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The Journal of Immunology, 2000, 165: 6133-6141.
Copyright © 2000 by The American Association of Immunologists

The Role of CD4+ T Cell Help and CD40 Ligand in the In Vitro Expansion of HIV-1-Specific Memory Cytotoxic CD8+ T Cell Responses

Mario A. Ostrowski*, Shawn J. Justement*, Linda Ehler*, Stephanie B. Mizell*, Shuying Lui*, Joan Mican*, Bruce D. Walker{dagger}, Elaine K. Thomas{ddagger}, Robert Seder* and Anthony S. Fauci*

* Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; {dagger} Partners AIDS Research Center and Infectious Disease Division, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129; and {ddagger} Immunex Corporation, Seattle, WA 98101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD4+ T cells have been shown to play a critical role in the maintenance of an effective anti-viral CD8+ CTL response in murine models. Recent studies have demonstrated that CD4+ T cells provide help to CTLs through ligation of the CD40 receptor on dendritic cells. The role of CD4+ T cell help in the expansion of virus-specific CD8+ memory T cell responses was examined in normal volunteers recently vaccinated to influenza and in HIV-1 infected individuals. In recently vaccinated normal volunteers, CD4+ T cell help was required for optimal in vitro expansion of influenza-specific CTL responses. Also, CD40 ligand trimer (CD40LT) enhanced CTL responses and was able to completely substitute for CD4+ T cell help in PBMCs from normal volunteers. In HIV-1 infection, CD4+ T cell help was required for optimal expansion of HIV-1-specific memory CTL in vitro in 9 of 10 patients. CD40LT could enhance CTL in the absence of CD4+ T cell help in the majority of patients; however, the degree of enhancement of CTL responses was variable such that, in some patients, CD40LT could not completely substitute for CD4+ T cell help. In those HIV-1-infected patients who demonstrated poor responses to CD40LT, a dysfunction in circulating CD8+ memory T cells was demonstrated, which was reversed by the addition of cytokines including IL-2. Finally, it was demonstrated that IL-15 produced by CD40LT-stimulated dendritic cells may be an additional mechanism by which CD40LT induces the expansion of memory CTL in CD4+ T cell-depleted conditions, where IL-2 is lacking.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD8+ CTL play a central role in the control of a number of acute and chronic viral infections such as EBV (1, 2), CMV (3, 4), SIV (5, 6), and HIV-1 (7, 8, 9, 10). In certain viral infections, especially those that have a tendency to become persistent, CD4+ T cells have been shown to play an important role in the maintenance of an effective ongoing CD8+ CTL response (11). This has been most convincingly demonstrated in the lymphocytic choriomeningitis virus murine model (12, 13, 14, 15, 16) in which CD4+ T cells, although not required for the initial induction of primary CTL responses, are necessary to maintain CTL function during the chronic phase of infection (12, 13, 16). In fact, transient CD4+ T cell depletion at the time of infection with lymphocytic choriomeningitis virus can convert a self-limited viral infection to one of chronic persistence (15, 17). The mechanisms responsible for viral persistence in this model include exhaustion of specific CD8+ T cell clones as well as the appearance of circulating nonfunctional CD8+ T cell clones (13, 18).

HIV-1 infection is characterized by chronic, persistent viral replication in the face of ongoing detectable CD8+ CTL responses (19, 20). In addition, early qualitative and later quantitative abnormalities in CD4+ T cells are seen typically in HIV-1-infected individuals (21, 22, 23, 24). It is unclear whether the deficient CD4+ T helper function in HIV-1 infection is responsible for the inability of CD8+ T cells to completely contain HIV-1 replication. Progressive loss of CTL responses is observed with CD4+ T cell decline and the onset of AIDS (7, 25). Studies by Rosenberg et al. (26) have shown that individuals with strong HIV-1-specific CD4+ T cell-proliferative responses to HIV p24 Ag are able to better control their viremia than individuals with diminished or absent responses. Some of these former individuals have also been shown to have higher levels of circulating Gag-specific CTL precursors (27). Thus, these findings suggest that for HIV-1 infection, a strong CD4+ T cell immune response may be necessary to maintain an effective CD8+ CTL response to HIV-1.

