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Prepublished online as a Blood First Edition Paper on July 5, 2002; DOI 10.1182/blood-2001-11-0136.
IMMUNOBIOLOGY
From the Department of Microbiology and Immunology,
Walther Oncology Center, Indiana University School of Medicine, and the
Walther Cancer Institute, Indianapolis, IN.
The CD28 The cytotoxic T lymphocyte (CTL)-mediated
immune response plays an essential role in the protection of the host
against infectious diseases and cancer. Antigen (Ag) recognition by
naive CD8+ T cells triggers a program of proliferation and
differentiation that leads to the production of effector lymphocytes
able to directly lyse Ag-bearing cells. The lytic mechanism primarily
involves release of cytoplasmic granules loaded with perforin (a
pore-forming protein) and granzyme B (a serine protease) at the contact
site between a CTL and the target cell.1 A large fraction
of Ag-specific CTLs are thereafter eliminated to maintain homeostasis.
Some cells differentiate into memory cells that are either cytolytic or
not cytolytic.2
CD28, a primary costimulatory molecule, is constitutively expressed in
naive CD4+ and CD8+ T cells and is involved in
initiating T-cell activation. However, the CTL function of human
CD8+ T cells has been linked to the loss of CD28 or
CD27,3-8 as is often observed during the course of a virus
infection9-11 or in tumor development.4 This
suggests that CD28 may not be directly involved in CTL functions. This
possibility is further suggested by apparent CTL responses in
CD28-deficient mice.12 The CD28 Alternative costimulatory molecules, such as 4-1BB,
OX40, CD30, and CD40 ligand (CD40L), all members of
the tumor necrosis factor receptor (TNFR) superfamily, that are induced
on activated CD4+ or CD8+ T cells have been
studied as potential candidates responsible for various effector
responses.16-19 Among those costimulatory molecules
belonging to the TNFR superfamily, 4-1BB is unique for primarily
augmenting CD8+ T cell-mediated
antiviral20-22 and antitumor responses,23-31
as well as allograft rejection.22,32-34 The effects
mediated by 4-1BB are abrogated in mice depleted of CD8+ T
cells,33 indicating that CD8+ T cells are
important targets for 4-1BB-mediated responses.18,35 Although 4-1BB-mediated costimulation has been shown to sustain long-term T-cell survival,16,35,36 its exact role in the
developmental pathway of effector or memory CD8+ T cells
has not been defined.
In this study, we address the question of how the expression of CD28
and 4-1BB, 2 costimulatory molecules, is coordinated during development
of effector or memory CD8+ T cells. Umbilical cord blood
(CB) offers a unique source of starting cells for studying the
developmental pathway for effector or effector memory CD8+
T cells, because CB is composed exclusively of naive CD8+ T
cells with few or no cytotoxic CD28 Reagents
Cell stimulation
Flow cytometry analyses For multiple-color staining, 5 to 10 × 105 cells were incubated with anti-CD8-Tri-Color plus the indicated antibodies conjugated with either FITC or PE in 50 µL staining buffer (PBS containing 1% bovine serum albumin) for 30 minutes at 4°C. The cells were washed 2 times with staining buffer and analyzed in a FACSCalibur cytofluorograph (Becton Dickinson, Mountain View, CA). Three-color staining was performed to detect CD28, 4-1BB, CD45RO, and CCR6 on CD8+ T cells with the use of FITC- or PE-conjugated corresponding antibodies with