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*
Institute of Pathology, Division of Immunopathology, University of Bern, Bern, Switzerland; and
Ontario Cancer Institute/Amgen Institute, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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TCRß IELs, CD8ß
IELs, and splenocytes from lymphocytic choriomeningitis virus
(LCMV)-specific TCR transgenic mice exert Ag-specific cytotoxic
activity in a long-term, but not in a short-term, cytotoxicity assay.
This cytotoxic activity is mainly Fas-Fas ligand mediated and is
significantly reduced in the presence of 20 µg/ml
Fas-Fc
1 fusion protein. Both CD8ß IELs and
CD8ß splenocytes isolated from LCMV-infected C57BL/6 mice exert
potent perforin-dependent cell-mediated cytotoxicity. CD8
TCRß IELs from LCMV-infected animals, however, show only minimal
Ag-specific cytotoxicity. The potent cytotoxic activity of in vivo
activated CD8ß IELs is not affected by the addition of
Fas-Fc1. Nevertheless CD8ß IELs from LCMV-infected
perforin-deficient mice exert Ag-specific cytotoxicity in a short-term
cytotoxicity assay, and this cytotoxicity is almost completely blocked
by the addition of Fas-Fc1. These results demonstrate
that naive CD8ß IELs exert Ag-specific, Fas-Fas ligand-mediated,
constitutive cytotoxic activity in a long-term cytotoxicity assay,
whereas primed CD8ß IELs primarily use the perforin-dependent
exocytosis pathway to exert their potent cytotoxic activity.
Furthermore, these results clearly illustrate the requirement for
Ag-specific determination of IEL-mediated cytotoxicity, because the
elevated, but variable, frequencies of memory-type T cells in this
compartment may lead to ambiguous results when polyclonal activation or
redirected assays are used. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
and CD8 TCRß T cells and of TCR
T cells that
preferentially express CD8 (1). Whereas CD8ß
TCRß IELs represent intrathymically differentiated T cells
(2), compelling evidence suggests that CD8 TCRß
IELs differentiate extrathymically in the intestinal mucosa, possibly
in the recently described cryptopatches (3, 4). In mice
the cellular composition of the IEL compartment varies greatly with age
and conditions of maintenance (5, 6). In general, the
number of TCRß CD8ß IELs increases with age and upon transfer
of the mice from a germ-free environment to conventional conditions of
maintenance, whereas the relative frequencies of TCR IELs and
CD8 IELs decrease under these conditions (7).
Concomitant with the transfer of mice from germ-free to conventional
conditions of maintenance, an increase in the constitutive cytotoxic
activity of CD8ß TCRß IELs is observed (5). The
IELs show a granular morphology due to their high content of
cytoplasmic granules, presumably containing molecules involved in
cell-mediated cytotoxicity such as perforin and granzymes
(8). In comparison with CD8 splenocytes, ex vivo isolated
CD8 TCRß IELs show strong constitutive cytotoxic activity when
assayed in TCRß- or CD3-redirected cytotoxicity assays
(9, 10, 11, 12). The availability of both naturally occurring and experimentally derived mouse strains deficient for molecules crucially involved in cell-mediated cytotoxicity led to the elucidation of two major pathways of cell-mediated cytotoxicity and their relative importance in vivo. The first pathway requires the exocytosis of several proteins, including the pore-forming protein perforin and one or several members of a serine protease family called granzymes. The importance of granule exocytosis in vivo in general and that of perforin in particular in cell-mediated cytotoxicity have been demonstrated in perforin-deficient (perforin-/-) mice infected with lymphocytic choriomeningitis virus (LCMV). In contrast to their heterozygous littermates, perforin-/- mice are unable to efficiently clear the infected host, thus leading to elevated virus titers in the serum of perforin-/- mice despite the presence of normal numbers of CD8 T cells that become normally activated upon infection (13). With the availability of perforin-/- mice it became possible to directly assess the extent of additional, nonperforin-dependent mechanisms of cytolysis. These studies revealed that the perforin-independent cytotoxic capacity can be attributed mainly, if not completely, to a Fas (CD95)-mediated induction of apoptosis in target cells (14, 15).
