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International Immunology Advance Access published online on October 31, 2007
International Immunology, doi:10.1093/intimm/dxm106
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Co-stimulation with 4-1BB ligand allows extended T-cell proliferation, synergizes with CD80/CD86 and can reactivate anergic T cells

Mojtaba Habib-Agahi1,2, Thanh T. Phan1,3 and Peter F. Searle1

1 Cancer Research UK Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
2 Present address: Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz 71345-3119, Iran
3 Present address: Department of Cardiovascular Medicine, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

Correspondence to: Correspondence to: P. F. Searle; E-mail: p.f.searle{at}bham.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Activation of T cells requires co-stimulation, in addition to signals through the antigen–receptor complex. Antigen encounter without adequate co-stimulation results in T-cell desensitization or anergy, a mechanism of peripheral tolerance and an apparent obstacle to cancer immunotherapy. One important co-stimulatory pathway involves CD28 engagement by CD80 or CD86. However, other ligand–receptor pairs can also provide co-stimulation and may have important functions modulating the immune response. Previous reports indicated that co-stimulation using 4-1BB ligand (4-1BBL) or agonistic anti-4-1BB antibodies could prolong T-cell responses, avoid activation-induced cell death and promote anti-tumour responses in mice. To further investigate the potential for cancer immunotherapy, we studied the effects of CD80/CD86 and 4-1BBL in repeated stimulation of human T cells and asked whether 4-1BBL might be capable of reversing anergy. We expressed CD80, CD86 and 4-1BBL in A549 lung carcinoma cells using adenovirus vectors and co-cultured these with human T cells stimulated with anti-CD3 antibody. Proliferation co-stimulated by CD80 or CD86 was transient; however, 4-1BBL-co-stimulated cultures continued to proliferate for up to 5 weeks, with repeated stimulation. Combined co-stimulation with CD80/CD86 and 4-1BBL also allowed continuous proliferation at a faster rate than either signal alone. Co-stimulation with 4-1BBL did not suppress expression of the inducible, inhibitory CD80/CD86R, CTLA-4. Significantly, we show that T cells that had become non-responsive to anti-CD3, either alone or together with CD80/CD86 co-stimulation, and thus were anergic, could be reactivated to proliferate when costimulated with 4-1BBL, either alone or combined with CD80/CD86.

Keywords: anergy, 4-1BB ligand, CD80, co-stimulation, human T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Activation of T cells requires both engagement of the TCR with a cognate peptide–MHC complex and an additional co-stimulatory signal. The best known co-stimulatory ligands are members of the B7-family, B7-1 (CD80) or B7-2 (CD86), that are expressed on professional antigen-presenting cells (APCs) such as dendritic cells (DCs); these act through their receptor CD28 on T cells (1). CTLA-4 (CD152) is a second receptor for CD80 and CD86, which is not expressed on most resting T cells but is induced upon T-cell activation. The interaction of CD80/CD86 with CTLA-4 has higher affinity than that with CD28, and down-regulates T-cell activation (2). Thus, CTLA-4 effectively competes with CD28 for CD80/CD86 at later stages of the immune response to suppress the activation and bring about activation-induced non-responsiveness (AINR) and/or activation-induced cell death (AICD) (3). Down-regulation of T-cell function by CTLA-4 engagement appears to play a key role in development of T-cell tolerance or anergy towards self- or tumour antigens (4–6), while antigenic stimulation in the absence of co-stimulatory signals is also tolerogenic (7–9).

A number of alternative ligand–receptor pairs capable of providing co-stimulatory signals to T cells have been identified, including 4-1BB ligand (4-1BBL). 4-1BBL is a member of the tumour necrosis factor family (10–12). Its receptor 4-1BB (CD137) is absent from resting T lymphocytes but rapidly expressed upon antigenic stimulation. 4-1BB can transmit co-stimulatory signals to T cells, which appear to modulate the magnitude and duration of the immune response and the size of subsequent immune memory compartments (13, 14). In mice, 4-1BB engagement was shown to inhibit AICD following T-cell stimulation by superantigen (13). In view of the role of CTLA-4 in curtailing T-cell activation, this raises the possibility that one mechanism by which signalling via 4-1BB exerts its effects might be to down-regulate expression of CTLA-4.

Early studies implied that 4-1BB stimulation acted principally upon CD8+ lymphocytes (15); however, others reported equivalent effects on both CD4+ and CD8+ subsets (16). Studies using knockout mice found that 4-1BBL deficiency only caused a modest reduction in the primary CD4+ T-cell response and had no effect on the magnitude of primary responses of CD8+ T cells, although co-stimulation via 4-1BB could substitute for CD28 signalling in CD28-deficient mice (17, 18). In contrast, the secondary response of CD8+ T cells was highly dependent upon 4-1BBL, which was also the case for some, though not all secondary CD4+ T-cell responses (14, 17–19).

Co-stimulation via 4-1BB permits responses at lower levels of signalling through the TCR–CD3 complex and CD28 and promotes TH1 differentiation/cytokine profiles in CD4+ cells (20, 21). This and the increased duration and magnitude of immune responses co-stimulated via 4-1BBL would appear beneficial in the context of cancer immunotherapy, and indeed stimulation of this pathway in mice can eliminate large, established, poorly immunogenic tumours (22–25). Immune stimulation via 4-1BB promoted eradication of tumours when CD80 was ineffective (26); this required only CD8+ cells, although CD4+ cells were necessary for a protective, memory response (27). In such assays, 4-1BBL acted synergistically with either CD80 (23) or IL12 (28, 29), or an antigenic peptide (25). The efficacy of 4-1BBL or agonistic anti-4-1BB antibodies in promoting immune rejection of poorly immunogenic tumours could be ascribed simply to enhancing the co-stimulatory signal to naive, tumour-specific T cells (25). However, the efficacy even in a diversity of models involving pre-established tumours suggests that 4-1BBL co-stimulation may be able to overcome anergy, to reactivate tumour-specific T cells that have been tolerized through prior, poorly immunogenic encounter with tumour antigens.