Recent studies in murine systems have demonstrated that CD4+ T cells help CD8+ T cells through interactions with dendritic cells (28, 29, 30, 31). After contact with their cognate Ag, CD4+ T cells are activated to express CD40 ligand, which then induces a signal through the CD40 receptor on dendritic cells. This interaction activates dendritic cells to become more efficient inducers of CD8+ CTL responses, probably due to the up-regulation of costimulatory molecules such as B7-1 and B7-2, as well as to the induction of cytokines including IL-12 (32, 33, 34, 35, 36). Two recent studies have used in vivo CD40 receptor ligation in conjunction with vaccination to break tolerance against tumor and viral Ags in mice (37, 38, 39). These findings suggest that CD40 ligation may represent a novel strategy to induce effective antiviral CTL responses in CD4+ T cell-deficient states such as HIV-1 infection.

Using a peptide-pulsed, dendritic cell-based coculture system in which endogenous cytokines produced by dendritic cells allow expansion of memory CTL responses, this study defines the role of CD4+ T cells in the ex vivo expansion of memory CD8+ CTL responses in two human viral infections, namely, influenza-specific responses in recently vaccinated HIV-1-uninfected individuals and HIV-1-specific responses from HIV-1-infected individuals at various stages of disease. We also address whether CD40 ligand trimer (CD40LT; Ref. 40) can enhance CTL responses or replace CD4+ T cell help in CD4+ T cell-depleted culture conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study subjects

Two HIV-1-uninfected individuals, who were HLA-A*0201 positive and had been vaccinated to influenza virus (A/H3N2) within the previous year, were recruited for apheresis to obtain large amounts of PBMCs. Ten HIV-1-seropositive individuals at varying stages of disease and treatment were also studied (Table IGo). Before the study, all ten HIV-1-infected individuals manifested CTL responses to HLA-restricted HIV-1 peptides using standard CTL assays after peptide stimulation in the presence of IL-7 and IL-2 or anti-CD3 and IL-2 (41, 42) (data not shown). All investigational protocols were approved by the National Institute of Allergy and Infectious Diseases and the Massachusetts General Hospital Institutional Review Boards.


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  		Table I. Profiles of HIV-1-infected patients

 
Peptide synthesis

Peptides were synthesized by F-moc chemistry using a Zinnser Analytical Synthesizer (Research Genetics, Huntsville, AL) and purity was established by HPLC. Peptides were dissolved in RPMI 1640 medium, and concentrations were determined using the Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA). The following peptides were used for CD8+ T cell expansion: 1) the HLA-A*0201-restricted matrix peptide of influenza, FLU (GILGFVFTL); and 2) the HIV-1-specific peptides HLA-A*0201-restricted P17 (SLYNTVATL), HLA-A*0201-restricted POL (ILKEPVHGV),HLA-A3-restricted P17 (KIRLRPGGK), HLA-B*3501-restricted NEF (VPLRPMTY), and HLA-B8-restricted NEF (FLKEKGGL). The peptide used as a T-helper epitope for CD4+ T cell stimulation was the universal tetanus helper epitope TET830–843 (QYIKANSKFIGITE) (43).

Preparation of monocyte-derived dendritic cells (MDDCs)2

MDDCs were prepared as previously described (44) with minor modifications. Briefly, PBMCs obtained by the Ficoll-Paque method (Organon Teknika, Durham, NC) were separated on multistep Percoll gradients (Sigma, Steinheim, Germany). The recovered monocytes were depleted of contaminating B and T cells using anti-CD19- and anti-CD2-conjugated magnetic beads (Dynal, Oslo, Norway). Monocytes were cultured at 1 x 106/ml in RPMI 1640, 10% FCS, 2 mM glutamine, 25 mM HEPES, and antibiotics supplemented with 50 ng/ml GM-CSF and 100 ng/ml IL-4 (PeproTech, Rocky Hill, NJ) for 7–9 days. MDDCs were then matured with 10 ng/ml TNF-{alpha} for 24 h (R&D Systems, Minneapolis, MN) before use.

Proliferation assays

Cells (5 x 105/well) were cultured in six replicate wells of 96-well U-bottom plates in the presence of test proteins including 1.0 µg/ml of p24 Ag (Protein Science, Meriden, CT) and 10 µg/ml tetanus toxoid Ag (Wyeth-Ayerst Laboratories, Marietta, PA). Six days later, cells were pulsed with [3H]thymidine at 1.0 µCi/well, and uptake was measured 12 h later with a scintillation counter.