To date the assessment of the cytotoxic potential of IELs has been mainly determined based on anti-CD3 and anti-TCR-redirected cytotoxicity assays. Hence, in the present study we compared the Ag-specific cytotoxic potential of CD8 IEL subsets and CD8 splenocytes from naive and in vivo-primed animals, isolated ex vivo without additional culture in vitro, before the assessment of cytotoxic activity. Furthermore, we determined the respective contributions of the two main pathways of cell-mediated cytotoxicity, i.e., perforin-mediated and Fas-FasL-mediated cytotoxicity in naive and in vivo-primed CD8 T cell subsets isolated from the intestinal epithelium and the spleen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were originally purchased from the Institute
für Labortierkunde, University of Zurich (Zurich,
Switzerland). LCMV gp33-specific TCRß tg (TCR tg) C57BL/6 mouse
lines 327 and 318 (16) were provided by H. Hengartner and
R. M. Zinkernagel (University Hospital Zurich, Zurich,
Switzerland), and H. P. Pircher (University of Freiburg, Freiburg,
Germany). Perforin-/- C57BL/6 mice
(17) were backcrossed with TCR tg mice (line 318). All
mice were bred and reared under conventional conditions in the animal
facility of the Medical Faculty, University of Bern. Mice were
regularly tested for the presence of pathogens. All strains were
consistently serologically negative for the presence of different
viruses, such as mouse hepatitis virus, parvovirus, reovirus 3,
sendaivirus, rotavirus, mouse pneumonia virus, or adenoviruses. The tg
TCR-specific H-2Db binding LCMV
glycoprotein-derived peptide gp33–41 (gp33) and the
H-2Db binding adenovirus-derived control peptide
adn5 were provided by H. P. Pircher.
Monoclonal Abs
Fluorescence- or biotin-conjugated mAbs used in this study were
either purchased from PharMingen (San Diego, CA; CD8 (53-6.7),
CD8ß (53-5.8), and V2 (B20.1)) or purified by protein G columns
from supernatants of hybridomas originally obtained from American Type
Culture Collection (Manassas, VA) and subsequently labeled according to
standard protocols (CD3 (145-2C11), CD4 (GK1.5), TCR (GL3), B220
(RA3-6B2), Mac1 (M1/70), and TCRß (H57-597)).
LCMV infection
C57BL/6 mice and perforin-/- C57BL/6 mice were injected i.v. with 103 PFU LCMV of the strain WE, provided by S. Oehen (University Hospital Zurich). Mice were sacrificed 8 days later, and IELs and splenocytes were isolated as described below.
IEL isolation
The IELs were isolated as described previously
(18). In brief, IELs were dissociated from small
intestinal tissue pieces in Ca2+- and
Mg2+-free HBSS containing 2% horse serum (Life
Technologies, Gaithersburg, MD), 1 mM DTT, and 0.5 mM EDTA. Isolated
cells were sequentially passed through 70- and 40-µm pore size nylon
mesh cell strainers (Becton Dickinson, San Jose, CA), and incubated for
30 min at 37°C in a 5% CO2 atmosphere. The
IELs were then purified from enterocytes by 40/70% discontinuous
Percoll (Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation
(15 min, 800 x g, room temperature). Enriched IELs
were stained with anti TCR-FITC, anti CD8ß-PE, and anti
CD8-Cy-Chrome and were subsequently separated on a FACS-Vantage
(Becton Dickinson) into
TCR-CD8+ß+
and
TCR-CD8+ß-
fractions.