The effects of 4-1BBL co-stimulation on T-cell activation, and on the magnitude and longevity of response, as well as the encouraging therapeutic activity in murine tumour models, suggest that manipulation of 4-1BBL co-stimulation could be beneficial for immunotherapy of human cancer. In this paper, we report the establishment of an in vitro system to study the effects of 4-1BBL and other co-stimulatory ligands on human T-cell responses, using replication-defective adenoviruses to express the ligands on A549 lung carcinoma cells. This system could be extended to investigate the effects of 4-1BBL and other co-stimulatory signals on antigen-specific responses, including tumour-specific T cells from cancer patients, and potentially also to therapeutic applications. Localized expression of immunostimulatory ligands using gene transfer approaches may reduce the risk of excessive, generalized immune activation seen in some clinical trials of systemically delivered antibodies (30, 31). Here, we investigate the proliferative response of normal human T cells to co-stimulation in vitro with CD80, CD86 and 4-1BBL. We show that 4-1BBL can synergize with CD80 or CD86 to increase T-cell proliferation and permits extended T-cell proliferation continuing over several weeks. Importantly, we demonstrate for the first time that co-stimulation with 4-1BBL can reactivate T cells that have been anergized by prior, sub-optimal antigenic stimulation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 Funding
 References
 
Recombinant adenoviruses for expression of co-stimulatory proteins
The E1-deleted, replication-defective adenoviruses (Ad-5) expressing human CD80 (Ad-CD80) or enhanced green fluorescent protein (GFP, used as a control) were described previously (32). The coding region of human 4-1BBL was obtained by reverse transcription–PCR from RNA from a lymphoblastoid cell line (LCL) and that for human CD86 using RNA from DCs. Both were initially cloned into the plasmid pxLNCX (32) and the correct sequence confirmed, before insertion into adenovirus vectors essentially similar to Ad-CD80, but with a larger E1 deletion (between bp 357 and 3525 of Ad5). Viruses were grown in HEK293 cells and purified by CsCl density gradient ultracentrifugation. The concentration of virus particles was determined by DNA assay using the fluorescent dye PicoGreen (Molecular Probes, Invitrogen, Paisley, UK).

The lung carcinoma cell line A549 was infected with the recombinant viruses at a multiplicity of infection (MOI) of 300 virus particles (vp) per cell (for co-infection with Ad-CD80 or Ad-CD86 and Ad-4-1BBL, 300 vp per cell of each virus), before plating in 96- or 24-well plates, depending on the experiment. Infections were performed 2 days before the A549 cells were required for co-culture with lymphocytes, to allow time for expression of the co-stimulatory molecules.

Lymphocyte cultures
Venous blood was obtained with informed consent from healthy human volunteers, and PBMCs were isolated by Ficoll density gradient centrifugation (Life Technologies, Paisley, UK). The isolated PBMCs (107 cells ml–1) were incubated in tissue culture flasks in complete medium (RPMI 1640 with 7% FCS and 2% human AB serum, 2 mM glutamine, 100 IU penicillin and 100 µg ml–1 streptomycin) at 37°C to allow attachment of plastic-adherent cells. The non-adherent cells were harvested after 2 h for use in the experiments.

3H-thymidine incorporation assay
A549 cells were infected 2 days previously as above with recombinant adenoviruses to express GFP, CD80, 4-1BBL or CD80 + 4-1BBL and irradiated at 80 Gy (Cs-137 Irradiator CIS IBL 437, France), before plating 5 x 104 cells per well in a 96-well plate. Lymphocytes were added (105 per well), in the presence of 0, 10 or 100 ng ml–1 OKT3 anti-CD3 mAb (JANSSEN-CILAG, High Wycombe, UK), and the plates were incubated at 37°C, 5% CO2 for 3, 7 and 14 days. Cultures were pulsed with 1 µCi per well of 3H-thymidine (Amersham, Little Chalfont, UK) during the last 10 h of incubation. Cells were harvested onto glass fibre filters and counted in a beta plate counter (Topcount NXT, PerkinElmer, Waltham, MA, USA).

Monitoring proliferation using carboxy fluorescein diacetate succinimidyl ester
After washing non-adherent PBMC in PBS, they were re-suspended at 2 x 107 cells ml–1 in PBS and gently mixed with an equal volume of 5 µM carboxy fluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Invitrogen) in PBS. The cell suspension was then incubated for 15 min at 37°C, before adding an equal volume of complete RPMI culture medium to stop the labelling. The cells were then washed three times and re-suspended in culture media. Typically, 1 x 106 lymphocytes were added per well to 24-well plates, seeded 2 days previously with 5 x 105 A549 cells infected with the recombinant adenoviruses, as above. OKT3 anti-CD3 antibody was included at 100 ng ml–1. The cultured lymphocytes were analysed at intervals by flow cytometry; the successive halving of fluorescent intensity per cell allows tracking of the number of divisions a cell has undergone since labelling (33). Calculations to estimate the proportions of input cells that had undergone divisions in culture were performed essentially as described (34).