Induction of peptide-specific CTL

The protocol for expanding circulating memory CTL ex vivo is illustrated (See Fig. 1Go). MDDCs (see above) were pulsed with the specific HLA class I-restricted peptide at 40 µg/ml for 1 h at 37°C. In addition, to provide a stimulus to CD4+ T cells, these MDDCs were also pulsed with the universal tetanus-specific helper epitope, TET830–843 (4 µg/ml). MDDCs were plated in 24-well plates (5 x 105 pulsed or nonpulsed MDDCs/well) in RPMI 1640 plus 10% FCS, 25 mM HEPES, 2 mM glutamine, and antibiotics. Freshly isolated or thawed autologous PBMCs were prepared both in unfractionated and CD4+ T cell-depleted conditions and added to MDDCs at a 10:1 ratio (5 x 106 cells/well in 2 ml medium). CD4+ T cells were depleted from PBMCs using two rounds of magnetic bead depletion (Dynal); the purity of depletion as tested by FACS analysis was always <0.1% CD4+ T cell contamination. The percentage of CD8+ T cells within total PBMCs and CD4+ T cell-depleted PBMCs was determined by FACS analysis so that equal input of CD8+ T cells could be plated in both unfractionated (total PBMCs) and CD4+ T cell-depleted conditions. Soluble CD40LT was added to certain cultures at a final concentration of 2 µg/ml. CD40LT was obtained as a gift from Immunex (Seattle, WA) at a stock concentration of 13.6 mg/ml dissolved in 25 mM Tris, 4% mannitol, and 1% sucrose buffer. Thus, the following three conditions were included in all experiments using both unfractionated (total) PBMCs and CD4+ T cell-depleted PBMCs: 1) MDDCs not pulsed with peptides; 2) peptide-pulsed MDDCs; and 3) CD40LT plus peptide-pulsed MDDCs. In certain experiments, CD40LT was also added to MDDCs that were not pulsed with peptides. In selected experiments, the effect of exogenous cytokines was also tested in CD4+ T cell-depleted, peptide-pulsed MDDC conditions, which included 20 U/ml IL-2, (Boehringer-Mannheim, Mannheim, Germany), 5 ng/ml IL-12, or 1 ng/ml IL-15 (R&D Systems). On days 3 and 5, medium was changed and supernatants were saved for cytokine analysis. On day 7, wells were pooled and cells were harvested and tested for CTL activity by standard 51chromium lysis assay and for intracellular IFN-{gamma} staining. Percentages of CD8+ T cells in both unfractionated or CD4+ T cell-depleted conditions were again determined by FACS analysis before CTL assays to assure for equal inputs of CD8+ T cells.



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  		FIGURE 1. Experimental protocol for ex vivo expansion of memory CD8+ T cells. HIV-1-uninfected vaccinees and HIV-1-infected volunteers were apheresed; the monocyte fraction was obtained and cultured in the presence of GM-CSF and IL-4 followed by TNF-{alpha} to produce MDDCs. MDDCs were pulsed with the HLA-A*0201-restricted influenza or HLA class I HIV-1-specific peptide and the universal tet830–843 peptide to stimulate CD8+ and CD4+ T cells, respectively. Autologous total and CD4+ T cell-depleted PBMCs were cocultured with peptide-pulsed MDDCs with or without CD40LT in medium. Effector function was measured 7 days later.

 
Cytotoxicity assay

Autologous B-lymphoblastoid cell lines (B-LCL) were labeled with sodium [51Cr] chromate and pulsed with the specific peptide at 10 µM. Control B-LCL were either pulsed with an irrelevant peptide or cultured in RPMI 1640 10% medium alone. Effector cells were added in triplicate at various E:T ratios. Supernatants were collected 4–6 h later and counted on a flatbed scintillation counter (Wallac, Gaithersburg, MD). Background chromium release was always <20%. Percentage of lysis was calculated from the formula 100 x (E - M/T - M), where E is experimental release, M is the release in the presence of RPMI 1640 10% medium, and T is release in the presence of 5% Triton X-100 detergent. Specific lysis was determined by subtracting lysis of control targets from peptide-pulsed targets.

Intracellular staining

Intracellular staining was performed to enumerate the number of IFN-{gamma}- or IL-12-producing cells, as previously described (45). Briefly, for peptide-specific IFN-{gamma} staining, 0.25 x 106 cells were cultured in U-bottom 96-well plates in the presence of peptide-pulsed (1–10 µM) autologous B-LCL or autologous CD8+ T cell-depleted PBMC as stimulator cells; nonpeptide-pulsed stimulator cells were used as background controls. Positive control cells were stimulated with PMA (10 ng/ml) and ionomycin (500 ng/ml). Cells were incubated with peptide-pulsed and nonpeptide-pulsed stimulator cells for 6 h at 37°C in 6% CO2. Monensin was added for the duration of the culture period to facilitate intracellular cytokine accumulation. After this period of culture, cell surface staining was followed by intracellular cytokine staining using the Cytofix/Cytoperm kit (PharMingen, San Diego, CA) in accordance with the manufacturer’s recommendations. For IL-12 staining, MDDCs were cultured in 96-well plates either in the presence of medium alone, or CD40LT (2 µg/ml) for 6 h in the presence of Monensin, and then harvested for staining. For intracellular staining, the following Abs were used: anti-IFN-{gamma} (clone 4S.B3) and anti-IL-12 (p40/p70) Ab (clone C11.5). All Abs were obtained through PharMingen.