Isolation and purification of CD8ß splenocytes
Spleens were mechanically disrupted and washed once with HBSS and 5% horse serum. After lysis of erythrocytes by osmotic shock treatment, splenocytes were resuspended at 2 x 106 cells/100 µl HBSS and 5% horse serum and incubated for 15 min. on ice in the presence of 0.5 µg biotinylated anti CD4 and anti-B220 Ab/106 cells. Incubation of stained splenocytes with avidin-coupled paramagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and subsequent magnetic cell separation were performed following the manufacturer’s instructions. The CD4- and B220-depleted splenocyte fraction was then stained with anti CD8ß-FITC and sorted on a FACS Vantage.
Cytotoxicity assay
The H-2Db-expressing thymoma cell line RMA
or the Fc receptor-positive mouse mastocytoma cell line P815 were used
as target cells. Target cells were labeled by incubation with 100 µCi
Na51Cr (Amersham, Aylesbury, U.K.) in a volume of
150–200 µl IMDM plus 2% FCS for 1 h at 37°C in a 5%
CO2 atmosphere. Effector cells were placed in a
V-bottom microtiter plate (Costar, Cambridge, MA) and serially diluted.
Thirty-five hundred labeled target cells pulsed with 1 µg/ml gp33 or
adn5 (RMA) or supplied with 1 µg/ml anti-TCRß mAb (P815)
were added per well of the assay plate. Maximum release of
51Cr from target cells was induced by addition of
Nonidet P-40 to specified wells to a final concentration of 0.5%.
After incubating the plate for 5 h or overnight (20 h) at 37°C
in 5% CO2 atmosphere, 40 µl of supernatant
from each well was harvested and measured for released
51Cr on a Top Count liquid scintillation counter
(Canberra Packard, Meriden, CT). Specific lysis was calculated as
follows: [(experimental counts - spontaneous counts)/(maximum
counts - spontaneous counts)] x 100.
Neutralization of Fas-FasL-mediated cytotoxicity
Cytotoxicity assays were performed as described above, and
different concentrations of Fas-Fc1 fusion
protein (19) were added to specified wells 30 min before
the addition of labeled and peptide-pulsed target cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CD8ß IELs
To assess the cytotoxic potential of unprimed CD8 IELs and splenic
T cells, C57BL/6 mice transgenic for an LCMV-gp33-specific TCRß
were used. The frequency of TCRß tg-positive CD8 T cells as
determined by the use of the transgenic V-chain (V2) varied
substantially among the different cell subsets analyzed (Table I
). Therefore, for assessment of the
Ag-specific cytotoxic activity of non-Ag-primed, naive T cells, only
TCRß tg-positive cells were considered to be effector cells for
the calculation of the E:T cell ratio. Differences in the relative
frequencies of TCRß tg CD8 T cells in the effector cell population
and, hence, in the total number of cells added to the target cells to
obtain identical E:T cell ratios did not affect the outcome of the
cytotoxicity assay (data not shown).
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and CD8ß
TCRß IELs or splenic CD8ß TCRß T cells (Fig. 1A). Upon extended incubation
of the ex vivo isolated, sorted effector cells with the target cells
for up to 20 h, however, significant killing activity was observed
in all three CD8 T cell subsets. This long-term cytotoxic activity was
consistently higher in CD8ß than in CD8 IELs and splenic
CD8ß T cells when normalized to the frequency of the TCRß
tg-positive cells in the respective T cell subsets. No cytotoxic
activity against an irrelevant, Db binding
peptide was found in these long-term (20-h) cytotoxicity assays (Fig. 1B).
