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The Journal of Immunology, 2007, 179, 8252 -8263
Copyright © 2007 by The American Association of Immunologists, Inc.
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4-1BBL Induces TNF Receptor-Associated Factor 1-Dependent Bim Modulation in Human T Cells and Is a Critical Component in the Costimulation-Dependent Rescue of Functionally Impaired HIV-Specific CD8 T Cells1

Chao Wang*, Tao Wen2,*, Jean-Pierre Routy{dagger}, Nicole F. Bernard{ddagger}, Rafick P. Sekaly§ and Tania H. Watts3,*

* Department of Immunology, University of Toronto, Toronto, Canada; {dagger} Immunodeficiency Service and Division of Hematology, Royal Victoria Hospital, McGill University Health Centre, Montreal, Quebec, Canada; {ddagger} Research Institute of the McGill University Health Center, Montreal, Quebec, Canada; and § Departement de Microbiologie et Immunologie, Universite de Montreal, Montreal, Quebec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During chronic infection, HIV-specific CD8 T cells exhibit progressive signs of functional impairment, attributed to persistent antigenic stimulation, up-regulation of the inhibitory receptor PD-1, and declining T cell help. Strategies that directly improve CD8 T cell function offer the potential of restoring immune control of HIV. Although PD-1 expression has been identified as a cause of functional impairment in HIV, in this study, PD-1 expression was observed on only a subfraction of HIV-specific CD8 T cells in a subfraction of donors, whereas HIV-specific CTL from all donors exhibited a limited repertoire of effector functions. CD137L (4-1BBL) is emerging as an important stimulator of antiviral CD8 T cell responses. Regardless of the PD-1 status of the donors, here we show that 4-1BBL, when combined with CD80 or CD70, expands a population of Ag-specific CD8 T cells expressing multiple markers of effector function, from the functionally impaired starting population. In contrast, CD70 in combination with CD80 was insufficient for these effects and the related TNF family ligand, LIGHT, had negligible activity. The unique contribution of 4-1BBL correlated with down-regulation of the proapoptotic molecule Bim in activated CD8 T cells. Decreasing the level of TNFR-associated factor 1 in T cells using small interfering RNA resulted in increased levels of Bim in the 4-1BBL-stimulated T cells. Thus, costimulation via 4-1BBL leads to TNFR-associated factor 1-dependent Bim down-modulation in T cells, resulting in increased T cell expansion. These studies identify 4-1BBL as a critical component in therapeutic strategies aimed at improving CD8 T cell function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cytotoxic T lymphocytes are important mediators of immune control of viral infection (1, 2, 3, 4, 5, 6, 7). However, HIV persists and disease progresses in most subjects. The inability to control chronic viral infections is associated with the functional impairment of virus-specific CTL, which in HIV infection is exacerbated by the progressive loss of CD4 T cell help (8, 9, 10, 11, 12, 13, 14, 15, 16). In support of a role for CD8 T cells in viral control, the function of HIV-specific CD8 T cells is intact in long-term nonprogressors (17, 18, 19, 20). Although it is arguable whether the loss of CTL function is the cause or the effect of viral persistence, recent evidence shows that restoration of CTL function through the blockade of PD-L1, the ligand for the coinhibitory receptor PD-1, results in significant viral load reduction during chronic lymphocytic choriomeningitis virus infection in mice, even in the absence of CD4 T cells (21). PD-1 is also up-regulated on HIV-specific CTL and its expression has been positively correlated with viral load (22, 23, 24, 25). Indeed, PD-L1 blockade can restore CTL function of HIV-specific T cells ex vivo (23, 24, 25). However, lymphocytic choriomeningitis virus challenge is lethal in PD-L1-deficient mice (21) and there are concerns that systemic PD-L1 blockade might lead to autoimmunity (26). Thus, Ag-specific strategies for improvement of CTL function during chronic viral infection in the context of high PD-1 expression remain an important therapeutic target for HIV.

CD137L (4-1BBL) is emerging as a prominent candidate for enhancing CD8 T cell function in HIV. A wealth of studies in mice have shown that 4-1BBL or anti-4-1BB Abs improve antiviral and antitumor CD8 T cell responses (27, 28, 29). 4-1BBL stimulation can prolong the survival of CD8 T cells following anti-CD3/CD28 stimulation (30, 31). 4-1BBL is also a potent adjuvant for memory antiviral responses of human CD8 T cells ex vivo (32, 33), with more potent effects than CD80 (B7.1) or OX40L (34). However, to date, the functional capabilities of HIV-specific CD8 T cells expanded by costimulation in the absence of T cell help have not been evaluated.

Recent studies have shown that control of viremia correlates with the ability of CD8 T cells to produce multiple cytokines (10, 16, 21, 35, 36). CD8 T cells which coproduce IL-2 and IFN-{gamma} can function in the absence of CD4 help, whereas the presence of CD8 T cells that produce only IFN-{gamma} is associated with functional impairment (9). In this study, we report the ability of costimulation with 4-1BBL in combination with CD80 or CD70 to induce an expanded population of CTL with multiple effector functions from a functionally impaired starting population of memory T cells in the absence of CD4 T cells. The critical role of 4-1BBL in the rescue of HIV-specific CTL function is explained at least in part by the ability of 4-1BBL-stimulated cells to maintain low levels of the proapoptotic molecule Bim via TNFR-associated factor 1 (TRAF1)4 signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Donors and consent

PBMC were obtained from HIV+ and HIV– volunteers and cryopreserved until use. For HIV+ donors, PBMC were obtained by leukapheresis. All donors gave informed consent as approved by the research ethics boards of all participating institutions. HIV+ donors were HLA typed and ELISPOT analysis was used to determine their CD8 T cell specificities using a panel of overlapping HIV peptides as previously described (33) and the dominant epitopes selected for further study. Donor HPI9 had three dominant reactivities and all three were studied (Table I). Table I summarizes the properties of the starting population of HIV-specific CD8 T cells. Healthy donors were identified as HLA-A2 positive by flow cytometry and studied using the immunodominant influenza matrix M1 peptide.