anti-CD8-Tri-Color. For staining intracellular granzyme B, cell-surface CD28 and CD8 were stained with anti-CD28-FITC and anti-CD8-Tri-Color and subsequently fixed and permeabilized with a kit (Fix and Perm; Caltag) according to the manufacturer's manual, followed by a PE-conjugated anti-granzyme B monoclonal antibody (mAb). Apoptosis assay by annexin V-FITC surface staining was undertaken according to the manufacturer's manual. Expression of 4-1BB ligand on Epstein-Barr virus (EBV)-transformed B cells was measured by staining the cells with 4-1BB-Fc and subsequently detecting the 4-1BB-Fc bound to the 4-1BB ligand with PE-conjugated anti-human Fc. Human IgG1 was used as a control for 4-1BB-Fc.Anti-CD3 redirected cytotoxicity assay Cytotoxicity was determined in a standard 4-hour 51Cr release assay using an EBV-transformed B-cell line, kindly provided by Dr Karen Pollok (Indiana University, Indianapolis), as target cells. Target cells (2 × 106 cells) were cultured 2 hours in 0.4 mL RPMI culture medium containing 500 µCi (18.5 MBq) Na2 51CrO4 and washed 3 times with PBS to remove free Na2 51CrO4. The target cells were then plated in 96-well round-bottom plates at 5000 cells per well. CD8+ T cells were mixed with target cells at effector-to-target cell ratios of 10:1, 5:1, 2:1, and 1:1. In some cases, 4-1BB-Fc was added to the target cells at the designated concentrations for 15 minutes at room temperature before the target cells were mixed with effector cells in 96-well plates. Human IgG1, a negative control for 4-1BB-Fc, was added to the target cells in a similar fashion to determine a specific effect of 4-1BB-Fc on CD8+ T cell-mediated cytotoxic activity. The plates were briefly centrifuged at 200g and incubated at 37°C in the presence or absence of anti-CD3 (1 µg/mL). After 4 hours, supernatants were counted in a gamma counter. The percentage of specific lysis was calculated by the following formula:
Cytotoxic defect of CD8+ T cells from CB can be overcome by stimulation with IL-12 and IL-15 In contrast to CD8+ T cells from adult PB, CD8+ T cells from CB are composed almost exclusively of naive cells, with few effector or memory cells. Exogenous cytokines, such as IL-12 and IL-15, are known to promote the effector functions of CB CD8+ T cells.39,40 We asked whether the lower amount of cytotoxic activity by CB CD8+ T cells might be related to the lack of effector CD8+ T cells in CB, and whether proinflammatory cytokines might induce the development of effector CD8+ T cells. To test this possibility, we compared the cytotoxic activity of CD8+ T cells obtained from adult PB and CB before and after stimulation with a combination of IL-12 and IL-15 for 5 days in a reverse antibody-dependent cell-mediated cytotoxicity (ADCC) assay using an EBV-transformed B-cell line as target cells. Cytotoxicity was initiated by adding anti-CD3 mAb, which will bind to the FcR on the B-cell line and stimulate the CD8+ T cells. Consistent with our previous report,37 fresh CB CD8+ T cells have less cytotoxic activity as compared with those obtained from PB (Figure 1A). In contrast to freshly isolated cells, CB CD8+ T cells stimulated with IL-12 and IL-15 demonstrated markedly enhanced cytotoxic activity that was almost indistinguishable from that mediated by stimulated PB CD8+ T cells (Figure 1B). Therefore, naive CD8+ T cells in CB can be differentiated into cytotoxic effector cells by proinflammatory cytokines such as IL-12 and IL-15.