Antibody staining and flow cytometry
Labelled mouse mAbs used for cytometry studies were sourced as follows: anti-CD4–FITC, anti-CD8–FITC, anti-CD25–PE, anti-CD45RA–PE, anti-CD45RO–PE, anti-CD62L–PE, anti-CD4–PE and anti-CD8–PE from Beckman Coulter (High Wycombe, UK); anti-CD8–TriColor from Invitrogen and anti-CD80–FITC, anti-CD80–PE, anti-CD86–PE, anti-CD137–PE, anti-CD137L–PE and anti-CD152–PE from BD PharMingen (Oxford, UK). Appropriate isotype controls were used to indicate levels of background staining.

After washing cells with PBS containing 2% FCS, samples were stained with ~1 µg ml–1 of appropriate fluorochrome-conjugated antibodies on ice in the dark for 30 min. Cells labelled with antibodies and/or CFSE were analyzed using a four-colour Beckman Coulter XL flow cytometer using Coulter System II software for data acquisition and WinMDI software for data presentation.

Since the majority of CTLA-4 (CD152) is located in intracellular vesicles and traffics to and from the cell surface (35), lymphocytes were permeabilized to allow detection of intracellular CTLA-4. Following staining as above for CD4 and CD8 surface markers, PFA solution (2%) and Saponin (Sigma) solution (0.1%) in PBS were used to fix and permeabilize cells, respectively, before incubation with anti-CTLA-4 or isotype control antibodies.

Magnetic depletion of CD4+ or CD8+ lymphocytes
Isolated lymphocytes at 1 x 107 cells ml–1 in RPMI medium were mixed with ice-cold, washed anti-CD4 or anti-CD8 antibody-coated Dynabeads (Invitrogen) and rotated for 30–60 min at 4°C. Using a magnetic separator, non-bound cell fractions were harvested and transferred to fresh tubes. The purity of the depleted fractions was verified by flow cytometry using anti-CD4 or -CD8 antibodies.

Extended cultures
Isolated lymphocytes were added (1–1.5 x 106 per well) to a 24-well plate in medium containing 100 ng ml–1 OKT3 anti-CD3 antibody. Depending on the different co-stimulation conditions required, some wells were pre-seeded 2 days previously with A549 cells infected with recombinant adenoviruses expressing GFP, CD80, 4-1BBL, CD80 + 4-1BBL, CD86 or CD86 + 4-1BBL. At intervals as specified in the figure legends, the lymphocytes were resuspended, an aliquot was mixed 1:1 with trypan blue stain and the cell density was determined using a haemocytometer. The density of lymphocytes was re-adjusted, either by dilution or by combining cells from more than one well, to 5 x 105–1 x 106 cells per well, when the remaining lymphocytes were transferred in fresh, OKT3-containing medium to new wells pre-seeded with A549 cells, expressing the appropriate co-stimulatory molecules or GFP as control. The cumulative expansion or shrinkage of lymphocyte populations over the extended cultures was calculated taking into account the fold increase or decrease in cell number between passages.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 Funding
 References
 
Expression of CD80 and 4-1BBL on A549 cells following adenoviral gene transfer
In order to investigate the effects of native, cell-associated CD80 and 4-1BBL on T-cell responses, we used replication-defective adenovirus vectors to express these co-stimulatory ligands on A549 lung carcinoma cells. Uninfected A549 cells did not express significant levels of these ligands, but infection with 300 virus particles per cell of Ad-CD80 (Fig. 1A) or Ad-4-1BBL (Fig. 1B) resulted in expression of CD80 or 4-1BBL, respectively, on most of the cells. In subsequent experiments (Supplementary Figure 1 available at International Immunology Online), expression of the co-stimulatory proteins on infected A549 cells was compared with that on in vitro-matured DCs (36) and an LCL (activated B cells). The mean level of CD80 expression on DCs was ~3-fold higher than on A549 cells at this MOI, and the distributions overlapped. Expression of 4-1BBL was higher on A549 cells infected with Ad-4-1BBL than on DCs or the LCL.


Figure 1
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  		Fig. 1. Expression of CD80 and 4-1BBL on A549 cells can provide co-stimulation to co-cultured lymphocytes. A549 cells were infected with 300 vp of Ad-CD80 (A) or Ad-4-1BBL (B). After 2 days, expression of the co-stimulatory proteins on the cells was determined by flow cytometry after staining with the respective FITC-labelled antibody (filled area) or an isotype control antibody (solid line). Dotted line—staining of uninfected cells for CD80 or 4-1BBL. Lymphocytes were co-cultured with irradiated A549 cells expressing GFP, CD80, 4-1BBL or CD80 + 4-1BBL and stimulated with 0, 10 or 100 ng ml–1 anti-CD3. Incorporation of 3H-thymidine into lymphocyte DNA was determined after (C) 3, (D) 7 and (E) 14 days, as an indicator of proliferation. Data show mean and standard deviation of triplicate wells.

 
CD80 and 4-1BBL enhance response to anti-CD3 stimulation
PBMCs were obtained from healthy human volunteers, and to minimize possible co-stimulation by monocytes, they were depleted of plastic-adherent cells before use in all experiments. Initial experiments compared the rates of lymphocyte proliferation in response to 0, 10 or 100 ng ml–1 OKT3 anti-CD3 antibody, when co-cultured with irradiated A549 cells that expressed CD80, 4-1BBL, both ligands or GFP (as a control). Proliferation was assessed by incorporation of 3H-thymidine after 3, 7 and 14 days of co-culture (Fig. 1C–E). On day 3, the proliferation of lymphocytes co-stimulated with 4-1BBL was very similar to the low, anti-CD3-dependent background of the control cultures with no co-stimulation (Fig. 1C). Cultures co-stimulated with CD80 showed ~4-fold greater thymidine incorporation than the non-co-stimulated cultures. The combination of CD80 and 4-1BBL caused a further marked increase in thymidine incorporation, to ~8 times the level of the non-co-stimulated cultures.