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ß tg CD8ß and
CD8 IELs and splenic CD8 T cells never expressed the T cell
activation marker CD25. Splenic TCRß tg CD8 T cells were mainly
CD62L positive; TCRß tg IELs, however, were mainly
CD62Llow. To directly assess whether the
down-regulation of CD62L expression may be due to the presence of an
additional, endogenous TCRß, we analyzed CD8-positive IELs and
splenic T cells from a RAG2-/- mouse transgenic
for the MHC class I restricted, OVA-specific TCRß OT-1
(20) for CD62L expression. Intriguingly, all IELs
(expressing the tg TCRß alone) were
CD62Llow, whereas all splenic CD8 T cells were
CD62Lhigh (data not shown). This finding thus
suggests that CD8 TCRß IEL are already preactivated in a
non-Ag-specific way. The Ag-specific cytotoxicity of naive IELs is mainly mediated by Fas-FasL interaction
To assess the contributions of distinct cytotoxic effector
mechanisms to the observed cell-mediated cytotoxicity in naive T cell
subsets, CTL activity was subsequently measured in CD8 and
CD8ß IELs and splenocytes from
perforin-/-, anti-LCMV-gp33 TCRß tg
mice. The cell-mediated cytotoxic activity of CD8ß IELs and
CD8ß splenic T cells measured in a long-term cytotoxicity assay
was only slightly lower in these perforin-/-
mice than in perforin+/+ mice (Fig. 2). Due to the low frequency of TCRß
tg CD8 IELs (Table I
), the cytotoxic activity of naive CD8
IELs could not be determined in perforin-/-
TCRß tg mice. To assess the contribution of FasL-mediated
cytotoxicity to the cytolytic activity of CD8ß IELs and splenic
CD8ß T cells, soluble Fas-Fc1 fusion
protein was added during the incubation of perforin-competent (Fig. 3), and
perforin-/- (Fig. 4) effector cells with target cells. As
shown in Fig. 3, blocking of Fas-FasL interactions greatly reduced the
cytotoxic activity of unprimed perforin-competent CD8 and
CD8ß IELs and splenic CD8ß T cells in a dose-dependent way.
As expected, most of the cytotoxic activity observed in a 20-h
cytotoxicity assay with unprimed CD8ß IELs and splenic T cells
from perforin-/- TCRß tg mice was
abrogated in a dose-dependent way by the addition of soluble
Fas-Fc1 fusion protein (Fig. 4). These results
clearly demonstrate that the Ag-specific cytotoxic activity of naive
IELs as well as splenocytes is mainly mediated by Fas-FasL interactions
that can be measured only in a long-term cytotoxicity assay.
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ß IELs exert potent Ag-specific cytotoxic activity during
LCMV systemic infection
For determination of the Ag-specific cytotoxic activity of in
vivo-primed IEL subsets and to assess the molecular mechanisms
involved, C57BL/6 mice were infected with 103 PFU
LCMV-WE. On day 8 postinfection CD8 and CD8ß IELs and
CD8ß splenic T cells were isolated for subsequent determination of
the Ag-specific activity in a short-term (5-h)
51Cr release assay; as shown in Fig. 5A, both CD8ß IELs and
CD8ß splenocytes exerted potent cytotoxic activity against
Ag-pulsed 51Cr-labeled RMA target cells. In
contrast, Ag-specific cytotoxic activity of in vivo-primed CD8
IELs was only minimal and became detectable only at a high E:T cell
ratio. Addition of soluble Fas-Fc1 did not
affect cytolysis of target cells by in vivo-primed CD8ß IELs and
CD8ß splenocytes (Fig. 5B). To determine whether in
vivo-primed IELs may also employ a perforin-independent (Fas/FasL)
pathway of Ag specifically killing of target cells,
perforin-/- cells were infected with LCMV-WE.
Although no cytotoxic activity was detected in CD8 IELs of
LCMV-WE infected perforin-/- mice, both
CD8ß IELs and splenic CD8ß T cells exerted cytotoxic activity
in a short-term cytotoxicity assay (Fig. 6A). This residual cytotoxic
activity of perforin-/-, in vivo-primed IELs
and splenic CD8ß T cells was almost completely blocked by the
addition of soluble Fas-Fc1 (Fig. 6B). These results indicate that in addition to perforin,
Fas-FasL-mediated cytotoxicity can also be used as an alternative
pathway by in vivo-primed CD8ß T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß, i.e., LCMV-gp33,
and from mice that were primed in vivo by a systemic infection with
LCMV-WE 8 days earlier. Furthermore, we attempted to determine the
respective contributions of the two main molecular pathways of
cell-mediated cytotoxicity, i.e., perforin-mediated and
Fas-FasL-mediated cytotoxic activity in these two instances.