The subset of CD8+ T cells generated in cord blood by IL-12 plus IL-15 lacks CD28 but expresses 4-1BB Most CD8+ and CD4+ T cells freshly isolated from CB express CD28, a primary costimulatory molecule for T-cell activation.3,5 Because the loss of CD28 is correlated with the effector function of CD8+ T cells,4,5 we asked whether treatment of CB CD8+ T cells with a combination of IL-15 and IL-12 down-regulated expression of CD28. We also examined the possible reciprocal induction of 4-1BB, an inducible costimulatory molecule, known to promote various CD8+ T cell-mediated effector functions, such as antiviral20-22 or antitumor responses.23-31 Expression of CD28 and 4-1BB on the CB CD8+ T cells was assessed by 2-color flow cytometry after incubation of CB CD8+ T cells for 5 days in the presence or absence of IL-12 plus IL-15. CD8+ T cells cultured in the absence of cytokines were essentially all CD28+ (Figure 1C). As shown in Figure 1D, more than half the CB CD8+ T cells cultured in IL-12 plus IL-15 were CD28 or
CD28low cells (51.1%). However, under the same culture
conditions, CD28CD4+ T cells were not induced
(data not shown). The 4-1BB induction is known to be dependent on CD3
stimulation.16 Interestingly, these proinflammatory
cytokines alone could also induce 4-1BB without CD3 engagement (8.6%
of the CD8+ T cells). The 4-1BB was detected exclusively in
the CD28 population, suggesting that the
CD28 CTLs are a primary cell type where 4-1BB may be
involved in activating effector function. In contrast, very little
4-1BB was detected in the CB CD8+ T cells cultured in the
absence of IL-12 plus IL-15 (fewer than 2% of CD8+ T
cells). Our results suggested the possibility that the lower cytotoxic
activity of CB CD8+ T cells compared with that of PB
CD8+ T cells might be related to lack of CD28
effector cells and that the gain of cytotoxicity by IL-15 and IL-12 might be attributed to an induction of the CD28
effector T-cell population. During culture with IL-15 and IL-12, there
was little cell proliferation or death of cord blood CD8+ T
cells unless anti-CD3 was added. Therefore, induction of the CD28CD8+ T cells after IL-15 and IL-12
stimulation was probably due to the down-regulation of CD28 expression
and not to selective expansion or survival of a CD28
population existing but not detected in the starting population of CB
CD8+ T cells.
Ability to induce 4-1BB by CB CD8+ T-cell is coupled to IL-15 and IL-12 stimulation The cytotoxicity induced by IL-15 and IL-12 in CB CD8+ T cells was driven by anti-CD3 stimulation. Naive and effector/memory CD8+ T cells may have different responsiveness to anti-CD3; naive CD8+ T cells may require more prolonged or stronger anti-CD3 stimulation compared with effector/memory cells in order to activate a program for killing target cells.41 To determine whether the differential cytotoxicities in CB and PB CD8+ T cells were correlated with the ability to induce 4-1BB upon anti-CD3 stimulation, we compared 4-1BB expression on CB and PB CD8+ T cells after stimulation with or without IL-15 and IL-12 for 5 days under conditions similar to those used for the cytotoxicity assay. We employed a suboptimal anti-CD3 stimulation condition as described in "Materials and methods" to better distinguish the distinctive responsiveness to anti-CD3 in 4-1BB induction in naive and effector/memory CD8+ T cells. Under this anti-CD3 stimulation condition, CB CD8+ T cells, consisting exclusively of naive cells, failed to induce 4-1BB (1.7%) (Figure 2). However, CB CD8+ T cells stimulated with IL-15 and IL-12 responded to anti-CD3 and expressed 4-1BB (42.2%). In contrast, PB CD8+ T cells expressed 4-1BB without such cytokine treatment in response to anti-CD3 stimulation (29.8%). However, the induction of expression of 4-1BB on PB CD8+ T cells was further enhanced after IL-15 and IL-12 stimulation (66.1%). The induction of the differential cytotoxic activities of PB and CB CD8+ T cells shown in Figure 1A-B occurred essentially parallel with the induction of 4-1BB. These results indicated that IL-15 and IL-12 induced the maturation process toward acquisition of cytotoxic activity as well as the expression of 4-1BB.
4-1BB costimulation induces a subset of CD28+ CD8+ T cells with greater cytotoxic activity Because cytotoxic activities were correlated with the expression of 4-1BB, we asked whether 4-1BB costimulation could cause expansion of the CD28 subset of CD8+ T cells, a prototype
CTL, or elicit any phenotypic changes in this population. To this end,
we analyzed the CD8+ T-cell population for
CD28 cells after stimulation of naive CB CD8+
T cells with IL-15 and IL-12 followed by anti-4-1BB costimulation with
anti-CD3 for 3 days. We included anti-CD28 and isotype control IgG as
controls to evaluate the specificity of anti-4-1BB costimulation. The
resulting cells consisted largely of 3 populations based on the
relative intensities of expression of CD28 and CD8 (Figure 3A). We designated the 3 populations as
subsets I, II, and III. Subset I corresponded to naive CD8+
T cells that expressed CD28 but no 4-1BB. Subset II identified cells
that expressed little or no CD28. This population was similar to that generated after stimulation with IL-12 and IL-15, as seen in
Figure 1D. Anti-CD28 costimulation increased numbers of subset II
cells. Interestingly, anti-4-1BB costimulation induced an additional population of CD28+ cells, designated subset III cells
(26.7%). Subset III cells were barely detected after incubation with
control IgG (1.8%) or anti-CD28 (1.4%). Subset II and III cells
showed brighter CD8 staining compared with cells in subset I. Thus, costimulation with CD28 and 4-1BB contributed to an expansion of
phenotypically different CD8+ T-cell subpopulations and
increased the proportion of cells in subsets II and III, respectively.