Flow cytometry

PBMCs or MDDCs were stained in PBS/1% FCS/0.02%NaN3 using flourochrome-conjugated Abs. The Abs used were anti-CD80, anti-CD86, anti-CD1a, anti-HLA-ABC, anti-CD4, anti-CD8, and anti-CD3. All Abs were obtained through PharMingen. After staining, cells were fixed in PBS/2% paraformaldehyde, and events were acquired using a FACScalibur flow cytometer (Becton Dickinson, San Diego, CA). Dead cells were excluded on the basis of forward and side light scatter. For intracellular IFN-{gamma} assays, a total of 50,000–100,000 events were collected for each sample, and CD8+ T cells were enumerated after gating on CD3-positive cells. Data were analyzed using CELLQuest (Becton Dickinson).

Measurement of cytokine production

ELISAs specific for IL-2, IL-15, and IL-10 were performed on cell culture supernatants in duplicate according to the manufacturer’s guidelines (R&D Systems).

Statistical analysis

Data were compared using the Wilcoxon signed rank test for paired samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of CD4+ T cells and CD40LT in the expansion of influenza-specific memory CTL in normal volunteers

To study the role of CD4+ T cell help on the expansion of virus-specific memory CTL, we used a coculture method in which peptide-pulsed dendritic cells stimulate CD8+ T cells in the absence of exogenous cytokines (Fig. 1Go). After 1 wk of culture, CTL effector activity was assessed by two assays: direct cytolysis of peptide-pulsed targets and intracellular IFN-{gamma} production after exposure to peptide-pulsed PBMCs or B-LCL. We have found excellent correlation between these two assays. Of note, intracellular IFN-{gamma} production yielded a greater degree of sensitivity because significant cytolysis was generally seen only when a frequency of 100 IFN-{gamma}-producing cells/10,000 CD8 cells was detected by flow cytometry (our unpublished observations). We believe that our protocol predominantly measures memory rather than naive CD8+ T cell responses, as we have not been able to induce detectable HIV-1-specific responses in two uninfected individuals using our culture conditions (data not shown). Because HIV-1 infection is associated with multiple immune function defects (21, 22, 24), we initially studied influenza-specific CD8+ T cell responses in uninfected individuals to establish a baseline for comparison. Two HIV-1-uninfected individuals who recently received the standard influenza vaccine were studied. They had previously demonstratable CTL activity against an HLA-A*0201-restricted epitope to the influenza matrix protein using conventional in vitro methods (41, 42) (data not shown). Coculture of PBMCs with peptide-pulsed dendritic cells promoted expansion of CTL because the frequency of influenza-specific CD8+ T cells in ex vivo PBMCs from Subject 1 was 8:10,000 CD8+ T cells by IFN-{gamma} staining before coculture, but 101:10,000 CD8+ T cells after 7 days of coculture (data not shown, and Fig. 2GoA). We found greater influenza-specific CTL responses as measured by cytolysis or IFN-{gamma} staining of influenza-specific CD8+ T cells when CD4+ T cells were included in the cocultures (Fig. 2Go). CD4+ T cell help has been proposed to operate though CD40 ligation on dendritic cells (28, 29, 30, 31). Addition of CD40LT to the cultures not only substituted for CD4+ T cells but increased CTL activity in both total PBMCs and CD4+ T cell-depleted conditions (Fig. 2Go).



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  		FIGURE 2. CD4+ T cell help is required for the induction of influenza-specific CTL. CD40LT dramatically enhances CTL responses and can substitute for CD4+ T cell help. Total or CD4+ T cell-depleted PBMC from two recently vaccinated normal volunteers were cocultured with autologous MDDCs that were either not pulsed or pulsed with an HLA-A *0201-restricted epitope of the influenza matrix protein and cultured with or without CD40LT to give six culture conditions. On day 7, CTL (A) and flow cytometric analysis (B) of IFN-{gamma}-producing cells directed against the influenza matrix epitope were measured. For intracellular cytokine flow cytometric analysis, cells were gated for CD3 and CD8 to enumerate IFN-{gamma}-producing CD8+ T cells only. The experiment from subject 1 was repeated with similar results. DC, Nonpulsed dendritic cell; DCp, dendritic cells pulsed with peptides.