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  		Table I. Characterization of starting population of T cells from HIV-infected subjectsa

 
Recombinant adenoviruses (AdV) and characterization of modified monocytes expressing costimulatory molecules

Replication-defective AdV encoding 4-1BBL and CD80 were constructed previously using the two-plasmid rescue method (32). Recombinant AdV expressing human CD70 and LIGHT were generated by the same approach. Fidelity of the sequence of the cDNAs in comparison with the published sequences was confirmed. Large scale virus production used 293N3S cells. Following cell lysis, viruses were purified by two rounds of cesium chloride gradient ultracentrifugation and titers were measured by plaque assay. Protein expression was verified by flow cytometry (data not shown). Infection of monocytes with a mixture of AdV carrying either the 4-1BBL or the CD80 gene resulted in substantial coexpression of the two costimulatory molecules (33). Similar results were observed for 4-1BBL and CD70 coexpression (data not shown). Control AdV or 4-1BBL-AdV treatment of the monocytes led to similar increases in the levels of MHC class I and II (MHC I and II) on the monocytes and in the transient induction of CD86 expression. AdV infection did not influence the endogenous levels of CD80 or 4-1BBL. HVEM-Fc and lymphotoxin β receptor (LTβR)-Fc, used to confirm integrity of LIGHT, were provided by C. Ware (La Jolla Institute for Allergy and Immunology, La Jolla, CA).

Tetramers and peptides

Biotinylated MHC I/peptide monomers were prepared as previously described (32, 37). Monomers were conjugated with extravidin-PE (Sigma-Aldrich). The HIV tetramers used include: A2-Gag (SLYNTVATYL), A2-Pol (ILKEPVHGV), A2-p15 (FLGKIWPSYK), B7-p17 (RPGGKKKYKL), B7-Nef (TPGPGVRYPL), and B8-Nef (FLKEKGGL), and the influenza tetramer is A2-M1 (GILGFVFTL). Peptides were obtained from the Sheldon Biotechnology Center (McGill University, Montreal, Quebec, Canada) or from the Alberta Peptide Institute (Edmonton, Alberta, Canada).

Costimulation assays

PBMC were suspended at 8 x 106 cells/ml and plated at 0.1 ml/well of a 96-well plate. Adherent monocytes were enriched at 37°C for 1 h and then infected with AdV overnight. Nonadherent cells were removed and stored overnight at 37°C, to be used as a source of T cells. 4-1BBL-AdV, CD80-AdV, CD70-AdV, LIGHT-AdV alone or their combinations, or control AdV were added to the adherent monocytes at a multiplicity of infection of 100, as determined by titration experiments (data not shown), followed by centrifugation at 3000 rpm at 37°C for 1 h. The influenza M1 peptide was added to the monocytes at a concentration ranging from 0.0008 to 25 µM for titration experiments. For subsequent experiments, the peptide dose that gave the highest response over background for each donor was used. For HIV experiments, peptide titrations were performed between 0.5 and 3 µM (data not shown) and the peptide concentration that gave the highest response for each donor was used for additional experiments. An irrelevant HLA-A2-restricted melanoma peptide was used for all A2 donors or no peptide was added for other HLA types. The "no Ag control" cultures included monocytes modified by both 4-1BBL- and CD80-AdV. Following overnight incubation, the activated monocytes were washed to remove excess AdV and peptide.

CD8 T cells were purified from the nonadherent fraction using the negative selection kit from Miltenyi Biotec (MACS). CD8 T cell purity was routinely ~92%. For CFSE experiments, purified CD8 T cells were labeled with 0.5 µM CFSE. CD8 T cells were added to modified monocytes at a concentration of 7.5 x 105 cells/ml. A small amount (0.04 U/ml) of exogenous IL-2 (eBioscience) was added to the pure CD8 T cell cultures. The low dose of IL-2 added to the CD8 T cells had been predetermined by titration on a subset of healthy donors as the minimal amount required for Ag- and costimulation-dependent CD8 T cell expansion. For several HIV donors, the addition of exogenous IL-2 was not required to achieve functional expansion in response to dual costimulation including a PD-1exp donor (data not shown); however, for consistency we added 0.04 U IL-2/ml for all CD8 T cell cultures, both healthy and HIV, a dose that is far below the level required to overcome T cell anergy (10, 16, 21, 35, 36).

T cells and activated monocytes were coincubated at 37°C for 7–9 days. Kinetic studies were done to identify the peak of expansion of Ag-specific CD8 T cells for every HIV donor reported (data not shown).

CTL functional analysis by 51Cr-release assay

The T2 cell line and the EBV-transformed BL7 cell lines (provided by Dr. M. Ostrowski, University of Toronto, Toronto, Canada) were used as targets. For HLA-A2-restricted donors, T2 cells were pulsed with 5 µM of either M1 peptides, relevant HIV peptides, or irrelevant HLA-A2-restricted melanoma peptides overnight. For HLA-B8-restricted donors, BL7 cells were pulsed with 5 µM relevant HIV peptides overnight. The next day, targets were labeled with 200 µCi of Na251CrO4 (Amersham) and incubated with effector T cells for 5 h. Supernatant was collected for analysis of radioactive chromium release.

Flow cytometry

Cell fluorescence was measured using a FACSCalibur (BD Biosciences) and analyzed with FlowJo software (Tree Star). The following mAbs were used (human (h) specific): anti-h4-1BBL, -hCD70, -hCD107a, -h4-1BB, and their respective isotype controls (BD Biosciences); anti-hLIGHT and -hHVEM (R&D Systems), anti-hCD8{alpha}, -hCD28, -hCD27, -hPD-L1, -hPD-L2, -hIFN-{gamma}, -hTNF-{alpha}, -hIL-2, -hBcl-xL, and -perforin (eBioscience), anti-Bim (Alexis Biochemicals). For intracellular cytokine staining, samples were restimulated with 5 µM peptide for 6 h in the presence of GolgiPlug (BD Biosciences). Restimulated cells were first stained with surface markers, fixed, and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences), followed by intracellular staining. In all figures, gates were drawn based on staining with isotype controls.

