		Figure 2. HIV-1 envelope engages CD4 on T cells in a manner similar to CD40L-down-regulating anti-CD4 antibodies. As described by Sattentau et al. (76), certain anti-CD4 monoclonal antibodies (especially OKT4a and OKT4e) block HIV-1 infection. In a syncytium-forming assay (x-axis), a lower 50% inhibitory dose (ID50) for infection indicates a more powerfully protective antibody. In entirely separate experiments, Rogozinski et al. described the ability of these same anti-CD4 antibodies to down-regulate the "help" provided by anti-CD3-activated CD4+ T cells for B cell IgG production (75), which is now known to be a result of CD40L (y-axis). The graph indicates that those anti-CD4 antibodies that strongly block the interaction of HIV-1 gp120 with CD4 also strongly inhibit CD40L expression. Of the several possible ways in which HIV-1 gp120 might interact with CD4, it interacts with the face of the CD4 molecule that conveys a negative signal for CD40L expression.

It is now understood that HIV uses a chemokine receptor as its main cellular receptor, and that binding to CD4 is of secondary importance [77 ]. Why, then, is HIV so committed to interacting with CD4? One explanation has been that HIV targets CD4+ cells to hide within the immune system and produce a persistent infection. A second explanation is that the portion of Env that binds to the chemokine receptor needs to remain inaccessible for neutralizing antibodies and that binding to CD4 is simply a means of inducing the conformational change needed to expose this chemokine receptor-binding domain. However, the suppressive effects of CD4 binding on CD40L expression suggest an additional explanation: HIV binds to CD4 to down-modulate CD40L expression, which in turn blunts the body’s anti-HIV ß-chemokine and CTL responses and permits the virus to replicate more efficiently.

No detailed information is available to indicate how much gp120 is required for CD40L down-regulation nor where in the body this down-regulation might be occurring. gp120 at the picomolar level (1–100 ng/ml) has been reported in sera from patients with late-stage HIV infection. This circulating gp120 occurs mainly as an immune complex with anti-gp120 antibody [78 , 79 ]. Complexes of gp120 bound to CD4 have also been detected on the surfaces of circulating CD4+ T cells [80 ]. Anti-gp120 antibodies may cross-link gp120 bound to CD4 on T cells, thereby promoting its suppressive effects [81 , 82 ]. One possibility is that the high level of gp120 present in the vicinity of an HIV-producing cell down-regulates CD40L expression on nearby CD4+ T cells and thereby prevents them from successfully activating APCs, creating a locally immunosuppressed microenvironment. Such a scenario may contribute to the striking absence of CD4+ T cells that respond to gp120 as an antigen (in contrast to the frequent detection of CD4+ T cells that respond to Gag or Nef antigens) [83 ]. The presence of high-titer anti-gp120 antibodies may be a result of B cell stimulation through a CD40L-independent pathway, such as contact with membrane TNF{alpha} [84 ].

It is conceivable that this gp120 suppression of CD40L expression defeats some crucial aspect of the immune response against HIV, accounting for the incomplete control of viral replication and the eventual viral conquest of the immune system. Experiments in LCMV-infected mice showed an expansion of proliferating MHC class I tetramer-positive CD8+ T cells, yet these LCMV-specific CD8+ T cells were unable to function [85 ]. Similarly nonfunctional HIV-specific CD8+ T cells have been identified in HIV-infected patients [86 , 87 ]. This situation is similar to experiments in which transgenic T cells were adoptively transferred into tumor-bearing mice. Although all of the transgenic T cells were able to recognize a tumor antigen, the tumor was not rejected. However, if CD40 stimulation was provided, the tumor was rapidly eliminated [88 ]. By analogy, deficient CD40L expression may contribute to the functional deficiencies of HIV-specific CD8+ T cells, which may underlie the incomplete control of HIV replication by the immune system.


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CONSEQUENCES OF IMPAIRED CD40L EXPRESSION FOR SUSCEPTIBILITY TO OPPORTUNISTIC INFECTIONS
 
Although the above concepts provide a rationale for believing that HIV has specifically evolved a means to down-regulate CD40L expression by CD4+ T cells, a susceptibility to AIDS-defining opportunistic infections may be another consequence of deficient CD40L expression. Children born with inactivating mutations in the CD40L gene have the X-linked HyperIgM syndrome (X-HIM). Despite adequate CD4+ T cell numbers, X-HIM patients have a high incidence of Pneumocysts carinii pneumonia (PCP), mycobacterial infections, cryptosporidiosis, cryptococcal meningitis, post-transfusion CMV, and other infections.

For PCP, a common infection in AIDS, elegant studies in mice have shown that CD40L is essential to activate immune cells (probably macrophages) to eliminate this organism [89 , 90 ]. The role of CD40L in mycobacterial infections is more controversial. Out of 57 patients in the international X-HIM registry, 2 contracted BCGosis after inadvertent BCG vaccination, 2 developed infection with M. bovis, and 2 developed tuberculosis (1 case was limited to the lung and the other case was disseminated) [91 ] (L. Notarangelo, personal communication). Although this suggests that CD40L is needed to resist mycobacterial diseases, CD40L knock-out mice differed very little from wild-type mice in their ability to control M. tuberculosis infection [92 ]. However, a study of M. avium infection in mice injected with anti-CD40L antibody showed that CD40L plays a role in the containment of this organism. In addition, CD40L treatment of human macrophages in vitro induces mycobacteriostatic activity against M. avium [93 ], M. bovis BCG, and M. tuberculosis (unpublished results). Because tuberculosis is the major opportunistic infection and cause of death in HIV-infected persons on a worldwide basis, this opportunistic infection may be the most serious consequence of the suppressive effects of HIV upon CD40L expression.


