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,
*
Division of Pulmonary and Critical Care Medicine, University of Washington, Seattle, WA 98195;
Infectious Disease Research Institute, Seattle, WA 98104;
Corixa Corp., Seattle, WA 98104; and
§
Immunex Corp., Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The second pathway requires direct effector cell-target signaling through CD95 ligand (CD95L)3 and its receptor CD95. CD95 (Fas, APO-1) is a member of the TNF receptor family and is expressed on both hemopoietic and nonhemopoietic cells (10, 13). On some cells, the binding of CD95L to CD95 leads to rapid and irreversible apoptosis. CD95L is expressed by T cells after exposure to either Ag or anti-CD3 Abs (14, 15). CD95/CD95L-mediated cell death occurs in the absence of extracellular calcium (10).
The mechanism by which CD4+ CTL lyse their targets has important implications for their biologic function. The use of perforin/granzyme potentially allows for lysis of all MHC class II-positive targets. In contrast, the CD95/CD95L pathway requires the target cell to both express CD95 and be susceptible to CD95-induced apoptosis. The majority of freshly isolated lymphoid and nonlymphoid cells do not undergo CD95-mediated apoptosis. As a result, CD4+ T cell dependence on the CD95/CD95L pathway would suggest a much more restricted role for these cells. Data generated in the mouse model have suggested that CD95 plays a major role in CD4+ T cell-mediated cytotoxicity (16, 17, 18). Because murine CD4+ CTL were shown to lyse only CD95-susceptible targets, these data have been used to argue that the primary role of CD4+ CTL is in the modulation of the immune response. On the other hand, recent data support a role for perforin/granzyme in CD4+ cytotoxicity. Williams and Engelhard (19) reported that short term CD4+ mixed lymphocyte cultures exhibited Ag-specific cytotoxicity that was CD95 independent and was diminished in perforin-deficient mice.
At present, little is known about the role of CD4+ CTL effector mechanisms in intracellular pathogenesis in humans. In this study we have investigated the relative contributions of the CD95 and perforin/granzyme cytolytic pathways in human PPD-specific CD4+ killing of a variety of cell targets. We have found that human CD4+ CTL efficiently lyse Ag-pulsed targets using both CD95-dependent and CD95-independent mechanisms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture medium consisted of RPMI 1640 supplemented with 10% FBS (BioWhittaker, Walkersville, MD) and 50 µg/ml gentamicin sulfate (BioWhittaker). For the generation of primary T cell lines and clones, RPMI was supplemented with 10% human serum (Trimar Hollywood, Reseda, CA). Agonistic anti-human CD95 mAb (clone CH-11) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). CH-11 is an IgM Ab and is able to cross-link CD95 in solution and induce an apoptotic signal in susceptible cells. The antagonistic CD95 Ab Fas M3 (Immunex Corp., Seattle, WA) is an IgG1 Ab that does not cross-link CD95 in solution and blocks CH-11- and CD95L-induced apoptosis (14). MgEGTA (0.1 M EGTA/0.2 M MgCl; Sigma Chemical Co., St. Louis, MO; pH 7.4) was prepared by the method of MacLennan (20). Recombinant soluble human CD40 ligand (CD40L) was expressed and purified from the supernatant of Saccharomyces cerevisceae (21). Human IL-4 was produced at Immunex Corp.
Cell lines
The T lymphoma cell line Jurkat was obtained from American Type Culture Collection (Rockville, MD). EBV-transformed B cell lines were generated in our laboratory using supernatants from the cell line 9B5-8. Cell lines were maintained by continuous passage in RPMI culture medium supplemented with 10% FCS.
Flow cytometry
Cells to be analyzed for CD95 expression were first incubated at 4°C in a blocking solution of PBS containing 2% normal rabbit serum and 2% normal goat serum to prevent nonspecific binding of mouse Ig. Cells were washed in FACS buffer (PBS containing 0.5% FBS and 0.02% sodium azide) and incubated with either the anti-CD95 Fas M3 Ab (5 µg/ml) or an IgG1 control (Becton Dickinson Immunocytometry Systems, San Jose, CA; 5 µg/ml) for 30 min at 4°C in a total volume of 50 µl. Cells were then washed and incubated in 50 µl of a 1/100 dilution of goat anti-mouse IgG-phycoerythrin (Tago, Burlingame, CA) for 30 min at 4°C. Flow cytometry was performed using a FACSCalibur (Becton Dickinson), and data were collected on 104 viable cells.