RNA interference to decrease TRAF1 levels in T cells

Purified CD8 T cells were transfected with 300 nM of either TRAF1 siRNA or scrambled RNA (Integrated DNA Technologies) using the Amaxa Human T cell Nucleofector kit. Transfected cells were rested overnight at 37°C and stimulated with modified monocytes as described in the costimulation assays. Cells were harvested at day 8 of coculture for both Western blot and FACS analysis. TRAF1 knockdown was verified by Western blot using anti-TRAF1 Ab (Cell Signaling Technology). Five of the eight donors showed significant knockdown (50%) and were followed by FACS analysis for Bim expression and expansion of tetramer-positive cells.

Statistical analysis

Statistics were performed using the paired Student t test for parameters measured in the same donor and the unpaired Student t test otherwise. Values of p < 0.05 were considered significant. Correlation was analyzed using the two-tailed bivariate test with Spearman coefficients.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Functional status of HIV-specific CD8 T cells

To assess the effect of costimulation on the expansion and function of HIV-specific CTL, we first examined the surface phenotype and functionality of the starting population of HIV-specific CD8 T cells from a cohort of HIV-infected subjects. Consistent with literature (38) for most donors, the majority of HIV-specific CD8 T cells were CD27+CD28– (Table I). After a brief restimulation of donor PBMC with relevant HIV-peptide, we found that, without exception, the HIV-specific CTL from this cohort are very poor producers of IL-2, and few cells express the degranulation marker CD107a (Fig. 1, A and B). As expected (39), T cells from most donors are capable of producing IFN-{gamma}. However, none of the HIV-specific T cells coexpressed IL-2 or CD107a with IFN-{gamma} and only one donor (HPI4, see Table I) showed coexpression of TNF and IFN-{gamma} by 26% of their HIV-specific T cells. The failure to detect coexpression of IFN-{gamma} with CD107a or IL-2 is not due to a technical limitation, as such populations are detected following costimulation (see Fig. 2). Thus, HIV-specific CD8 T cells from this HIV-infected cohort show a limited repertoire of effector molecules, consistent with the functional impairment of the vast majority of the HIV-specific CD8 cells.


Figure 1
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  		FIGURE 1. HIV-specific CD8 T cells show a limited repertoire of effector functions regardless of PD-1 expression. A, Left, Representative intracellular staining for cytokine production by CD8 T cells from an HIV-infected donor after peptide restimulation (see Materials and Methods). Right, HIV-tetramer staining on CD8 T cells before peptide restimulation. B, Left, Summary of responses of nine donors. Right, Cytokine production per tetramer+ CD8 T cell was obtained by dividing the percent of cytokine+ of CD8 by the percent of tetramer+ of CD8. C, PD-1 expression on HIV-specific CTL was measured by staining unfractionated PBMC and gating on the HIV tetramer+ populations. Left, Summary of the percent of PD-1-expressing tetramer+ cells. The five donors with >10% PD-1+tetramer+ cells were classified as PD-1exp and those with <10% as PD-1nil. Right, Representative FACS analysis of PD-1 expression from one PD-1exp and one PD-1nil donor (gated on HIV-tetramer+ CD8 T cells).

 

Figure 2
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  		FIGURE 2. Expansion and functional rescue of HIV-specific CTL by costimulation. Purified (at least 92%) CD8 T cells from HIV-infected subjects were cultured with peptide-pulsed or control-treated autologous monocytes that had been modified with either control AdV or recombinant AdV expressing the costimulatory ligands indicated in the figures. Controls included Ag with control AdV and costimulation without Ag for each donor. Cells were harvested at days 7–9 and surface stained directly or, for intracellular cytokine analysis, stimulated for 6 h with relevant peptide. Results are shown for each donor at the peptide concentration and time point that gave maximal expansion (see Materials and Methods). A, Representative data from donor HPI1 at day 8. i, MHC I tetramer analysis. ii, Analysis of CD107a+ and IFN-{gamma}+ expression on CD8 T cells. iii, Analysis of IL-2+ and IFN-{gamma}+ expression on CD8 T cells. iv, Isotype controls for ii and iii. v, Cytolytic effector function of T cells (reported as average ± SEM of triplicate cultures for donor HPI1 and representative of two donors). B, Summary of HIV-specific CTL expansion and function for each donor (n = 11 epitopes from nine donors). i, Fold-expansion is calculated by dividing the number of HIV-specific live T cells recovered after costimulation by the number of Ag-specific cells recovered in the cAdV + Ag-stimulated cultures. ii, CD107a+IFN-{gamma}+ coexpression. iii, IL-2+IFN-{gamma}+ coexpression. The line at 0.1 indicates the frequency of IL-2-producing cells below which the results cannot be distinguished from background. Statistical significance was analyzed relative to the control culture containing no Ag, but including costimulation (see Materials and Methods). All flow cytometry data are gated on CD8 T cells.

 
We next examined the levels of PD-1 on the HIV-specific (tetramer+) population of T cells from the same donors. Despite the functional impairment observed for virtually all the HIV-specific CTL in all donors analyzed, PD-1 was detected on HIV-specific CD8 T cells from only a subset of donors (Fig. 1C, right panel). For those PD-1+ donors with detectable PD-1 expression, PD-1 was detected on only a subset of HIV-specific CTL, ranging from 11.9 to 30.6% of tetramer+ T cells (Fig. 1C, left panel, Table I). Overall, as previously observed (24), the presence or absence of PD-1 did not correlate with the immediate peptide-specific recall responses of the T cells (Fig. 1B).