By day 7 (Fig. 1D), lymphocytes co-stimulated with 4-1BBL showed a modest increase in anti-CD3-dependent proliferation. Cultures with CD80 showed a modest reduction in thymidine incorporation compared with day 3, but this was still higher than with 4-1BBL. Cultures co-stimulated with both CD80 and 4-1BBL again showed the strongest anti-CD3-dependent proliferation, two to three times the level seen with CD80 alone.

By day 14 (Fig. 1E), proliferation of all the cultures was reduced; notably that of cultures co-stimulated with CD80 alone was now lower than cultures co-stimulated with 4-1BBL alone, while CD80 + 4-1BBL still resulted in greatest proliferation.

At all time points, there was minimal thymidine incorporation in cultures without anti-CD3, confirming that the co-stimulatory signals cannot stimulate lymphocyte proliferation in the absence of an antigenic signal and indicating that the allogeneic response to A549 cells was minimal. Furthermore, since significant lymphocyte proliferation was only obtained by co-culture with A549 cells expressing CD80 and/or 4-1BBL, it is clear that the level of co-stimulation provided by B cells present in the PBMC (or by trace contamination with other APCs not removed by prior plastic adherence) is insignificant in this assay. In subsequent experiments, co-stimulation with CD86 also resulted in lymphocyte proliferation, with a similar magnitude and timecourse to that obtained using CD80, and CD86 also demonstrated similar cooperativity with 4-1BBL (results not shown).

Dual co-stimulation increases the proportion of cells undergoing multiple divisions
To provide greater insight into the proportion of lymphocytes that proliferate, and the number of divisions they are induced to undergo, lymphocytes were labelled with the fluorescent dye CFSE before stimulation with 100 ng ml–1 anti-CD3 and co-culture with A549 cells expressing GFP or the co-stimulatory ligands. Use of CFSE allows tracking of the number of divisions a cell has been through since labelling as it is partitioned between daughter cells (33). Cultures were analysed by flow cytometry after 3, 7 and 14 days. As shown in Fig. 2, lymphocytes stimulated with anti-CD3 and co-cultured with GFP-expressing A549 cells remained almost entirely undivided. Cultures co-stimulated with CD80 showed clear peaks of cells that had divided up to three times by day 3; and by day 7, 75% of the lymphocytes had undergone between one and seven cell divisions. Little further cell division was apparent by day 14, and indeed there appeared to be some loss of the divided cells relative to the undivided cell peak, attributable to AICD.


Figure 2
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  		Fig. 2. Lymphocyte proliferation analysed by CFSE labelling and flow cytometry. Lymphocytes were labelled with CFSE on day 0, then stimulated with anti-CD3 mAb and co-cultured with A549 cells expressing GFP, CD80, 4-1BBL or CD80 + 4-1BBL as indicated. Cultures were analysed after 3, 7 and 14 days. The successive halving of cellular fluorescence as they divide results in a series of peaks corresponding to cells that have been through increasing numbers of divisions since labelling.

 
Cultures co-stimulated with 4-1BBL showed only a single, small peak of divided cells by day 3, but by day 7 ~40% of the cells had undergone one to four divisions. This response was appreciably less than that co-stimulated by CD80. However, proliferation continued in these cultures, so that by day 14 a detectable fraction of the cells had undergone up to seven cell divisions.

Compared with the cultures co-stimulated with CD80 alone, cultures co-stimulated with the combination of CD80 + 4-1BBL showed a slight increase in the proportion of cells undergoing two divisions by day 3. By day 7, the combined co-stimulation resulted in many more cells having undergone three or more divisions, with the peaks corresponding to three, four and five divisions each exceeding the number of undivided cells. Considerable further proliferation was evident by day 14. Later experiments including CD86 showed very similar results to those with CD80, including similar cooperativity with 4-1BBL (results not shown).

Calculation (34) of the number of input cells that gave rise to those in the peaks corresponding to one or more divisions at day 7 indicates that 30% of the input lymphocytes underwent one or more divisions in response to anti-CD3 combined with CD80 co-stimulation; 16% of input lymphocytes responded to anti-CD3 + 4-1BBL; while 45% responded to anti-CD3 + CD80 + 4-1BBL. (These calculations assume there was no lymphocyte death before day 7.) Despite the near additivity of these proportions, the number of cells that underwent three or more divisions in response to dual co-stimulation clearly greatly exceeds the sum of the responses to the individual ligands (Fig. 2). Therefore, the signals from CD80 and 4-1BBL must interact cooperatively on at least some of the responding cells to increase the number of lymphocytes that undergo three or more divisions, which constituted 68% of the culture by day 7 and a still greater proportion by day 14.

The receptor 4-1BB (CD137) is not expressed on resting T cells, but is induced upon anti-CD3 stimulation (11), peaking at up to ~15% positive cells after 3 days in our experiments (data not shown). In an analysis of receptor expression at day 4, co-stimulation with CD80 alone did not appear to influence CD137 expression, although in the cultures with CD80 + 4-1BBL, the CD137+ population expanded in line with their proliferation. Reciprocally, while co-stimulation with CD80 down-regulated CD28 expression from ~98 to ~20% of CD4+ cells and from ~40 to ~15% of CD8+ cells by day 4, 4-1BBL did not appear to affect the expression of CD28 (Habib-Agahi, unpublished data).