Separation of target cell subsets
PBMC from healthy PPD-positive individuals (>10-mm Mantoux skin test) were isolated from heparinized blood by centrifugation over Ficoll-Hypaque (Sigma) and washed three times with culture medium.
T cell-depleted targets were isolated from human PBMC by rosetting with 2-aminoethyl-isothiouronium bromide hydrobromide-treated sheep erythrocytes. These cells were then layered on Histopaque (Sigma), and the cell pellet was discarded. Following hypotonic lysis of sheep erythrocytes, cells were washed twice in PBS.
To generate B cell blasts, T cell-depleted PBMC were further enriched for CD19+ cells by positive immunomagnetic sorting (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol. These cells were then incubated in the presence of CD40L (3 µg/ml) and IL-4 (30 ng/ml) for 3 days in medium containing 10% FBS.
Monocytes were prepared from fresh PBMC by countercurrent elutriation (Beckman J6-MI, Palo Alto, CA). Wright’s Giemsa-stained cytospin preparations of these cells were >90% monocytes. Monocytes were maintained in polypropylene tubes to minimize adherence.
Adherent monocytes (macrophages) were prepared from fresh PBMC that had been cultured in 162-cm2 tissue culture flasks (Costar, Cambridge, MA) for 2 h, and the nonadherent cells were removed. Cell dissociation medium (Sigma) was used to remove the adherent cells, which were subsequently washed and maintained in polypropylene tubes.
Generation of PPD-specific T cell lines and clones
PBMC from healthy PPD-positive individuals were isolated from heparinized blood by centrifugation over Ficoll-Hypaque and washed three times with culture medium. One million cells were cultured in the presence of PPD (Lederle, PA) at a concentration of 10 µg/ml in 16-mm wells (Costar no. 3524) in 2 ml of culture medium. Cultures were incubated at 37°C in 5% CO2 for 5 to 7 days. At this time, FACS analysis revealed that these cells were >85% CD4+.
To generate a panel of CD4+ T cell clones reactive to PPD, a cell line was incubated for 12 days in 5% CO2 as described above. T cells were then cloned by limiting dilution in the presence of 2 x 105 irradiated (3500 rad using a 137Cs source) heterologous PBMC in the presence of anti-CD3 (10 ng/ml) and rIL-2 (10 ng/ml). Cell culture media consisted of 200 µl of RPMI supplemented with 10% FBS. T cell clones were selected based upon their proliferative responses to PPD.
Cytotoxicity assay
A standard 4- to 5-h 51Cr release assay was used to assess cytolytic activity. Briefly, target cells were labeled with 50 µCi of 51Cr for 16 h at 37°C in 1 ml of culture medium in the presence of either PPD (25 µg/ml) or secreted proteins from Mtb strain H37Rv (25 µg/ml; Colorado State University, Fort Collins, CO). T cell lines or clones to be assessed for cytolytic activity were washed twice in culture medium and serially diluted in 96-well V-bottom plates (Costar no. 3896). For anti-CD3 stimulation, the plates were coated for 16 h with anti-CD3 at 10 µg/ml and washed twice with PBS before using. Where indicated, CH11 (1 µg/ml), Fas M3 (5 µg/ml), and MgEGTA (2 mM EGTA; 4 mM Mg2+) were added in a volume of 25 µl to achieve the final concentration indicated above. 51Cr-labeled targets (2 x 103) were added immediately thereafter to make a final volume of 0.2 ml/well. After incubation for 4 h at 37°C, plates were harvested using a Skatron SCS harvesting system (Skatron, Sterling, VA). The 51Cr content of supernatants was determined using a 9600 gamma scintillation counter (Packard, Meriden, CT). The percent specific 51Cr release was calculated according to the formula: 100 x ((experimental cpm) - (spontaneous cpm))/[(maximum cpm) - (spontaneous cpm)), where spontaneous cpm is the counts per minute released in the absence of effector cells, and maximum cpm is the counts per minute released in the presence of 1% Triton X-100 (Sigma).