 
Role of CD4+ T cells and CD40LT in the expansion of HIV-1-specific memory CTL in infected individuals

Ten HIV-1-infected individuals at various stages of disease, with previously demonstrable CTL activity against a dominant HLA class 1-restricted HIV-1-specific epitope (41, 42), were examined in this study (see Tables IGo and IIGo). Again, coculture of PBMCs with peptide-pulsed dendritic cells promoted expansion of HIV-specific CTL. For example, the baseline frequency of HIV-1-specific CD8+ T cells in ex vivo PBMC from patient 4 was 21:10,000 CD8+ T cells by IFN-{gamma} staining and 185:10,000 CD8+ T cells after 7 days of coculture (data not shown, and Table IIGo). After coculturing total (unfractionated) PBMCs with peptide-pulsed MDDCs for 1 wk, HIV-1-specific memory CD8+ T cell responses ranging from 19 to 1300 IFN-{gamma}-producing CD8+ T cells/10,000 were detected (Table IIGo). In comparison to total PBMC conditions, CD4+ T cell depletion reduced the ability of peptide-pulsed dendritic cells to expand memory HIV-1-specific CTL responses in 9 of 10 patients (Fig. 3Go and Table IIIGo). The frequency of HIV-1-specific IFN-{gamma}-producing cells was, on average, reduced by 91% (±15%, geometric mean) in CD4+ T cell-depleted conditions (p < 0.05). Of note, in one long-term nonprogressor (patient 7), memory CD8+ T cells were able to expand slightly without CD4+ T cell help (Fig. 3Go). This expansion was markedly enhanced in the presence of CD40LT (Fig. 3Go). We found variable effects of CD40LT on CD8+ memory T cell expansion when CD4+ T cells were present in the cultures (i.e., total PBMCs), from suppression to no change to enhancement (Fig. 3Go). Taken as a group, there was no significant effect of CD40LT on CD8+ T cell responses in total PBMC conditions (p = 0.6). The addition of CD40LT to CD4+ T cell-depleted cultures significantly enhanced memory CD8+ T cell expansion in 9 of 10 patients (p < 0.05); however, the degree of enhancement was variable among patients (Fig. 3Go and Table IIIGo). In 6 of 10 HIV-1-infected patients, addition of CD40LT in CD4+ T cell-depleted conditions was able to expand IFN-{gamma}-producing HIV-1-specific cells to within at least 50% of the levels produced in total PBMC conditions (Fig. 3Go; patients 1, 2, 5, 6, 7, and 9). An example of the ability of CD40LT to fully restore CTL responses in CD4+ T cell-depleted conditions to those of total PBMC conditions is illustrated in patient 5, a long-term nonprogressor (Fig. 4GoA). In this patient, when unfractionated PBMCs were cocultured with peptide-pulsed MDDCs, 11% of CD8+ T cells produced IFN-{gamma} in response to the A2/p17 epitope, whereas only 1.0% of CD8+ T cells in CD4+ T cell-depleted PBMC cultures produced IFN-{gamma} (Fig. 4GoB). The addition of CD40LT to CD4+ T cell-depleted cultures increased the frequency of A2/p17-specific CD8+ T cells to that found in unfractionated cultures (Fig. 4GoB). In patients 3, 4, 8, and 10 (Fig. 3Go and Table IIIGo), CD40LT was unable to fully compensate for CD4+ T cell help (HIV-1-specific IFN-{gamma}-producing CD8+ T cells <50% of that in total PBMC conditions). These four patients also had poor p24 Ag-proliferative responses (Table IIIGo). Of note, CD40LT did not appreciably enhance HIV-1-specific CD8+ T cell responses in those cultures in which MDDCs were not pulsed with HIV-1-specific peptide (data not shown).