Similar and independent initial proliferative responses of CD4+ and CD8+ cells
The proportion of CD4+ and CD8+ lymphocytes was monitored in the CFSE-labelled cultures. This showed broadly similar responses of CD4+ and CD8+ cells under all co-stimulation conditions at day 7, with just a slight tendency for the CD8+ cells to undergo more divisions. By day 14, this trend was more pronounced particularly in the cultures co-stimulated with both CD80 and 4-1BBL, where the modal number of divisions for activated CD4+ cells was four to five, while that for CD8+ cells was seven (data not shown). Figure 3(A) summarizes the proportions of CD4+ and CD8+ lymphocytes from five donors, stimulated with 100 ng ml–1 anti-CD3 under the different co-stimulation conditions for 3, 7 and 14 days. While at day 7 the proportions of both CD4 and CD8 cells tended to increase in all experimental conditions, indicating expansion of both populations relative to other PBMC, by day 14 there was an apparent tendency for the proportion of CD8+ cells to increase, and CD4+ cells to decline, in cultures co-stimulated with 4-1BBL alone or in combination with CD80.


Figure 3
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  		Fig. 3. Comparison of responses of CD4+ and CD8+ lymphocytes. (A) Non-adherent PBMCs were stimulated with 100 ng ml–1 anti-CD3 mAb and co-cultured with A549 cells expressing GFP, CD80, 4-1BBL or CD80 + 4-1BBL. The proportions of CD4+ and CD8+ cells were determined by flow cytometry after 3, 7 and 14 days. The lines connect datapoints from each of the five samples analysed, from different donors. (B) Non-adherent PBMCs, without depletion or magnetically depleted of CD4+ or of CD8+ lymphocytes, were labelled with CFSE on day 0, then stimulated with anti-CD3 and co-cultured with A549 cells expressing GFP, CD80, 4-1BBL or CD80 + 4-1BBL as indicated. The cultures were analysed by flow cytometry on day 5.

 
In order to determine whether both CD4+ and CD8+ cells respond directly and independently to both CD80 and 4-1BBL, similar co-cultures were established after immunomagnetic depletion of either CD4+ or CD8+ cells. The CD4+ cells were depleted from 70 to 0.2% of lymphocytes and CD8+ cells from 31 to 0.5% (data not shown). Analysis of the CFSE-labelled cells on day 5 after stimulation (Fig. 3B) again showed very similar responses of CD4+ and CD8+ cells, and of the undepleted non-adherent cells, to stimulation with anti-CD3 and CD80, 4-1BBL or both, confirming that during the early stages of the response, each subtype responds similarly and independently to the co-stimulatory signals, in agreement with the 7-day time point in Fig. 3(A).

Analysis of activation markers and CTLA-4 expression
A selection of T-cell activation markers was monitored on lymphocytes stimulated with anti-CD3 and the different conditions of co-stimulation. Lymphocytes stimulated with anti-CD3 but without co-stimulation showed little change in the expression of CD45RA or -RO of CD25 or of CD62L (Supplementary Figure 2A–D is available at International Immunology Online). Co-stimulation with CD80, 4-1BBL or both reduced the proportion of CD45RA, and increased the proportion of CD45RO-positive cells, reflecting the levels of proliferation in these conditions. Up-regulation of CD25 was apparent particularly with dual co-stimulation. There appeared little change in CD62L expression, apart from a drop from 73 to 29% CD62L+ cells between days 7 and 14, in the cultures co-stimulated with both CD80 and 4-1BBL.

We also monitored expression of the inducible, inhibitory receptor CTLA-4 (2, 3, 37) (Supplementary Figure 2E–G is available at International Immunology Online). Prior to stimulation, only ~5% of CD4+ T cells and 1% of CD8+ T cells showed positive staining for CTLA-4. Anti-CD3 stimulation increased the frequency of CTLA-4-positive cells to ~35–70% after 3 days. Notably, neither the levels of CTLA-4 induced nor the proportion of CTLA-4-positive cells showed any significant reduction in cultures co-stimulated with 4-1BBL (with or without CD80) relative to cultures co-stimulated with CD80 alone.

Co-stimulation with 4-1BBL prolongs T-cell response
In the above experiments, the A549 cells expressing GFP or co-stimulatory ligands were added to the cultures on day 0; however, in the co-stimulated cultures, the A549 cells were largely eliminated during the first week, possibly due to activation of CTLs. To investigate the effects of repeated co-stimulation on the T-cell response, similar lymphocyte cultures were passaged at intervals, and fresh A549 cells expressing GFP or the co-stimulatory ligands were added, along with 100 ng ml–1 anti-CD3. Viable lymphocytes were counted at intervals, and Fig. 4 charts the cumulative expansion/contraction of the lymphocyte populations. Stimulation with anti-CD3 either alone or in the presence of GFP-expressing A549 cells led to a progressive decline in the number of viable lymphocytes. Co-stimulation with CD80 led to a 2.8-fold increase in total lymphocyte number by day 8. Re-stimulation at this time did not result in any further change in cell number by day 17, after which the number of lymphocytes decreased despite further re-stimulation, dropping below the starting level between days 23 and 31. As expected, cultures co-stimulated with 4-1BBL initially lagged behind those with CD80; however, by day 8 the lymphocyte number exceeded that with CD80 alone, and continued to increase up to day 31, reaching a 19-fold expansion from the initial lymphocyte number. Cultures co-stimulated with both CD80 and 4-1BBL showed the greatest cell number at all time points and expanded continuously to 92 times the starting cell number after 31 days. Thus, whereas CD80 co-stimulation allows a transient activation followed by non-responsiveness and cell death, co-stimulation with 4-1BBL allows a more sustained activation lasting >4 weeks. Combined co-stimulation with 4-1BBL and CD80 allows a continuous response that greatly exceeds that obtained with either ligand individually.