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CD40L EFFECTS RELEVANT TO AN HIV VACCINE
 
CD40L is important for antiviral immunity. Studies of CD40L knock-out mice and humans with X-HIM have shown that CD40L is critical for the production of anti-viral IgG and IgA antibodies. CD8+ T cells are also important defenses against viruses. In macaques, the depletion of CD8+ T cells by anti-CD8 antibody revealed an essential role for CD8+ T cells in limiting SIV replication [94 , 95 ], and it is likely that CD8+ T cells are equally important in anti-HIV immunity [96 ]. Because CD40L is essential for CD8+ CTL activities against allogeneic transplants and vaccine-induced CTLs against tumor cells [5 ], it would seem obvious that CD40L is necessary for antiviral CD8+ T cells as well. However, this assumption has been challenged by studies showing that CD40L is not required for the generation of early antiviral CTLs against LCMV, pichinde virus, and VSV [97 98 99 ]. These findings were interpreted to show that, unlike other microbial pathogens, "viruses know the trick" for stimulating early CD8+ CTL without CD4+ T cell help, probably because these inflammatory viruses directly induce the expression of costimulatory molecules on dendritic cells [100 ]. However, CD40L appears to be required for the generation of memory CD8+ T cells [97 98 99 ] and CD40L is important to the outcome of viral infection in vivo. In studies on macrophage-tropic strains of LCMV, which produce a more chronic viremia, Ahmed’s group found that CD40L was absolutely required for the eventual clearance of LCMV from the blood [32 ]. Similarly, a study of VSV infection in mice showed 50% mortality in CD40L knock-out mice versus no mortality in wild-type controls [101 ]. Using tetramer analysis, mice lacking the CD40 receptor and challenged with VSV developed only 10% of the normal number of anti-VSV mucosal CD8+ T cells in their intestinal lamina propria [102 ].

CD40L stimulation also induces other TNFSF molecules that support cellular immune responses, such as OX40L and 4-1BBL. For OX40L, studies using knock-out mice [103 , 104 ] or an agonistic anti-OX40 antibody [105 ] demonstrated that an OX40L/OX40 interaction is necessary to maintain a large population of antigen-specific CD4+ T cells. Because CD40- stimulation induces the appearance of OX40L on dendritic cells [103 ] and B cells [106 ], OX40L may mediate some of the supportive effects of CD40L on CD4+ T cell responses. Similarly, 4-1BBL has been shown to be important for maintaining strong CD8+ T cell responses. 4-1BBL knock-out mice have impaired generation of CTLs [107 ] and, conversely, an agonistic anti-4-1BB antibody enhanced the development of CD8+ T cells [108 , 109 ]. Because CD40-stimulated B cells and mature dendritic cells (formed after CD40L stimulation) both express 4-1BBL [110 ], this is another pathway through which CD40L promotes cellular immunity.

For HIV, CD4+ T cell help is thought to be essential for anti-HIV CD8+ T cell activity [111 112 113 114 ]. Because CD40L provides the molecular basis for CD4+ T cell help [115 ], it seems very likely that CD40L is important for both the humoral and cellular aspects of anti-HIV immunity. Recently, a study in mice showed that the co-injection of a CD40L expression plasmid along with a plasmid for the HIV p55 Gag protein led to a marked enhancement in antibody and CTL responses [116 ]. This suggests that CD40L could be a useful component in an anti-HIV vaccine.


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CONCLUSIONS
 
CD40L is established as a crucial regulator of cellular as well as humoral immunity. The activation of T cells to express CD40L leads to effects on APCs that both protect CD4+ T cells from HIV infection (e.g., through ß-chemokine production) and activate these cells for enhanced HIV replication. CD40L stimulation can also both protect dendritic cells and macrophages from HIV infection and activate already infected dendritic cells to express HIV. With the development of AIDS, CD40L-expressing CD4+ T cells are disproportionately reduced, perhaps as a result of CD40L down-regulation secondary to gp120 interactions with CD4. Because CD40L is needed for strong cellular immunity, the reduced expression of CD40L on CD4+ T cells may be a factor in the progression of HIV infection. Similarly, by creating an acquired CD40L deficiency, HIV infection may predispose the host to those opportunistic infections for which CD40L expression confers protection. These considerations suggest that therapies designed to prevent or reverse the decline in CD40L-expression by CD4+ T cells might be useful in HIV infection. In addition, the effectiveness of an HIV vaccine may depend upon its ability to generate CD4+ T cells capable of expressing CD40L in response to HIV antigens.


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ACKNOWLEDGEMENTS
 
Supported by grants from the Elizabeth Glaser Pediatric AIDS Foundation (50944-PG-24), the National Institutes of Health (HL57911), the UCSD Center for AIDS Research (supported by NIH grant AI36214), the Department of Veterans Affairs, and the Research Center on AIDS and HIV Infection of the VA San Diego Healthcare System.


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