At this time, it was not clear whether subset III cells originated from
the subset II population.
Because of a positive correlation between the level of 4-1BB expression and cytotoxic activity, we postulated that subset III specifically induced by 4-1BB costimulation would be the most cytotoxic. To test this hypothesis, we separated each subpopulation by cell sorting and compared the cytotoxic activity against an EBV-transformed B-cell line as target cells (Figure 3B). As shown, subset III cells were the most cytotoxic, subset II cells were intermediate, and subset I cells were the least cytotoxic. We did not detect distinguishable differences in the cytotoxic activity of populations I and II when comparing treatment with anti-4-1BB and treatment with anti-CD28 (data not shown). Because 4-1BB has been shown by others to promote various cytotoxic effector functions, such as antitumor and allogeneic responses, subset III cells may be likely candidate cells for those 4-1BB-mediated CTL responses.21,22,27,33 Subset III cells may represent effector memory CTLs As subset III cells were the most potent cytotoxic subpopulation of CD8+ T cells, we analyzed the phenotypes of subset III cells in comparison with subset I and II cells (Figure 4). As expected, subset III cells demonstrated the highest expression of 4-1BB (87.4%), compared with subset II cells, which exhibited somewhat less 4-1BB (67.4%). Little 4-1BB was detected in the subset I population, which had elicited the least cytotoxic activity. We noticed several other unique phenotypes for subset III, as compared with subset I and II cells. CD45RO and CCR6 are known as prototype memory cell markers for T cells.42 These memory markers were expressed predominantly on subset III, compared with subset I and II cells, suggesting that the subset III population may represent memory-type cytotoxic CD8+ T cells. Granzyme B is an important cytolytic mediator for natural killer (NK) and CTL cells. The highest levels of granzyme B were detected in the subset III cells. This abundance of granzyme B in subset III may account for the strong cytotoxic activity of these cells, as shown in Figure 3B. Notably, subset III cells were extraordinarily large as estimated by a high mean fluorescence intensity of FSC (506 for subset III versus 368 and 384 for subsets I and II, respectively). These results indicate that anti-4-1BB costimulation gave rise to interesting phenotypic changes in the cells present in subset III. These changes correlated with the induced functional activities of these cells.