Figure 4
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  		Fig. 4. Tracking the number of lymphocytes in long-term cultures. Lymphocytes were stimulated with anti-CD3 either alone or in co-culture with A549 cells expressing GFP, CD80, 4-1BBL or CD80 + 4-1BBL. On days 5, 8, 17, 23 and 31 viable lymphocytes were counted, and on days 8, 17 and 23 (arrowheads) cultures were re-stimulated with fresh media containing anti-CD3 and co-cultured with pre-infected A549 cells as before, after adjusting culture densities. The graph plots on a log scale the cumulative expansion or shrinkage of the lymphocyte populations relative to the starting number.

 
4-1BBL reactivates non-responsive, anergic T cells
Exposure of T cells to antigenic ligands in the absence of co-stimulatory signals can induce a state of unresponsiveness or clonal anergy (8, 9, 37, 38). Although co-stimulation via CD80/CD86 allows initial response to an antigen, this can nonetheless also lead to anergy, either as a result of inhibitory signals mediated via CTLA-4 or in some situations by CTLA-4-independent mechanisms (5, 6, 9, 39).

Figure 5(A) shows an example of a culture of non-adherent PBMC stimulated with anti-CD3 alone that expanded by ~30% during the first week, before becoming unresponsive and declining to one-third the initial number of cells after 3 weeks, despite weekly replenishment of the medium containing anti-CD3. After 3 weeks, some of the remaining cells were placed in co-culture with A549 cells expressing either 4-1BBL or CD80 + 4-1BBL, still in the presence of anti-CD3. Over the following week, the rate of decline was slowed by 4-1BBL co-stimulation, and after 2 weeks the cell number had increased to >25% more than when transferred to 4-1BBL co-stimulation. More dramatically, lymphocytes transferred after 3 weeks of anti-CD3 exposure without co-stimulation, to cultures with anti-CD3, CD80 and 4-1BBL, had expanded by ~25% within 1 week, and the cultures expanded 4.6-fold after 2 weeks of dual co-stimulation. At this time, the parallel culture maintained with anti-CD3 alone had declined by a further factor of ~16 to ~2% of the starting cell number.


Figure 5
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  		Fig. 5. Reactivation of non-responsive lymphocytes by co-stimulation with 4-1BBL. Non-adherent PBMCs were stimulated with anti-CD3 either (A) alone or co-cultured with A549 cells expressing either (B) CD80 or (C) CD86. Viable lymphocytes were counted after 3, 7, 14, 21, 28 and 35 days, and cultures were adjusted for cell density and re-stimulated at weekly intervals (indicated by small arrowheads). On day 21 (large double arrowhead), some of the lymphocytes were transferred to co-culture with A549 cells expressing 4-1BBL, either alone or together with (A and B) CD80 or (C) CD86. The cumulative expansion or shrinkage of the lymphocyte populations was calculated and normalized relative to the cell number when the co-stimulation was switched, on day 21 (indicated by the horizontal dotted line).

 
As shown in Fig. 5(B), in the same experiment, lymphocytes initially activated using anti-CD3 and CD80 co-stimulation showed an initial expansion of ~70%, but subsequently became unresponsive to these stimuli and the cultures declined to below the input cell number by 3 weeks, despite weekly re-stimulation with anti-CD3 and CD80. Switching some of these lymphocytes to co-cultures with A549 cells expressing 4-1BBL, with or without CD80, resulted in a doubling of cell number within 1 week and a 6- to 8-fold increase in cell number after 2 weeks. This contrasts with the cultures that continued to receive anti-CD3 and CD80 co-stimulation alone, which declined 8-fold over the same 2 weeks, to below 10% of the input cell number. Similarly, as shown in Fig. 5(C), the proliferation induced by anti-CD3 and CD86 co-stimulation was also transient, with the cells becoming unresponsive and diminishing in number after the first few days, despite weekly restimulation. Again, switching to 4-1BBL co-stimulation was able to halt the decline and initiate further proliferation, and more rapid proliferation was obtained by dual co-stimulation with CD86 + 4-1BBL. Thus, exposure to 4-1BBL was able to reactivate T cells from an unresponsive, anergic state brought about by treatment with anti-CD3 antibody either alone or with CD80/CD86 co-stimulation, and the rate of proliferation of these reactivated cells could be further increased by dual co-stimulation with both CD80/CD86 and 4-1BBL.


   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
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 References
 
The recognition that T-cell activation required two signals—one antigenic, the other costimulatory—was an important step in understanding how the immune system can maintain peripheral tolerance to self-antigens, while allowing robust responses towards foreign or pathogen-associated antigens. The main pathway of co-stimulation involves the engagement of CD28 on T cells with B7-family ligands on APCs (1). Delivery of antigenic signals alone was shown cause to T-cell unresponsiveness or anergy, whereas simultaneous engagement of CD28 on T cells with B7-1/CD80 allowed optimal T-cell proliferation and IL-2 secretion (8). Using a similar model system involving adenovirus-mediated gene transfer to A549 lung carcinoma cells, we have confirmed that co-stimulation of human T cells with CD80 (or the related B7-2/CD86) allows them to proliferate in the presence of anti-CD3 antibody, whereas there was little or no response to anti-CD3 alone. In the typical experiment shown in Fig. 2, ~30% of the input lymphocytes went through between one and seven divisions, and the responding fraction expanded by ~7-fold, resulting in a 2.8-fold increase in total lymphocyte number. DNA synthesis was much reduced by day 7, and there was no significant further increase in cell number by day 14. Even in cultures replenished with fresh media and re-stimulated with anti-CD3 and CD80-expressing A549 cells, the lymphocytes ceased to proliferate and, after 2–3 weeks in culture, the number of viable cells declined (Fig. 4). These observations are consistent with the reported up-regulation of the inhibitory CD80R, CTLA-4/CD152 upon T-cell activation, bringing about AINR and AICD (3–6, 37). Thus, while exposing T cells to an antigenic signal alone can cause unresponsiveness and anergy, simultaneous co-stimulation with CD80/CD86 leads only to a transient activation and proliferation, followed by AINR and AICD. The importance of CTLA-4 in the natural down-regulation of the T-cell response was illustrated by the lethal lymphoproliferation in CTLA-4-deficient mice (40) and by the induction of autoimmunity in human cancer patients treated with CTLA-4-blocking antibodies (30).