Effect of 4-1BBL and CD80 costimulation on expansion of HIV-specific CD8 T cells

To test the role of costimulation in expanding HIV-specific CD8 T cells, we took advantage of a previously described model in which autologous donor monocytes are pulsed with antigenic peptides and modified with replication defective recombinant AdV to express costimulatory ligands (see Materials and Methods (32)). Costimulation of isolated CD8 T cells with monocytes overexpressing 4-1BBL increased the expansion of HIV-specific CD8 T cells for most donors (Fig. 2, Ai and Bi). In contrast, CD80 had a relatively minor effect on CD8 T cell expansion, with only two donors showing a significant effect (Fig. 2, Ai and Bi). Not surprisingly, the two CD80-responsive donors were those with the highest frequency of CD28+CD8+tetramer+ T cells (Table I, HPI4 and HPI7). However, the combination of CD80 and 4-1BBL resulted in enhanced CD8 T cell expansion as compared with controls, for all donors tested (Fig. 2Bi). Thus, dual costimulation with 4-1BBL and CD80 increases the proportion of donors that respond to costimulation and increases the overall level of HIV-specific CTL expansion.

Following costimulation, expanded HIV-specific CD8 T cells express multiple markers of effector function upon restimulation

The surface expression of CD107a upon peptide stimulation is indicative of degranulation and cytotoxic activity (40). The starting population of HIV-specific CD8 T cells expressed little or no CD107a, and a negligible population coexpressed CD107a and IFN-{gamma} following peptide stimulation (Fig. 1B). In contrast, brief restimulation of the 4-1BBL-expanded cultures with specific HIV peptide revealed a significant IFN-{gamma}/CD107a coexpressing CD8 T cell population in most donors and the response was increased in all donors when both 4-1BBL and CD80 were included in the initial cultures (Fig. 2, Aii and Bii). Not surprisingly, the CD80-costimulated cultures, which contained few Ag-specific CD8 T cells, also contained a much lower frequency of peptide-specific CD107a+/IFN-{gamma}+ CD8 T cells. Furthermore, the levels of cytokine production per cell were increased with 4-1BBL/CD80 costimulation in most donors, as demonstrated by a higher median fluorescence intensity (MFI) for IFN-{gamma} staining (see Fig. 2Aii). Analysis of CTL killing in a 51Cr-release assay confirmed the correlation between CD107 expression and CTL effector function and showed that 4-1BBL overexpression alone could induce some CTL function, with enhanced effects when combined with CD80 (Fig. 2Av).

Although the IL-2-expressing populations observed are quite small, costimulation with 4-1BBL alone, and even more so 4-1BBL together with CD80 resulted in a population of CD8 T cells capable of producing both IL-2 and IFN-{gamma} upon peptide restimulation (Fig. 2, Aiii and Biii). Overall, these data show that independently of CD4 T cells, HIV-specific CD8 T cells capable of expressing multiple markers of effector function upon restimulation can be generated from a functionally impaired starting population of CD8 T cells. 4-1BBL is a potent mediator of these effects, with enhanced effects when combined with CD80 costimulation.

Comparison of 4-1BBL, CD70, and LIGHT in the expansion and activation of virus-specific CD8 T cells

We next asked whether 4-1BBL is unique in its ability to rescue CD8 T cell responses from functionally impaired HIV-specific precursors. Previous studies have shown that 4-1BBL is more potent than CD80 in stimulating influenza-specific responses of isolated CD8 T cells and that the related TNF family ligand, OX40L had minimal direct effects on isolated human CD8 T cells (34). 4-1BBL is closely linked on human chromosome 19 with two other TNF family members, CD70 and LIGHT, which bind to their respective TNFR family members CD27 and HVEM (41). Both CD70 and LIGHT have been shown to have costimulatory activity for CD8 T cells (29). Of note, CD27 expression on adoptively transferred HIV-specific T cells correlates with their increased survival in vivo (42). We generated recombinant AdV expressing human CD70 and human LIGHT and initially compared their efficacy in costimulation of healthy donor T cell responses to influenza A virus using the modified monocyte assay. All three costimulatory molecules were similarly expressed on monocytes following their delivery by recombinant AdV (data not shown). Furthermore, resting human T cells express CD27, whereas HVEM and 4-1BB are inducible on the Ag-specific T cells (data not shown); thus, receptors to all three costimulatory ligands are present on activated CD8 T cells early in the culture period.

As previously reported (32), 4-1BBL is a potent stimulator of influenza-specific CD8 T cell expansion for all donors tested (Fig. 3, A and B). In contrast, CD70 allowed expansion of Ag-specific CD8 T cells in about half of the donors tested (Fig. 3, A and B). Furthermore, 4-1BBL was effective at lower peptide doses than CD70 (Fig. 3A and data not shown). The failure of some donors to respond to CD70 was not due to a generalized defect in the responsiveness of the memory T cell population, as donors who failed to respond to CD70 exhibited the full range of responses to 4-1BBL (data not shown). Of note, the donors that respond to CD27 have a higher frequency (p = 0.0087) of CD28– T cells in their starting population, whereas the A2/M1 tetramer+ T cells in the starting population of the nonresponders were almost all CD28+, perhaps reflecting a more recently activated or more differentiated effector memory population in the responder population (data not shown).


Figure 3
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  		FIGURE 3. Comparative analysis of 4-1BBL, CD70, and LIGHT in stimulating influenza-specific CD8 T cell responses. Purified CD8 T cells from healthy donors were stimulated with influenza A2/M1 peptide as done for the HIV responses in Fig. 2. A, Recovery of influenza-specific CD8 T cells from day 8 cultures as a function of peptide dose. Representative data are shown for four donors. The peptide concentration that gave the peak expansion for each donor was used for additional experiments. B, Data from 12 donors analyzed as in A, are summarized as fold expansion, calculated as the number of influenza-specific CD8 T cells generated in response to costimulatory molecules divided by the number obtained in the cAdV and Ag-stimulated cultures, shown for the maximal response point in the peptide titration curve. C, IFN-{gamma}/TNF-{alpha} coproduction in response to peptide restimulation of T cells from the cultures of CD70 responders (fold expansion ≥3, see A and B). D, Analysis of Perforin expression of CD70-responders from B. E, Cytolytic effector function of T cells from CD70 and 4-1BBL-stimulated cultures. Left panel, The frequency of tetramer-positive cells in the cultures used for the 51Cr-release assay in the right panel (n = 3). For details, see Materials and Methods.