Our analyses reveal that naive TCRß tg CD8ß IELs do not exert
significant Ag-specific cytotoxic activity in a short-term assay upon
isolation ex vivo. On the other hand, however, significant cytotoxic
activity is measured when total cell-mediated cytotoxicity of IEL
subsets, isolated ex vivo, is assessed, in a short-term (5-h)
anti-TCRß-redirected cytotoxicity assay (Fig. 7). In this assay, cytotoxic activity of
CD8ß T cells isolated from small intestinal epithelium is
severalfold higher than that of splenic CD8ß T cells (Fig. 7).
Similar results have been previously reported when lymph node T cells
were compared with IEL subsets in an anti-TCRß-redirected
killer assay (23). These results possibly reflect the
higher frequencies of previously activated, memory CD8 T cells in the
intestinal epithelium than in spleen or lymph nodes. This assumption is
supported by the observed lower frequencies of TCRß tg-positive
CD8ß T cells in IELs than in splenocytes (Table I) and by the
presence of cytoplasmic granules, presumably containing perforin and
granzymes that are released upon TCR-redirected activation in IELs
(10). A recent report indicates that FasL is also
integrated into the membrane of these granules, thus providing the
possibility that upon exocytosis of the content of the cytoplasmic
granules, the density of FasL on the cell surface is increased at the
site of TCR-MHC/Ag interaction (24, 25).
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ß IELs, unprimed intestinal CD8 IELs
show a slightly reduced cytotoxic activity in a 20-h cytotoxicity
assay. CD8 IELs are putatively extrathymically derived and seem
to be positively selected against self-Ags, as demonstrated in male
mice transgenic for an HY-specific TCRß (26, 27) and
in LCMV-gp33 transgenic mice double transgenic for the LCMV
glycoprotein and the gp33-specific TCRß (S. Müller and C.
Mueller, unpublished observations). This may explain the low frequency
of TCRß tg CD8 in the TCRß tg animals used in this
study (Table I). Due to the low frequency of TCRß tg CD8
IELs in perforin-/- mice, the relative
contributions of perforin and Fas-FasL interactions to the Ag-specific
cell-mediated cytotoxicity of naive CD8 IELs could not be
assessed. Upon in vivo priming, CD8 exert only limited
Ag-specific cytotoxic activity that seems to be mainly mediated by
exocytosis of cytolytic granules, because in
perforin-/- mice no specific cytotoxic activity
is observed (Fig. 6). Although the observed low cytotoxic activity of
CD8 may be due to intrinsic regulatory pathways that prevent the
generation of potent cytotoxic effector mechanisms, other explanations,
including a reduced precursor frequency of CD8 IELs specific for
non-self MHC class I-restricted Ags or enhanced susceptibility of
CD8 IELs to activation-induced cell death, should also be
considered. In contrast to CD8 IELs, in vivo priming by an LCMV
infection leads to the generation of a potent, Ag-specific,
cell-mediated cytotoxicity in CD8ß TCRß IELs. The analysis of
the molecular pathways of cytotoxicity involved reveals a certain
degree of redundancy, because addition of
Fas-Fc1 fusion protein does not affect the
cytotoxic activity of these in vivo-primed CD8ß IELs and CD8ß
splenocytes (Fig. 5) (15). In the absence of perforin,
however, the contribution of FasL-mediated cytolysis becomes apparent
even in short-term cytotoxicity assays (Fig. 6).