CD28+ CTLs induced by 4-1BB costimulation are a direct
precursor of CD28 subset II cells, no direct evidence for this was
shown. To address this possibility, we started with FACS-purified
subset II cells from which we attempted to induce subset III cells
after anti-4-1BB costimulation. We also included anti-CD28 or control
IgG in this study to evaluate the specificity of anti-4-1BB
costimulation. Purified subset II cells were cultured with anti-CD3
under different costimulatory conditions. The resulting cells were
subjected to FACS analysis for induction of subset III cells. We chose
up-regulation of CD28 as a marker for subset III cells. As shown in
Figure 5A, a significant increase of CD28
expression was detected in the purified population of subset II cells
after anti-4-1BB costimulation (66.2%) as compared with those
cultured with isotype control IgG (15.2%) or anti-CD28 (21.3%). To
rule out the possibility that the failure of CD28 induction after
stimulation with anti-CD28 might be caused by residual anti-CD28
released from the plates, which could block our anti-CD28 staining
process, we repeated the experiments with biotin-conjugated anti-CD28
to test for the presence of biotin-conjugated anti-CD28 on the cell
surface by staining with streptavidin-PE. Our flow cytometry results
indicated that there was no detectable amount of biotin-conjugated
anti-CD28 on the cell surface when compared with control cells
simultaneously incubated in the plates coated with biotin-conjugated
isotype control IgG (data not shown). From these experiments, we
confirmed that anti-4-1BB, but not anti-CD28 costimulation, induced
CD28 expression in subset II cells generating a subset III-like
phenotype. Subset III cells were also characterized by a remarkably
high content of granzyme B. The level of granzyme B induced by
anti-4-1BB costimulation was comparable to that seen in the subset III
cells shown in Figure 4. Granzyme B content was not increased by either control IgG or anti-CD28. Up-regulation of CD28 and granzyme B expression suggests that the CD28 subset II cells are a
direct precursor of CD28+ subset III cells. These results
also suggest a unique role for 4-1BB in the process of differentiation
from CD28 CTLs to CD28+
CTLs.
Consistent with reports by others,13 the majority of
CD28 4-1BB may be directly involved in target cell lysis Thus far, our data suggested a role for 4-1BB in the CTL differentiation process. Next, we tested the possibility that 4-1BB might actually be involved in the killing mechanism. To this end, we established a cytotoxic assay system in which we could block 4-1BB signals delivered by 4-1BB ligands expressed on the target cells to test a potential role for 4-1BB in killing activity. We used 4-1BB-Fc, a soluble form of 4-1BB consisting of the extracellular part of 4-1BB fused with the Fc portion of human IgG1. We first tested whether 4-1BB-Fc was capable of binding to 4-1BB ligands on the EBV-transformed B-cell line used in our cytotoxic assay. Bound 4-1BB-Fc on the target cells was detected with a PE-conjugated goat F(ab')2 anti-human Fc. We found that more than 90% of target B cells were positive for 4-1BB ligand (Figure 6A). In contrast, human IgG1 gave rise to only a background level of fluorescent intensity, indicating that detection of 4-1BB ligand on the target cells resulted from 4-1BB-Fc's binding the 4-1BB ligand, but not the Fc receptor. This information allowed us to use 4-1BB-Fc to block the majority of 4-1BB ligands on target cells from triggering 4-1BB on effector cells. We then determined whether treatment of target cells with 4-1BB-Fc affected cytotoxic activity by subset III cells, which expressed a high level of 4-1BB. As seen in Figure 6B, target cells pretreated with 4-1BB-Fc at a concentration of 10 µg/mL were almost completely resistant to cytolysis by effector cells. This inhibitory 4-1BB-Fc effect was dose-dependent. In contrast, human IgG1 used as a negative control did not affect cytolysis of the target cells. These results strongly suggest that 4-1BB signals delivered by 4-1BB ligands on the target cells are involved in cytolytic activity. At this time, the exact mechanisms through which 4-1BB signals might trigger the cytolytic process remain unclear. Because EBV-transformed B cells are known to be resistant to Fas-mediated cell death,43 we favor the possibility that 4-1BB-costimulation may be involved in promoting a granule enzyme-mediated cytotolytic pathway.
The down-regulation of CD28 on CD8+ T cells often
occurs during an acute virus infection or during tumor
development.5 Recently, low expression of CD28 on the cell
surface has been used for the identification of cytotoxic effector T
cells.3,44 CD28 The differentiation pathway from CD28 Our results strongly suggest that 4-1BB plays an important role in
maintaining survival during the CTL differentiation pathway, particularly from CD28 One basic pathological problem for HIV-infected patients is defective
HIV-specific CTL responses, although most HIV-infected patients have
sustained HIV-specific cytotoxic T-cell expansion. We speculate that
this immune incompetence may be related to impaired CTL
differentiation. Many of these HIV-specific CD8+ T cells
are not functional, even with a high viral burden.53-56 Importantly, the balance between CD28 In conclusion, 4-1BB specifically regulates the differentiation of a
CD28
We thank Dr Karen E. Pollok for providing the EBV-transformed B-cell line. We are also grateful to Ortho Biotech and Immunex for providing anti-CD3 and 4-1BB-Fc, respectively.