 
For those donors that did respond to CD70, the ability of the expanded CD8 T cells to produce cytokines upon restimulation with peptide was at least as good as that of the 4-1BBL-expanded T cells (Fig. 3C). In contrast, 4-1BBL was superior to CD70 in inducing perforin expression (Fig. 3D) and target cell killing under conditions where cultures had similar frequencies of Ag-specific T cells (Fig. 3E). Thus, 4-1BBL improves the level of effector function per T cell.

In contrast to the effects of 4-1BBL and CD70, LIGHT had negligible activity in stimulating Ag-specific CD8 T cell responses in 12 of 12 healthy donors with demonstrable A2/influenza M1-specific CD8 T cells (Fig. 3, A–D). This was not due to the absence of its receptor on the T cells and is unlikely due to misfolding of LIGHT, as anti-LIGHT Ab as well as, HVEM-Fc and LTβR-Fc, the two known receptors for LIGHT, each bound preferentially to the LIGHT-expressing monocytes (data not shown). Thus, 4-1BBL and CD70, but not LIGHT, are each capable of expanding functional human influenza-specific memory CD8 T cells, with 4-1BBL inducing a higher proportion of donor responses at a lower peptide dose, as well as increased cytolytic effector function per T cell.

Activation of HIV-specific CD8 T cells by 4-1BBL and CD70

The finding that CD70, although not as potent as 4-1BBL, could stimulate influenza-specific T cell responses from donors with a high proportion of CD28– tetramer+ CD8 T cells suggested it might be a good candidate for restoring functions of HIV-specific CD8 T cells, which are largely CD28 negative (Table I). However, CD70 alone was found to have marginal activity in stimulating HIV-specific CD8 T cells for all donors tested (data not shown). We therefore tested CD70 in combination with CD80 or 4-1BBL (Fig. 4). The results show that the combination of 4-1BBL with CD70 is as least as good, if not better than 4-1BBL plus CD80 in generating HIV-specific CD8 T cells capable of producing multiple cytokines and degranulating upon restimulation (Fig. 4). Again, the increased expression of CD107 correlated with increased CTL activity in a 51Cr-release assay, with 4-1BBL/CD70 or 4-1BBL/CD80 costimulated cultures showing improved CTL function compared with control cultures. In contrast, the combination of CD80 and CD70 showed much weaker activity in these assays.


Figure 4
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  		FIGURE 4. Differential effects of costimulatory combinations in rescuing functionally impaired HIV-specific CTL. Purified CD8 T cells were stimulated with the indicated costimulatory molecule combinations, expressed on autologous monocytes as described in Fig. 2. Values of p above a single set of data indicate significance relative to control with no Ag plus costimulation; those between two groups indicate significance between the different costimulatory conditions plus Ag. The filled symbols indicate T cells from donors in which there was a significant population (>10%) of PD-1-expressing HIV-specific CTL in the starting population, whereas PD-1nil represents donors with minimal PD-1 expression on the starting T cell population. The p values next to the vertical bars are a comparison between the PD-1exp and PD-1nil donors. A, Fold expansion of Ag-specific T cells. B, Frequencies of IFN-{gamma}/CD107a coproducers. C, CTL lytic activity as measured in a 51Cr-release assay (reported as average of triplicates ± SEM and representative of two donors). D, IFN-{gamma}/IL-2 coproducers from the cultures in A, were determined as in Fig. 2.

 
As shown in Fig. 1, a subset of donors exhibited PD-1 expression on a proportion of the starting population of HIV-specific CD8 T cells. To determine whether the presence of PD-1 in the cultures was indicative of greater functional impairment of the CTL, we identified the donors in Fig. 4 by the PD-1 status of their starting population of HIV-specific T cells (<10% PD-1+ = PD-1nil or >10% PD-1+ = PD-1exp). There was a trend to decreased expansion (Fig. 4A) and decreased production of IL-2 (Fig. 4D) in response to suboptimal costimulation with CD80 plus CD70 in the PD-1exp cultures, although this did not reach statistical significance (p = 0.08 and p = 0.07, respectively). Cultures which included 4-1BBL showed no evidence of segregation between the PD-1nil and PD-1exp cultures.

Analysis of PD-1 expression in the presence and absence of costimulation

To determine whether 4-1BBL could influence the expression of PD-1 or its ligand directly, cultures from PD-1exp donors were monitored for the expression of PD-1 and PD-L1 during the first few days of culture, before the onset of cell division, which begins at about day 4 (based on kinetics of CFSE dilution, data not shown). Consistent with the literature (24), PD-L1 is expressed at low levels on unstimulated monocytes from HIV+ subjects (data not shown). By day 1, PD-L1 expression was further up-regulated on the monocytes which had been modified with 4-1BBL, CD80, and to a lesser extent cAdV (data not shown). PD-L1 expression persists through day 3 of culture (data not shown). PD-1 expression on PD-1+ HIV-specific CTL was maintained up to and including day 3 of coculture with AdV-modified monocytes regardless of whether the AdV expressed additional costimulatory ligands (data not shown). Thus, PD-1 expression is independent of overexpression of costimulatory molecules in the cultures.