In conclusion, we provide evidence that similar to splenic CD8ß T
cells, ex vivo isolated, naive CD8ß and, to a lesser extent,
CD8 intestinal IELs exert cytotoxic activity in a 20-h, but not
in a short-term, cell-mediated cytotoxicity assay. This cytotoxic
activity of unprimed IELs is mainly mediated by Fas-FasL interactions.
Upon in vivo priming, potent cytotoxic effector cells are induced in
CD8ß, but not CD8 IELs. The cytotoxic activity of CD8ß
IELs equals or even exceeds the activity of splenic CD8ß T cells
from the spleen of the same animals. The cytotoxic activity of in
vivo-primed CD8ß IELs cannot be blocked by soluble
Fas-Fc1 fusion protein. However, in
vivo-primed CD8ß IELs from perforin-/-
mice show a significant residual cytotoxic activity that is completely
blocked by Fas-Fc1 fusion protein, thus
demonstrating a limited redundancy in the cytotoxic effector mechanisms
operative in in vivo-primed CD8ß IELs. Furthermore, the results of
the present study illustrate the requirement for an Ag-specific
determination of the cytotoxic activity of IELs, since due to the
elevated frequency of previously activated, memory T cells in this
compartment, assays using polyclonal T cell activation may yield
ambiguous results.
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Acknowledgments |
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ß tg mice; and Prof. H. P.
Pircher for gp33 and adn5 peptides and for helpful discussions. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Christoph Mueller, Division of Immunopathology, Department of Pathology, University of Bern, Murtenstrasse 31, CH-3010 Bern, Switzerland. E-mail address:
3 Abbreviations used in this paper: IEL, intraepithelial lymphocytes; LCMV, lymphocytic choriomeningitis virus; FasL, Fas ligand; gp, glycoprotein-derived peptide.
Received for publication July 13, 1999. Accepted for publication October 21, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Proc. Natl. Acad. Sci. USA 88:43./ T cells to the intestinal epithelium is independent of normal microbial colonization. J. Exp. Med. 172:239. T-cell antigen receptors. Proc. Natl. Acad. Sci. USA 90:8591.-producing activities of T cells in the mouse intestinal epithelium are strain dependent. Proc. Natl. Acad. Sci. USA 90:8204.ß-CD8+, but not -CD8+, TCR-ß+ murine intestinal intraepithelial lymphocytes can mediate perforin-based cytotoxicity, whereas both subsets are active in Fas-based cytotoxicity. J. Immunol. 156:35.[Abstract]
/ co-receptors by self-antigen in the murine gut. Proc. Natl. Acad. Sci. USA 89:5336./ß in the intestinal epithelium. J. Exp. Med. 178:1947.This article has been cited by other articles:
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B. L. Shacklett, C. A. Cox, M. F. Quigley, C. Kreis, N. H. Stollman, M. A. Jacobson, J. Andersson, J. K. Sandberg, and D. F. Nixon Abundant Expression of Granzyme A, but Not Perforin, in Granules of CD8+ T Cells in GALT: Implications for Immune Control of HIV-1 Infection J. Immunol., July 1, 2004; 173(1): 641 - 648. [Abstract] [Full Text] [PDF]
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L. Saurer, I. Seibold, S. Rihs, C. Vallan, T. Dumrese, and C. Mueller Virus-Induced Activation of Self-Specific TCR{alpha}{beta} CD8{alpha}{alpha} Intraepithelial Lymphocytes Does Not Abolish Their Self-Tolerance in the Intestine J. Immunol., April 1, 2004; 172(7): 4176 - 4183. [Abstract] [Full Text] [PDF]
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L. Musey, Y. Ding, J. Cao, J. Lee, C. Galloway, A. Yuen, K. R. Jerome, and M. J. McElrath Ontogeny and Specificities of Mucosal and Blood Human Immunodeficiency Virus Type 1-Specific CD8+ Cytotoxic T Lymphocytes J. Virol., December 16, 2002; 77(1): 291 - 300. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), CD8
), and CD8