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  		Table II. Frequency of HIV-1-specific IFN-{gamma}-producing cells when total PBMC are cocultured with peptide-pulsed MDDCs

 


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  		FIGURE 3. CD4+ T cell help is required to optimally expand HIV-1-specific CD8+ T cell memory responses in most HIV-1-infected patients. CD40LT has variable effects on its ability to replace CD4+ T cell help in HIV-1-infected individuals. Total or CD4+ T cell-depleted PBMC from 10 HIV-1-infected patients were cocultured with autologous MDDCs that were either not pulsed or pulsed with the HLA-restricted epitopes of HIV-1 proteins (see Table IIGo) and cultured with or without CD40LT as outlined in Figs. 1Go and 2Go. HIV-1-specific IFN-{gamma}-producing CD8+ T cells were measured after a 7-day culture by intracellular flow cytometry. Data from six different coculture conditions are depicted as the number of HIV-1-specific IFN-{gamma}-producing CD8+ T cells/10,000. In those patients who had detectable cytolysis by chromium assay, similar trends were obtained. The experiments were repeated in patients 3, 8, and 10 with similar results. Statistical comparisons (Wilcoxon signed rank test) were performed between the following culture conditions: total PBMC vs CD4+ T cell-depleted, p < 0.05; total PBMC vs total PBMC + CD40LT, p = 0.6; total PBMC vs CD4+ T cell-depleted + CD40LT, p = 0.1; CD4+ T cell-depleted vs CD4+ T cell-depleted + CD40LT, p < 0.05. UF, Unfractionated PBMC; DC, unpulsed MDDCs; DCp, MDDCs pulsed with peptide; CD4-, CD4+ T cell-depleted PBMC; CD40LT, addition of CD40LT.

 

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  		Table III. Comparison of HIV-specific CTL from CD4+ T cell-depleted to total PBMC conditions

 


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  		FIGURE 4. CD4+ T cell help is required for optimal expansion of HLA-A*0201-restricted gag-specific HIV-1 memory CTL in an HIV-1-infected long-term nonprogressor (patient 5). CD4+ T cell help can be completely replaced by the addition of CD40LT. Total or CD4+ T cell-depleted PBMC from patient 5, a long-term nonprogressor, were cocultured with autologous MDDCs that were either not pulsed or pulsed with the HLA-A*0201-restricted Gag-p17 epitope of HIV-1 and cultured with or without CD40LT as outlined in Figs. 1Go and 2Go. HIV-1-specific cytolysis (A) and the frequency of HIV-1-specific IFN-{gamma}-producing CD8+ T cells (B) were measured after 7 days of culture. Numbers in upper right quadrant represent percentage of HIV-1-specific CD8+ T cells per total CD8+ T cells.

 
Possible explanations for the inability of CD40LT to fully restore the effects of CD4+ T cell help in some HIV-1-infected patients include 1) an inability of dendritic cells to become activated in the presence of CD40LT; 2) an inability of CD8+ T cells to respond appropriately to an activated dendritic cell; or 3) an aberrant response of dendritic cells to CD40LT with the production of an excess of counterregulatory cytokines such as IL-10. To address these possibilities, we examined the following: 1) dendritic cell activation after exposure to CD40LT; 2) the levels of endogenous cytokine production in cocultures; and 3) the effect of adding exogenous cytokines during coculture on the CTL response. CD40LT was able to activate MDDCs to a similar degree in all patients in terms of dendritic cell morphology, viability, surface expression of CD80 (B7-1), CD86 (B7-2), CD83, CD1a, or intracellular IL-12 production (data not shown and Fig. 5Go). The effects of CD40LT on MDDC activation in patient 3, who did not show enhancement of CTL to CD40LT is illustrated in Fig. 5Go. CD40LT activated the MDDCs from this patient as manifested by morphological changes, up-regulation of costimulatory molecules, and IL-12 expression (Fig. 5Go). Thus, the level of activation of MDDCs did not appear to explain the variable responses to CD40LT. The addition of individual exogenous cytokines such as IL-2, IL-12, or IL-15 was usually able to enhance CTL responses in CD4+ T cell-depleted conditions to the level of that seen in unfractionated conditions. This included those patients who gave poor in vitro responses to CD40LT. IL-2 tended to have a greater effect on in vitro CD8+ T cell expansion than IL-15 or IL-12. Representative experiments from two patients (patients 3 and 8) who showed poor in vitro responses to CD40LT are illustrated in Fig. 6Go. In patient 3 (Fig. 6GoA), the addition of CD40LT to CD4+ T cell-depleted cultures had, in fact, suppressed CD8+ CTL activity, whereas the addition of IL-2, IL-12, or IL-15 had expanded CD8+ CTL responses to the level of total PBMC conditions. In patient 8 (Fig. 6GoB), CD40LT could only minimally enhance CD8+ T cell responses in CD4+ T cell-depleted conditions, whereas the addition of IL-2 expanded CD8+ T cell responses exceeding those produced by total PBMC without cytokines. This indicates that although CD8+ T cells in some patients were not able to optimally expand in the presence of a CD40LT-conditioned dendritic cell, they could fully respond given the appropriate cytokine milieu. To further characterize which cytokines might be driving influenza-specific (i.e., in HIV-1-uninfected subjects) or HIV-1-specific CD8+ T cell (i.e., in HIV-1-infected patients) proliferation in vitro, supernatants from total PBMC, CD4+ T cell-depleted, and CD40LT-stimulated CD4+ T cell-depleted conditions were examined for IL-2, IL-10, and IL-15 by ELISA. IL-2 could only be detected in those cocultures which contained CD4+ T cells (total PBMC). Alternatively, IL-15 was only detectable in CD40LT-stimulated cocultures. IL-10 was always detectable; however, CD40LT tended to enhance IL-10 production to a greater degree in cultures from HIV-1-infected individuals compared with those from HIV-1-uninfected subjects (data not shown). However, we could not clearly correlate the level of IL-10 production with an impaired induction of CD8+ T cell responses by CD40LT in the HIV-1-infected cohort (data not shown). Thus, in 4 of 10 HIV-1-infected patients, CD40LT could not completely substitute for CD4+ T cell help, although MDDCs were activated by CD40LT, and the CD8+ T cells could be expanded with the addition of exogenous cytokines.