4-1BBL induces Bim down-regulation in activated virus-specific CD8 T cells

Data from HIV-specific and influenza-specific CD8 T cell responses identify 4-1BBL as a key costimulator for induction of CD8 T cells with multiple effector functions, even from a starting population of functionally impaired memory T cells. The effects of 4-1BBL on expansion of this population could be due to effects on cell division or survival or both. The rate of cell division of influenza-specific CD8 T cells from healthy donors was measured using CFSE. Analysis of the CFSE profiles shows that by day 4 of culture few cells have divided, whereas by day 8, based on MFI of undivided vs divided populations it appears that the cells have divided about four times (Fig. 5A). CD70-stimulated cells showed a slight delay in the kinetics of cell division compared with 4-1BBL, but by day 8, 4-1BBL- and CD70-stimulated T cells showed a similar proportion of divided cells (Fig. 5A). Both CD70- and 4-1BBL-stimulated T cells up-regulated the antiapoptotic Bcl-2 family member Bcl-xL, with CD70-stimulated T cells showing, if anything, marginally higher levels of Bcl-xL (Fig. 5B, left panel). However, by day 8 of culture, the 4-1BBL-stimulated cultures had accumulated 2- to 3-fold more Ag-specific T cells (Fig. 5B, right panel), suggesting a survival advantage not explained by Bcl-xL expression.


Figure 5
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  		FIGURE 5. Role of 4-1BBL and CD70 in cell division and survival of influenza-specific CD8 T cells. Isolated CD8 T cells from A2-positive healthy donors were stimulated for 4, 6, or 8 days with costimulatory molecule modified autologous monocytes and influenza M1 peptide. A, The CD8 T cells were first labeled with CFSE, and after culture, cells were stained with influenza-tetramer and CD8, and analyzed by flow cytometry. Representative FACS plots are shown gated on influenza-tetramer+ CD8 T cells (n = 6). B, Left panel, Representative Bcl-xL expression on influenza-specific CTL (n = 4). Right panel, Number of tetramer+ cells recovered from the same cultures. Error bar is based on SD for the same donor in two separate experiments. Similar results were obtained for three other donors. C, Bim expression in influenza-specific CTL. Left panel, Representative FACS plots. Right panel, The summary of differential Bim expression in response to 4-1BBL and CD70 costimulation after both 6 and 8 days of culture with data for each donor linked.

 
Bim is a proapoptotic BH3-only member of the Bcl-2 family that contributes to the death of activated (43) and memory (44) T cells. Previous studies (44) have shown that in activated T cells Bim is maintained at lower levels in the presence of TRAF1. Because 4-1BB recruits TRAF1 during signaling in T cells (45), we compared the levels of Bim in tetramer+ T cells stimulated by CD70 vs 4-1BBL. At both days 6 and 8 of coculture, the expression of Bim in influenza-specific CTL is lower (in both percent and MFI) for all donors examined in response to 4-1BBL compared with CD70 costimulation (Fig. 5C and data not shown). These data suggest that a unique aspect of 4-1BBL-mediated costimulation is its effectiveness in maintaining low levels of Bim in the activated T cells.

4-1BBL-induced Bim modulation is TRAF1 dependent

To determine whether the Bim modulation observed is directly linked to 4-1BBL-induced effects on the CD8 T cells, we used siRNA to knockdown TRAF1 expression in the CD8 T cells (Fig. 6A). As compared with transfection with scrambled RNA, the level of Bim expression is significantly higher in 4-1BBL-expanded influenza-specific CD8 T cells treated with siRNA to reduce TRAF1 expression (Fig. 6B). In contrast, TRAF1 siRNA had little effect on the level of Bim expression in CD70-induced tetramer+ cells (Fig. 6B). Furthermore, 4-1BBL-induced expansion of influenza-specific CD8s was significantly reduced in TRAF1 siRNA-transfected compared with control (scrambled siRNA) transfected T cells (Fig. 6C) for 4-1BBL-stimulated cultures. In contrast, the effect of TRAF1 siRNA on CD70-dependent T cell expansion was marginal (Fig. 6C).


Figure 6
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  		FIGURE 6. Decreasing TRAF1 by siRNA results in increased levels of Bim during 4-1BBL mediated costimulation. CD8 T cells from healthy donors were purified and transfected with either scrambled RNA (scRNA) or siRNA specific for TRAF1 (TRAF1 RNA interference). After resting overnight, transfected T cells were coincubated with modified monocytes for 8 days. A, Representative Western blot showing TRAF1 and β-actin expression in CD8 T cells stimulated with 4-1BBL-modified monocytes, harvested at day 8 of coculture. A total of 100 nM (1x) or 300 nM (3x) of either scRNA or siRNA were used in this experiment. 300 nM of siRNA was used for transfection in all other experiments. B, Upper panel, Representative Bim expression gated on influenza-tetramer+ CD8 T cells harvested from 8 days of coculture. Lower panel, Summary of Bim expression in influenza-specific CD8 T cells stimulated with 4-1BBL-modified monocytes (n = 5, data are representative of two separate experiments). C, Upper panel, Representative FACS plots from one donor, gated on CD8 T cells. Lower panel, Summary of the percent of influenza-tetramer+ CD8 T cells (n = 5, data are representative of two separate experiments).

 
Analysis of division and survival of costimulated HIV-specific CD8 T cells

As was the case for healthy donor influenza-specific CD8 T cells, HIV-specific CD8 T cells showed no evidence of cell division for the first 4 days of culture (data not shown). For HIV-specific T cells, recovery of cells was lower with individual costimulatory ligands (Fig. 7A). The combinations of 4-1BBL/CD80 and 4-1BBL/CD70 each induced comparable cell division (about two to four divisions between days 4 and 8, Fig. 7A and data not shown). In contrast, few cells were recovered from the CD70/CD80 cultures, although those CFSE-labeled cells that were recovered showed evidence of division (Fig. 7A). The absence of CFSE-negative tetramer+ T cells and the finding that the bulk of the costimulated cells showed evidence of two to four divisions (Fig. 7A and data not shown) argue that cells expanded only 4- to 16- fold in the cultures. This limited number of divisions makes it unlikely that the functional T cells observed in Fig. 2 arose from a rare functional cell that outgrew the nonfunctional starting population.