Engagement of the tumour necrosis factor receptor (TNFR)-family receptor 4-1BB (CD137) was shown to enhance the expansion and long-term survival of superantigen-activated T cells (13), suggesting this pathway can modulate the magnitude and duration of an immune response. Indeed, 4-1BB signalling has been shown to regulate cell cycle progression of CD8+ T cells by increasing expression of cyclins D2, D3 and E, while down-regulating expression of the CDK-inhibitor p27kip1 (41) and to promote T-cell survival via NF{kappa}B-mediated up-regulation of the anti-apoptotic genes bcl-xL and bfl-1 (42). The ability of 4-1BBL to provide a co-stimulatory signal in the absence of ligands for CD28 was evident in our model system. At early times, these cultures lagged behind those co-stimulated with CD80, which may be explained by the fact that CD28 is constitutively expressed on a high proportion of resting T cells, allowing an immediate response, whereas 4-1BB/CD137 only becomes expressed on a proportion of T cells upon stimulation (11). In our experiments, CD137+ cells increased from <1 to ~12% of PBMC with 24 h, peaking at ~15% after 3 days of anti-CD3 stimulation (data not shown). Importantly, co-stimulation with 4-1BBL allowed a continuous proliferative response to anti-CD3 stimulation, showing a further substantial increase in lymphocyte number between 7 and 14 days after initial stimulation (Fig. 2), a period during which cultures co-stimulated with CD80 (or CD86) were static or showing onset of AICD. In cultures that were passaged and re-stimulated (Fig. 4), co-stimulation with 4-1BBL allowed continuous expansion of the T cells for as long as the cultures were maintained (at least 4–5 weeks).

At all time points, simultaneous co-stimulation with both CD80 (or CD86) and 4-1BBL allowed greater levels of T-cell proliferation than either signal alone. After 7 days, the number of cells that had undergone three or more divisions clearly exceeded the sum of the responses to CD80 or 4-1BBL individually, and the trend continued to day 14 (Fig. 2). Thus, the results cannot be explained by different populations of cells responding independently to either CD80 or 4-1BBL; a significant proportion of T cells must respond more strongly to both co-stimulatory signals than to either alone. With passage and restimulation, cultures with both CD80 + 4-1BBL expanded continuously, corresponding to a ~40- to 90-fold increase from the starting lymphocyte population after 4–5 weeks (Fig. 4). Thus, these results are consistent with previous observations that 4-1BB-mediated co-stimulation can function independently of CD28 (17, 43) or synergize with this pathway (20, 23). Many studies have relied upon 3H-thymidine incorporation to compare relative rates of lymphocyte proliferation in different conditions; however, this does not directly indicate the actual increase in cell number. Our flow cytometric analysis of CFSE-labelled lymphocytes provides a more detailed picture of the fraction of responding cells and the number of divisions undergone over 7–14 days of stimulation. Because we have monitored the actual number of viable cells in the extended, re-stimulated cultures, tracking the cumulative expansion (or shrinkage) of the lymphocyte population, our results particularly highlight the extended timescale over which the responses can continue and the large expansion of the responding cell population that can result from dual co-stimulation with CD80 and 4-1BBL. Our recent preliminary results (data not shown) using autologous fibroblasts to express co-stimulatory ligands and display peptide antigens have shown similar cooperativity between CD80 and 4-1BBL for expanding antigen-specific T cells, supporting the validity of the above conclusions drawn using anti-CD3 to provide the TCR signal.

We found that both CD4+ and CD8+ lymphocytes initially showed similar responses to anti-CD3 stimulation with CD80, 4-1BBL co-stimulation or both, in either unfractionated or magnetically depleted cultures, as previously reported (16, 44). However, by day 14 following stimulation, there appeared a distinct trend for the frequency of CD4+ cells to decline and for CD8+ cells to increase in cultures co-stimulated with 4-1BBL alone or with CD80, consistent with the report that the inhibition of AICD by 4-1BB ligation was selective towards CD8+ lymphocytes (13).

In view of the role of the inducible receptor CTLA-4 in the normal down-regulation of T-cell responses co-stimulated by CD80/CD86 after the initial few days of activation, we considered the possibility that 4-1BBL might interfere with the up-regulation of CTLA-4. However, we found that 4-1BBL did not prevent the increased expression of CTLA-4 following stimulation, thus one effect of 4-1BB engagement must be to override the inhibitory actions of CTLA-4. This is likely to be at the level of intracellular signalling, although other possibilities, such as interference with the cycling of CTLA-4 to and from the cell surface or with its interaction with CD80/CD86, cannot be ruled out. In addition to the role of CTLA-4 in terminating T-cell responses, exposure to CD80 or anti-CD28 antibodies can down-regulate CD28 expression on T cells (45), thereby increasing their susceptibility to activation-induced apoptosis (46). We also observed down-regulation of CD28 in cultures co-stimulated with CD80 or CD86, and this down-regulation was unaffected by 4-1BBL (Habib-Agahi, unpublished results). The anti-apoptotic functions of 4-1BBL (42) could support the continued survival of T cells with down-regulated CD28, while also co-stimulating their proliferation.