Granzyme (N-acetylglucosaminidase) assay
Granzyme release was assessed by the modified method of Henkart (unpublished) (22). T cells (5 x 105) were incubated in HBSS without phenol for 4 h at 37°C in the presence of 5% CO2 in 16-mm culture wells previously coated with anti-CD3. Fifty microliters of supernatant was mixed with 30 µl of 8 mM p-nitrophenyl-N-acetyl-ß-D-glucosaminide (Sigma N9376) and 10 µl of 0.5 M acetate, pH 5.0, in 0.4% Triton X-100 (Sigma). After 4 h at 37°C, the reaction was stopped with 150 µl of 50 mM NaOH and read in an ELISA reader at 405 nm (650 nm reference). Maximum release was determined from T cells incubated in the presence of 0.1% Triton during the course of the assay.
Expression of FasL and perforin mRNA
T cells (2 x 106) were incubated in the presence or the absence of insoluble anti-CD3 for 90 min, and total RNA was isolated using the TRIzol Regent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. cDNA was synthesized and standardized to yield similar amounts of ß-actin PCR product within the linear range of amplification. cDNA specific for FasL and perforin was amplified by PCR and visualized by autoradiography. FasL and perforin primers were used as previously reported (23, 24). PCR was performed with AmpliTaq polymerase (25 U/ml; Perkin-Elmer/Cetus, Norwalk, CT) plus 2.5 mM MgCl2 for 30 s of denaturation at 94°C followed by 30 s of annealing and extension at 72°C for 1 min for 26 consecutive cycles. PCR products were resolved by agarose gel electrophoresis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze the role of CD95 in CD4+ T cell
cytotoxicity, PBMC from a healthy PPD-positive individual were
stimulated with PPD in vitro. After 5 days, these cells (>85%
CD4+) were assessed for their ability to lyse PPD-pulsed
targets that had been depleted of T cells by rosetting with SRBC. Lysis
of PPD-pulsed, but not nonpulsed, target cells was observed (Fig. 1
). Addition of the antagonistic CD95 Ab
Fas M3 resulted in substantial inhibition of the Ag-specific lytic
activity. In a series of three similar experiments, the Fas M3 Ab
inhibited 7 to 60% of the specific lytic activity. These experiments
suggested that in at least a subset of target cells the CD95/CD95L
mechanism was being used. To clarify the relative contribution of
CD95/CD95L in CD4+ cytolytic function, PPD-specific
CD4+ T cell clones were generated and assessed for their
ability to lyse purified target populations.
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CD4+ T cell clones reactive with PPD were
prepared as described in Materials and Methods. PPD-pulsed
adherent macrophages were efficiently lysed by two independent
CD4+ T cell clones in a 5-h 51Cr release assay
(Fig. 2). Addition of the anti-CD95
Ab Fas M3 did not result in significant inhibition of this lytic
activity. This experiment suggested that lysis of these targets was
probably occurring through the perforin/granzyme pathway. To date, a
total of eight PPD-specific clones from two donors have been analyzed,
and all are cytolytic. Of the four clones from two donors that have
been evaluated, all lyse Ag-pulsed macrophages in a
Ca2+-dependent manner (data not shown). We considered the
following possibilities: 1) the T cell clones were deficient in their
expression of CD95L; 2) these targets were not susceptible to
CD95-mediated lysis during the 4-h assay; and 3) the killing pathways
were redundant.