Figure 7
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  		FIGURE 7. 4-1BBL-mediated costimulation results in lower Bim expression in HIV-specific CTL. CD8 T cells from HIV-infected subjects were CFSE labeled and cocultured with AdV-modified autologous monocytes for 8 days. Cells were stained and analyzed as in Fig. 5. A, Representative CFSE labeling results for HIV-specific CD8 T cells. B, Upper panel, Representative histograms of differential Bim expression on dividing (CFSElow) HIV-specific CTL from two donors in response to 4-1BBL/CD80 and CD70/CD80 dual costimulation. Lower panel, Data from 7 HIV+ donors. C, The number of dividing HIV-specific CTL was plotted against the percent of Bim+CFSElow HIV-specific CTL. Only results from dual costimulation were included because single costimulation did not allow recovery of sufficient cells for analysis, as reflected in A. Statistics was calculated using SPSS software (negative correlation, R = –0.7909, p < 0.0001).

 
Ag-specific T cells from 4-1BBL/CD80-stimulated cultures showed lower levels of Bim expression than T cells stimulated by CD70/CD80, consistent with increased T cell survival in these cultures (Fig. 7B). Furthermore, there was an inverse correlation between Bim levels and the recovery of Ag-specific cells that had divided in culture (Fig. 7C, p < 0.0001). These data suggest that 4-1BBL contributes to CD8 T cell survival at least in part through maintaining lower levels of Bim in the activated T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the face of viral persistence, CD8 T cell effector function deteriorates over time (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). This functional impairment may reflect the delicate balance that the host must maintain between controlling viral infection and avoiding long-term tissue damage; improving CD8 function, however, can reduce viral load (21). In this report, we provide evidence that functional impairment in HIV-specific CD8 T cells can be overcome by costimulation. We found that overexpression of 4-1BBL in combination with CD70 or CD80 results in a population of HIV-specific CD8 T cells exhibiting key features of functional restoration, from a functionally impaired HIV-specific memory CD8 T cell population. In contrast, CD70 in combination with CD80 was much less effective than either combination involving 4-1BBL, identifying 4-1BBL as a key player in rescuing HIV-specific CTL from functionally impaired precursors.

The expansion of functional CD8 T cells following Ag plus 4-1BBL- mediated costimulation of a functionally limited starting population could be due to recruitment of naive T cells, the outgrowth of a small population of functional T cells that were below the limits of detection at the start of the culture, or could represent a gain of function in the functionally impaired T cell population following expansion in the presence of overexpressed costimulatory molecules. By gating on the tetramer-positive CD8 T cells combined with CFSE labeling, we observed that there was minimal cell division in either healthy donor cultures (Fig. 5) or in HIV-infected T cell cultures (data not shown) during the first 4–5 days of culture. By day 8 of culture, a substantial portion of the CFSE-labeled tetramer-positive cells in the costimulated cultures showed evidence of two to four divisions (Figs. 5 and 7, and data not shown), with very few cells observed in the undivided population and no evidence that there is a subpopulation of highly divided (CFSE negative) tetramer+ T cells. Thus, it is unlikely that the functional T cells recovered after costimulation arise from either naive precursors or from a rare subpopulation of functional memory T cells in the starting population, as such a population should have been detected in the starting tetramer+ population to account for its presence after only four divisions. Furthermore, culture of HLA-A2-positive healthy donor T cells with syngeneic monocytes expressing 4-1BBL and CD80 together with A2-restricted HIV-peptides for 8 days failed to induce any Ag-specific T cells (data not shown). Together, the evidence suggests that following division in the presence of overexpressed costimulators, the functionally impaired HIV-specific CD8 T cells gain multiple effector functions.

Although PD-1 has been shown to correlate with functional impairment during chronic viral infections and blockade of PD-1/PD-L1 interaction can rescue CTL from PD-1-expressing nonfunctional precursors (21, 23, 25, 46), in this study, we observed functional impairment of CD8 T cells even in the absence of detectable PD-1 expression. This suggests there may be other mechanisms causing functional impairment in HIV-specific CD8 T cells, and certainly one of these conditions may be the absence of CD4 T cells (47), which are progressively lost during HIV infection. A number of studies have shown that CD8 T cells primed or maintained in the absence of CD4 T cell help are impaired in their ability to respond to Ag and control infections (48, 49, 50, 51, 52, 53, 54). IFN-{gamma}/IL-2-secreting CD8 T cells have been reported to support proliferation of HIV-specific CTL independently of CD4 (35). The finding that 4-1BBL can stimulate isolated CD8 T cells to expand and acquire multiple effector functions argues that it may overcome deficits in CD8 T cells by contributing to IL-2 production by the T cells themselves, perhaps overcoming the absence of CD4 T cells.

Despite the limited expression of PD-1 in these donors, the data in Fig. 4, showed a trend toward greater functional impairment of T cells from donors with higher PD-1 expression: after suboptimal costimulation with CD70 plus CD80, donors with >10% PD-1+ Ag-specific T cells showed decreased expansion (p = 0.08) and a lower frequency of multifunctional T cells (p = 0.07), compared with T cells from donors with minimal PD-1 expression. In contrast, 4-1BBL-containing cultures showed enhanced expansion of T cells regardless of the PD-1 status of the starting population (Fig. 4). Because of the relatively low proportion of PD-1-expressing cells in the cultures, it is difficult to determine whether 4-1BBL can directly impact this population of cells.

To determine whether 4-1BBL was unique among TNF family ligands in its contribution to rescuing functionally impaired CTL, we compared 4-1BBL with its two closely linked relatives, CD70 and LIGHT. Our studies failed to show any efficacy of LIGHT in human T cell activation, even though it was highly expressed on recombinant AdV modified-monocytes and capable of binding anti-LIGHT as well as its known receptors. Recent evidence shows that in addition to acting as a receptor for LIGHT, HVEM can function as a ligand for the immunoreceptor tyrosine inhibitory motif-containing receptor, B and T lymphocyte attenuator (55, 56). Thus, it is possible that HVEM-B and T lymphocyte attenuator interaction predominates during T cell activation resulting in minimal impact of HVEM signaling in T cells. Soluble LIGHT is known to block the costimulatory activity of membrane-bound LIGHT (57). Indeed, soluble LIGHT was detectable by ELISA in the cultures containing T cells and LIGHT-modified monocytes, but not in the cultures lacking either T cells or LIGHT overexpression (data not shown). The presence of soluble LIGHT in the cultures may be another reason why monocyte expressed LIGHT has minimal efficacy in T cell costimulation in this model.