Submitted December 4, 2001; accepted June 4, 2002.
Prepublished online as Blood First Edition Paper, July 5, 2002; DOI 10.1182/blood-2001-11-0136.
These studies were supported by US Public Health Service grants RO1 HL56416, RO1 HL67384, RO1 DK53674 (H.E.B.), and RO1 AI46455 (R.R.B.) from the National Institutes of Health, and by a grant from the Phi Beta Psi Sorority (H.E.B.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hal E. Broxmeyer, Walther Oncology Center, Indiana University School of Medicine, Bldg R4, Rm 302, 1044 W Walnut St, Indianapolis, IN 46202-5254; e-mail: hbroxmey{at}iupui.edu.
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  G. Pulle, M. Vidric, and T. H. Watts IL-15-Dependent Induction of 4-1BB Promotes Antigen-Independent CD8 Memory T Cell Survival. J. Immunol., March 1, 2006; 176(5): 2739 - 2748. [Abstract] [Full Text] [PDF]
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M. Vidric, W.-K. Suh, U. Dianzani, T. W. Mak, and T. H. Watts Cooperation between 4-1BB and ICOS in the Immune Response to Influenza Virus Revealed by Studies of CD28/ICOS-Deficient Mice J. Immunol., December 1, 2005; 175(11): 7288 - 7296. [Abstract] [Full Text] [PDF]
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J. Bukczynski, T. Wen, C. Wang, N. Christie, J.-P. Routy, M.-R. Boulassel, C. M. Kovacs, K. S. MacDonald, M. Ostrowski, R.-P. Sekaly, et al. Enhancement of HIV-Specific CD8 T Cell Responses by Dual Costimulation with CD80 and CD137L J. Immunol., November 15, 2005; 175(10): 6378 - 6389. [Abstract] [Full Text] [PDF]
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N. L. Alves, F. A. Arosa, and R. A. W. van Lier IL-21 Sustains CD28 Expression on IL-15-Activated Human Naive CD8+ T Cells J. Immunol., July 15, 2005; 175(2): 755 - 762. [Abstract] [Full Text] [PDF]
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Y.-J. Kim, T. M. Stringfield, Y. Chen, and H. E. Broxmeyer Modulation of cord blood CD8+ T-cell effector differentiation by TGF-{beta}1 and 4-1BB costimulation Blood, January 1, 2005; 105(1): 274 - 281. [Abstract] [Full Text] [PDF]
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N. L. Alves, B. Hooibrink, F. A. Arosa, and R. A. W. van Lier IL-15 induces antigen-independent expansion and differentiation of human naive CD8+ T cells in vitro Blood, October 1, 2003; 102(7): 2541 - 2546. [Abstract] [Full Text] [PDF]
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CD8+ T cells
Top
Abstract
Introduction
-mercaptoethanol with or without a combination of IL-12 (2 ng/mL)
and IL-15 (10 ng/mL) for 5 days in 5% CO2 at 37°C. Cells
were then primed with anti-CD3 (1 µg/mL) in complete RPMI 1640 medium
on ice for 30 minutes, and washed twice with cold medium to remove free
antibodies, and immediately placed in 24-well plates (Costar,
Corning, NY). The resulting cells were harvested and analyzed for
surface antigens or sorted by FACS. For determining the effects of
anti-4-1BB costimulation, cells were incubated in plates that were
precoated with isotype control IgG (mouse IgG1, MOPC 21),
anti-CD28 (clone CD28.2), or anti-4-1BB (clone 4B4) at 10 µg/mL plus
anti-CD3 (1 µg/mL). After 3 days, cells were analyzed for the
expression of CD28, 4-1BB, granzyme B, and memory markers in
CD8+ T cells. These cells were also used for
51Cr release assays following cell isolation by FACS.
I, II, and III
T lymphocytes: antigenic and functional properties.
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.
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