While this manuscript was in preparation, another study has reported up to 104-fold expansion of CD8+ T-cell cultures within 3 weeks, by incubation with K562 cells transduced with lentiviral vectors to express CD80 and 4-1BBL, combined with anti-CD3 stimulation (47). That study supports the benefits of combing these two co-stimulatory ligands, as reported here. In our work, we chose to use a relatively low concentration (100 ng ml–1) of soluble anti-CD3 to provide a weak, sub-optimal signal through the TCR since this could be more representative of the weak stimulation provided by natural tumour antigens and increase the dependency on co-stimulation. One key difference in the approach of Suhoski et al. (47) is that the K562 cells were also engineered to express Fc receptors, allowing the anti-CD3 mAb to be bound at the surface of the ‘artificial-APCs’ and thus provide a much stronger signal through the TCR complex. This may account for the even greater magnitude of lymphocyte proliferation observed in their system.

A number of studies have shown that activation of 4-1BB signalling can promote tumour-specific immunity and tumour rejection in mice. These effects have been attributed to the roles of 4-1BB-mediated co-stimulation discussed above, i.e. enhancement of T-cell expansion and inhibition of AICD (48–50); or to polarization towards a TH1 immune response (50). However, the efficacy even against established tumours (51) suggested a further possibility that 4-1BB engagement may allow reactivation of T cells that have become anergic. While it has been shown that signalling via another TNFR family member, OX40, can overcome peripheral tolerance in CD4+ T cells (52, 53), to our knowledge it has not been reported whether 4-1BB signalling can reactivate anergic T cells.

T-cell anergy is a mechanism of peripheral tolerance, by which non-immunogenic antigen encounter results in a state of hypo-responsiveness, although the T cell remains viable for extended periods (i.e. longer than the 8–24 h characteristic of cells committed to apoptosis) (9). Thus, provision of ‘signal 1’ for T-cell activation, in the form of antigen or anti-CD3 antibodies and in the absence of costimulation, results in clonal anergy of the lymphocytes (7–9). Although co-stimulation via CD28 allows initial response to antigen and apparent prevention of anergy in some models (7, 8), in other situations it appears that anergy can be induced despite CD80 co-stimulation, depending upon signalling through CTLA-4 (4–6, 39). The inability of our lymphocyte cultures stimulated at weekly intervals with anti-CD3 and CD80 (or CD86) to expand after the first week indicates that non-responsiveness or anergy has been induced and would be consistent with a CTLA-4-dependent mechanism. Since co-stimulation with 4-1BBL, either alone or together with CD80/CD86, allowed continuous proliferation in response to anti-CD3 for at least 4–5 weeks (Fig. 4), this clearly prevented the induction of anergy that occurred in parallel cultures, identical but for the absence of 4-1BBL. We therefore asked whether co-stimulation with 4-1BBL might be able to reverse the anergy that had been established in cultures lacking 4-1BBL. Our results clearly show that T cells that had become non-responsive either to anti-CD3 antibody alone or to anti-CD3 + CD80/CD86 co-stimulation, and continued to show progressive AICD when re-treated with these same ligands, could be rescued from the unresponsive, anergic state and reactivated to further proliferation when co-stimulated with 4-1BBL (Fig. 5). As with the primary stimulation, combining 4-1BBL co-stimulation with either CD80 or CD86 resulted in a greater level of proliferation than obtained with 4-1BBL alone. Over the 2 weeks following the switch to co-stimulation with 4-1BBL + CD80, the treated lymphocyte populations expanded by ~4–8-fold relative to the cell number at the time the co-stimulation was switched or ~50-fold higher than the number of lymphocytes in parallel cultures that were maintained without 4-1BBL co-stimulation. We conclude that 4-1BBL is able to reactivate T cells from an anergic state and propose that the anti-tumour effects of 4-1BB ligation observed in a number of mouse tumour models (23, 49, 50), particularly those involving pre-established tumours (51), are likely to be due at least in part to reactivation of anergic, tumour-specific T cells.

This study reinforces the role of 4-1BBL as an important modulator of T-cell immune responses. Our demonstration that co-stimulation with 4-1BBL is capable not only of allowing greatly extended proliferation of T cells but also of reactivating unresponsive, anergised T cells, strengthens the rationale for developing applications in the immunotherapy of human cancer. The model system we have developed in this study is being extended to analyse the effects of 4-1BBL on antigen-specific immune responses, including studies of tumour antigen-specific responses of lymphocytes from cancer patients.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
Funding
 References
 
Supplementary data are available at International Immunology Online.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
References
 
Iranian Ministry of Health and Medical Education to M.H.-A. (12234).


   Acknowledgements
 
We thank Ankit Rao and Gordon Ryan for assistance with the experiment in Supplementary Figure 1.


   Abbreviations
 
Ad, adenovirus
AICD, activation-induced cell death
AINR, activation-induced non-responsiveness
APC, antigen-presenting cell
4-1BBL, 4-1BB ligand
CFSE, carboxy fluorescein diacetate succinimidyl ester
DC, dendritic cell
GFP, green fluorescent protein
LCL, lymphoblastoid cell line
MOI, multiplicity of infection
TNFR, tumour necrosis factor receptor
vp, virus particles

   Notes
 
Transmitting editor: M. Feldmann

Received 8 May 2007, accepted 28 September 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 

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