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  		FIGURE 5. CD40LT activates MDDCs. A, MDDCs were cultured for 3 days in GM-CSF/IL-4 medium with or without 2 µg/ml CD40LT. CD40LT induces morphological changes (e.g., cell clustering) indicative of activation and (B) increased expression of costimulatory molecules including CD80 and CD86, and increased HLA class I and II expression. Histograms indicate level of surface marker expression after gating for CD1a expressing MDDCs. C, MDDCs were cultured overnight with medium and 5 ng/ml IFN-{gamma} with or without CD40LT. CD40LT induces intracellular IL-12 expression. Data are taken from HIV-1-infected patient 3 and are representative of experiments with MDDCs made from HIV-1-infected and -uninfected patients.

 


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  		FIGURE 6. Exogenous cytokines can replace CD4 help when CD40LT is unable. A, CTL assays from cocultures taken from patient 3 showing effects of CD4+ T cell depletion and addition of either CD40LT, 20 U/ml IL-2, 5 ng/ml IL-12, or 1 ng/ml IL-15. B, HIV-specific intracellular IFN-{gamma} staining was performed on cocultures consisting of peptide-pulsed MDDCs and either total PBMC or CD4-depleted PBMC with or without addition of 2 µg/ml CD40LT or 20 U/ml IL-2. Data are taken from a representative experiment from patient 8. Numbers in upper right quadrant represent percentage of HIV-1-specific CD8+ T cells per total CD8+ T cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates the critical need for CD4+ T cell help in the expansion of influenza- or HIV-1-specific memory CD8+ T cells upon interaction with dendritic cells expressing cognate Ags. When examining influenza-specific responses in HIV-1-uninfected subjects and HIV-1-specific responses in HIV-1-infected patients, CD8+ T cell responses were significantly reduced on average by 90% in the absence of CD4+ T cells. The finding of the lack of requirement for CD4+ T cell help in one of our long-term nonprogressors is intriguing and will require further study. This study also establishes for the first time in human viral systems that CD40 ligation can substitute for CD4+ T cell help through activation of dendritic cells. CD40LT was able to replace CD4+ T cell help in the induction of influenza-specific T cell memory responses in PBMCs taken from two individuals with a normal immune system (HIV-1 uninfected). Of note, CD40LT was able to enhance influenza-specific CTL responses in both total PBMC and CD4+ T cell-depleted conditions. Study of a larger number of HIV-seronegative individuals will be required to definitively show that CD40LT consistently enhances influenza-specific CTL responses in PBMCs taken from normal individuals.