CD70 was efficacious in stimulating T cells from a subset of healthy donors with a higher frequency of CD28– T cells in the starting memory population, perhaps reflecting a more recent Ag exposure or a more differentiated state of the T cells. Despite its efficacy in stimulating influenza-specific CTL, CD70 was unable to rescue the expansion of HIV-specific CD8 T cells from any of the HIV-infected donors. However, in combination with 4-1BBL, CD70 was at least as good as CD80 in increasing the effects of costimulation through 4-1BBL (Fig. 4).

Why is 4-1BBL more effective than CD70 in rescuing HIV-specific CD8 T cells? Both 4-1BB and CD27 function by recruiting TRAFs, which link them to NF-{kappa}B activation, Bcl-xL induction, and the survival of T cells (29). 4-1BB has been identified as an important survival factor for mouse and human CD8 T cells (31, 58, 59). Similarly, CD27 provides survival signals to CD8 T cells subsequent to the effects of CD28 (60). In humans, CD70 can promote the proliferation and resistance to apoptosis of CD8 T cells and when adoptively transferred back into an HIV-positive donor, CD27+ HIV-specific CD8 T cells survived longer compared with CD27– HIV-specific CD8 T cells (42).

The survival of activated T cells is greatly affected by the balance between the pro- and antiapoptotic Bcl-2 family members (61). In particular, Bcl-2 and Bcl-xL function to oppose the effects of the upstream proapoptotic Bcl-2 family member Bim (61). Previous results have shown that 4-1BBL stimulation of human CD8 T cells results in Bcl-xL up-regulation, under conditions where Bcl-2 remains constant (32). Consistent with these findings, we found that T cells expanded using both 4-1BBL and CD70 showed evidence of Bcl-xL up-regulation. However, we observed greater recovery of cells from 4-1BBL-stimulated cultures, correlating with decreased levels of Bim for both HIV and influenza-specific CD8 T cells (Figs. 5 and 6). Recent work from our laboratory has shown that the presence of TRAF1 in activated and memory T cells plays a critical role in their survival, through maintaining low levels of Bim. Bim is transcriptionally activated by the FOXO3A transcription factor in cells following cytokine withdrawal (62). In addition, Bim can be down-regulated in cells by ERK phosphorylation, leading to its proteasome-dependent degradation (63). TRAF1 is recruited to the 4-1BB signaling complex in T cells (45) and 4-1BB signaling has been linked to ERK activation (30). Thus, a plausible model for the effects of 4-1BBL on Bim levels in activated T cells is that 4-1BB signaling leads to TRAF1 recruitment and Bim down-modulation via an ERK-dependent mechanism. Indeed, decreasing the levels of TRAF1 in CD8 T cells using siRNA resulted in increased levels of Bim in the T cells and decreased T cell expansion following 4-1BBL costimulation, compared with T cells treated with scrambled RNA (Fig. 6). This result suggests that TRAF1-dependent Bim down-modulation enhances T cell survival following 4-1BB signaling in T cells. CD27 is known to recruit TRAF2 and 5 in T cells (64, 65, 66) both of which are required for CD27 signaling (65, 66, 67), but to date, evidence that CD27 signaling recruits TRAF1 in T cells is lacking. Down-regulation of TRAF1 in T cells had minimal effects on CD70-mediated stimulation (Fig. 6). Thus, differential recruitment of TRAF1 may explain the different efficacy of 4-1BBL and CD70 in Bim down-regulation.

In addition to its effects on rescuing CTL via survival effects leading to a greater number of T cells, it is likely that 4-1BBL contributes more directly to increased effector functions. T cells from 4-1BBL-stimulated cultures show increased levels of perforin per cell (Ref. 32 and Fig. 3D) and, on a per cell basis, 4-1BBL-stimulated cells were found to be more efficacious in killing target cells than CD70-stimulated T cells (Fig. 3E). Thus, the effects of 4-1BBL appear to be more than just promoting survival of functional cells, rather 4-1BBL can improve the effector function of epitope-specific CTL on a per cell basis.

In sum, the data in this report show that costimulation by 4-1BBL in combination with other costimulatory ligands can expand fully functional HIV-specific CTLs from a starting population with limited functional capabilities. Although the effects of 4-1BBL were enhanced when combined with other costimulatory ligands such as CD80 or CD70, 4-1BBL was an essential component of the costimulation, at least in part through its effects on TRAF1-dependent Bim down-modulation in activated T cells. The finding that these effects are independent of the presence of CD4 T cells or PD-1+ T cells in the starting cultures raises the possibility of a therapeutic application for 4-1BBL to generate functional HIV-specific T cells.


   Acknowledgments
 
We thank Dr. Carl Ware for provision of anti-LIGHT, HVEM-Fc, and LTβR-Fc, Dr. Mario Ostrowski for provision of BL7 target cells, Birinder Ghumman for purification and plaque assays of the recombinant AdV, Dr. Yuanqing Liu for critical discussion, and Benoit Bessette (University of Montreal) for production of HIV/MHC I tetramers.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant 74492 from the Canadian Institutes for Health Research (to T.H.W.) and by the Canadian Network for Vaccines and Immunotherapeutics. Back

2 Current address: Sanofi-Pasteur, Toronto, Ontario, Canada. Back

3 Address correspondence and reprint requests to Dr. Tania H. Watts, Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada, M5S 1A8. E-mail address: tania.watts{at}utoronto.ca Back

4 Abbreviations used in this paper: TRAF1, TNFR-associated factor 1; LTβR, lymphotoxin β receptor; MHC I, MHC class I; MHC II, MHC class II; h, human; MFI, median fluorescence intensity; HVEM, herpes virus entry mediator; siRNA, small interfering RNA; AdV, adenovirus. Back

Received for publication June 9, 2007. Accepted for publication October 10, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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