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To test whether these CD4+ T cell clones were
able to express CD95L, clones were activated with immobilized
(plate-bound) anti-CD3 and assessed for their ability to lyse the
CD95-sensitive Jurkat cell line in a bystander assay. As shown in
Figure 3A, the
CD4+ CTL clones were capable of lysing Jurkat targets in a
manner that was completely blocked by the addition of the anti-CD95
Fas M3 Ab. Supporting a primary role for CD95 in this assay, the
removal of extracellular calcium with MgEGTA resulted in minimal
inhibition of cytolysis (Fig. 3B). Thus, we concluded
that these PPD-specific T cell clones were capable of using both the
perforin-granzyme and CD95 cytolytic pathways. As a result, utilization
of the CD95/CD95L pathway probably depends upon the susceptibility of
the target cell to CD95-mediated apoptosis.
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For a CD4+ CTL to kill its target via the CD95
apoptotic pathway, the target cell must both express CD95 and be
susceptible to CD95-mediated apoptosis. We initially analyzed a variety
of targets for CD95 expression. Flow cytometric analysis of purified
cell subsets revealed the presence of CD95 on a variety of targets
(Fig. 4). The Jurkat cell line, B cell
blasts, elutriated monocytes, and EBV-transformed lymphoblastoid cells
all exhibited high levels of CD95. Adherent macrophages expressed lower
levels of CD95. The agonistic anti-CD95 Ab CH-11 was used to assess
susceptibility to CD95-dependent lysis by 4-h 51Cr release
or by uptake of trypan blue after overnight incubation with the CH-11
Ab (Table I). Jurkat cells and B cell
blasts were both sensitive to CD95-mediated apoptosis, whereas
monocytes and adherent macrophages were relatively refractory to
cytolysis in these assays. Interestingly, the EBV-transformed
lymphoblastoid line that expressed high levels of CD95 was relatively
refractory to CH-11-mediated lysis. Similar data have been obtained
using lymphoblastoid cell line (LCL) generated from four different
individuals (data not shown). These data confirmed that CD95 expression
was necessary but not sufficient for susceptibility to CD95-mediated
lysis and predicted that among APC, only the B cell blast population
would be lysed in a CD95-dependent manner.
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To test the hypothesis that Ag-specific CD4+ T
cell-mediated lysis could use both the perforin/granzyme and CD95
cytolytic pathways, PPD-specific T cell clones were tested for their
ability to lyse a panel of target cells. As shown in Figure 5, these clones were capable of efficient
short term lysis of macrophages, elutriated monocytes, LCL, and B cell
blast targets. However, significant inhibition with the CD95 Fas M3 Ab
could only be demonstrated with the B cell blast targets (Table II). Addition of an isotype-matched
control Ab had no effect. In addition, we assessed the ability of the
PPD-specific T cell clones to lyse Mtb-infected monocytes. At an E:T
cell ratio of 20:1, we observed 25% specific lysis, which was
completely blocked by the addition of MgEGTA (data not shown).
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While utilization of the perforin-granzyme pathway is inferred
from the dependence of lytic activity on extracellular calcium, we
sought to directly demonstrate the release of granule contents of the
PPD-specific T cell clones upon activation. The CD4+ T
cell clones were incubated in the presence of immobilized anti-CD3
for 4 h, and the supernatants were assessed for their ability to
cleave the hexosaminidase substrate. As shown in Table III, PPD-specific CD4+ clones
released serine esterase when stimulated with anti-CD3.
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Finally, to confirm the ability of CD4+ T cell
clones to express both CD95L and perforin, reverse transcriptase-PCR
(RT-PCR) was employed. After induction with anti-CD3, both clones
expressed CD95L mRNA. In contrast, both resting and stimulated clones
expressed perforin mRNA (Fig. 6).
Collectively, these data confirm the potential for CD4+ CTL
to use both the CD95 and perforin/granzyme cytolytic pathways.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several experiments suggested that the short term cytolysis we observed was due to activation of the perforin/granzyme system. First, we could detect the release of serine esterase following stimulation of the T cell clones and the presence of perforin mRNA by RT-PCR. Second, addition of MgEGTA resulted in complete inhibition of lysis of the CD95-insensitive targets and only partial inhibition of the B cell blast targets.