HIV-1-infected individuals have numerous immunologic perturbations including chronic immune activation and CD4+ T cell depletion (21, 22, 23, 24). We observed variable but reproducible levels of enhancement of CD8+ memory T cell responses with in vitro CD40LT stimulation in our HIV-1-infected cohort. In total PBMC conditions, i.e., in the presence of CD4+ T cells, we saw considerable variability in the effects of CD40LT in HIV-1-infected individuals compared with HIV-1-uninfected controls. The reasons for this observation are not readily apparent and likely reflect complex interactions between CD40LT and CD4+ T cells in these cultures. It is possible that the combination of CD4+ T cells and excess CD40LT may provide excessive stimulation in some instances and result in the induction of counterregulatory signals to CD8+ T cells. In CD4+ T cell-depleted cocultures, CD40LT could enhance CD8+ T cell responses to some degree in most HIV-1-infected patients. CD40LT could not sufficiently substitute for CD4+ T cell help in 4 of 10 patients, despite the fact that CD40LT could activate the dendritic cells from these patients. However, the addition of exogenous cytokines such as IL-2 was able to enhance CD8+ T cell responses to the levels found in CD4+ T cell-containing conditions, even when CD40LT could not. These findings suggest that in some HIV-1-infected patients, memory CD8+ T cells are heavily dependent on cytokines such as IL-2 to proliferate despite receiving optimal signals from dendritic cells. A similar defect in CD8+ T cell effector function from later stage HIV-1-infected individuals has also been demonstrated by Trimble et al. (46). In these patients, CD3{zeta} down-regulation on T cells was observed, which could be reversed with exogenous IL-2.

It is also possible that a lack of CD4+ T cell help in vivo may be responsible for a lack of response to CD40LT in vitro. Of note was the fact that those patients with a suboptimal response to CD40LT also had absent proliferation to p24 Ag. Hay et al. (47) recently reported an HIV-1-infected rapid progressor who had absent HIV-1-specific CD4+ T cell-proliferative responses in association with detectable circulating but dysfunctional HIV-1-specific CD8+ T cells in the face of a high level plasma viremia. Their findings suggested that a lack of CD4+ T cell help in vivo in this patient prevented circulating memory CD8+ T cells from expanding to sufficient numbers that would contain viral replication. It is of interest that these four individuals were also receiving HAART. Recent studies have shown an inhibitory effect of therapeutic levels of ritonavir on in vitro CTL responses (48). Thus, we cannot rule out an alternative possibility that concurrent protease inhibitor use may have interfered with Ag processing and the in vitro response to CD40LT stimulation.

Examination of cytokine production in our cultures provided some insight into potential mechanisms of CD40LT action. Although IL-2 could be detected when CD4+ T cells were present, we did not detect IL-2 in CD40LT-stimulated, CD4-depleted cocultures, reflecting the inability of memory CD8+ T cells to produce appreciable IL-2 upon restimulation (49). However, IL-15 was detected in CD40LT, CD4-depleted conditions. IL-15 has been shown to share a number of biological activities with IL-2. Of note, IL-15 has been used to expand memory CTLs in vitro in an IL-2-independent fashion (50), and IL-15 has recently been shown to be induced in dendritic cells after CD40 ligation (R. Seder and J. McDyer, unpublished observations; Ref. 51). Thus, CD40LT may bypass the role of CD4+ T cell help in part through IL-15 induction. IL-10 production was also increased during CD40LT stimulation, especially in cocultures from HIV-1-infected patients. This indicates that CD40 ligation can also induce counterregulatory cytokines. Thus, the net result of CD40 ligation on expanding CD8+ T cell responses will reflect a balance of both positive and negative regulatory effects. This may also explain in part the variable effects of CD40 ligation in both CD4+ T cell-containing and CD4+ T cell-depleted conditions in our HIV-1-infected patients.

Although CD40LT was able to expand in vitro virus-specific responses in uninfected and most HIV-1-infected individuals, a similar effect could also be demonstrated with exogenous cytokines, such as IL-2, IL-12, and IL-15 in our culture system. This suggests that, in the absence of CD4+ T cell help, there may be more than one way to supplement CD8+ T cell function.

This study has potentially important implications for the use of CD40LT in vaccine strategies or as an effective immunotherapy. The incorporation of CD40LT in the design of current anti-viral vaccines that give poor CTL immune responses in vivo would be an important strategy to pursue. Furthermore, the use of CD40LT in association with HIV-1-specific vaccines as an immunotherapy in HAART-treated HIV-1-infected patients would also be an attractive approach for clinical application, with the caveat that HIV-1-specific CTL may not be expandable in all infected individuals.


   Acknowledgments
 
We thank John Ridge and Polly Matzinger for helpful discussions, Tae-Wook Chun and Susan Moir for critical reading of the manuscript, and our patients for their time and commitment.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Mario A. Ostrowski, Clinical Sciences Division, Room 6271, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada, M5S 1A8. Back

2 Abbreviations used in this paper: MDDCs, monocyte-derived dendritic cells; CD40LT, CD40 ligand trimer; B-LCL, B-lymphoblastoid cell lines. Back

Received for publication May 3, 2000. Accepted for publication August 29, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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