We show that the CD4+ T cell clones are capable of inducible CD95L expression by RT-PCR, and functionally in a bystander assay, as lysis of the CD95-sensitive Jurkat cell line was effectively inhibited by the antagonistic CD95 Ab, while addition of MgEGTA had little effect. Our experiments might underestimate the role of CD95 in two ways. First, chelation of extracellular calcium may inhibit T cell activation and thus the induction of CD95L expression, an obvious requirement for utilization of the CD95 lytic pathway. Second, if the kinetics of apoptosis induced by CD95 were slower than those of perforin-granzyme, our short term assays would favor detection of the former mechanism. Nonetheless, the efficient lysis of CD95-insensitive targets in a short term assay supports a dominant role for the perforin-granzyme mechanism of cytolysis.
Our data also do not exclude the possibility of CD4+ T cell subsets that differentially use either the CD95 or perforin granzyme pathways. However, we have found that all our PPD-reactive T cell clones are capable of efficient short term cytolysis of LCL targets. Additionally, it is possible that our CD4+ T cell clones are using an as yet undescribed, calcium-dependent mechanism that is independent of the perforin-granzyme pathway. However, experiments performed in the mouse model have suggested that the CD95 and perforin/granzyme mechanisms can account for all short term cytolysis.
These observations may have important implications for the biologic
role of CD4+ CTL. The ability of human CD4+ CTL
clones to lyse a variety of targets using the perforin-granzyme
mechanism would suggest a role for these cells in the recognition and
the elimination of APC. Thus, CD4+ CTL may be important in
host defense against intracellular pathogens, such as Mtb, whose host
is the macrophage (29). Lysis of infected cells, particularly those in
which containment has failed, might serve to release intracellular
organisms into the extracellular space, allowing fresh macrophages to
lyse the bacterium. Alternatively, cytotoxic T cells may play a unique
role in host defense to Mtb through the release of granular
constituents that promote the destruction of infected macrophages. The
directed exocytosis of cytolytic granules by T cells induces apoptosis
in the target cell. In this regard, it has been suggested that
apoptosis will kill intracellular mycobacteria (30). However, mice
deficient in the expression of perforin, granzyme, or CD95 (Fas) are
still able to contain infection with Mtb (31, 32), suggesting that in
the mouse model the secretion of macrophage-activating cytokines such
as IFN-
and TNF-
may be sufficient for protective immunity.
Alternatively, other components of the cytotoxic granule may have a
direct anti-mycobacterial effect (23, 33).
Since human CD4+ CTL are capable of using the CD95 lytic pathway, these cells may still have an important role in the modulation of the immune response. This role has been inferred from the development of a lymphoproliferative disorders both in the CD95-deficient lpr mouse (34, 35) and in patients with the Canale-Smith syndrome (36). An immunomodulatory role for CD95 is further supported by the observation that B cells become CD95 sensitive when activated in the absence of Ag (28) and by the observation that activated CD4+ T cells lyse CD8+ T cells in a CD95-dependent manner (37). Recently, Vergelli et al. (38) have described autoreactive myelin basic protein-specific CD4+ T cell clones, a subset of which kill through CD95, a subset of which are presumed to kill through perforin, and a subset of which kill through both. Since we have not found evidence for PPD-reactive T cell clones that lyse exclusively through CD95, it is interesting to speculate that CD4+ T cell clones that are capable only of CD95-mediated lysis may play a unique immunoregulatory role.
In conclusion, we have found that human PPD-specific CD4+ CTL use both CD95-dependent and CD95-independent mechanisms. Collectively, the data suggest that CD4+ CTL may play an important role in the immune response to intracellular pathogens.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David Lewinsohn, Infectious Disease Research Institute, Box 358080, 1124 Columbia St., Seattle, WA 98104.
3 Abbreviations used in this paper: CD95L, CD95 ligand; PPD, purified protein derivative; CD40L, CD40 ligand; Mtb, Mycobacterium tuberculosis. LCL, lymphoblastoid cell line; RT-PCR, reverse-transcriptase polymerase chain reaction.
Received for publication April 23, 1997. Accepted for publication November 4, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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