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* Department of Molecular and Cell Biology and Cancer Research Laboratory, University of California, Berkeley, CA 94720;
Department of Microbiology, University of Alabama, Birmingham, AL 35294; and
Emory Vaccine Center, Emory University, Atlanta, GA 30322
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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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Although termed "NK cell receptors," the expression of many of these molecules is not restricted to NK cells. We and others (3, 4) found that a significant fraction of murine CD8+ T cells in the periphery express inhibitory members of the Ly49 receptor family. By several criteria these cells appear to be memory CD8+ T cells (3), but it remains unclear under what circumstances Ly49 receptor expression is initiated. A subset of these cells also shows variable expression of other NK cell receptors, such as the CD94/NKG2A receptor (5), KLRG1 (6, 7), CD49b (recognized by mAb DX5), and NKR-P1 family members (3, 8). Similarly, NK cell receptors are expressed by some human CD8+ T cells, and evidence indicates that signaling by these receptors can modulate Ag-specific T cell responses (9, 10).
It is commonly hypothesized that CD8+ T cells
initiate the expression of NK cell receptors during an immune response
to fine-tune the TCR-mediated response. Indeed, several NK cell
receptors have been recently shown to be up-regulated on murine T cells
during viral infections. Looking specifically at the populations of
CD8+ T cells responding to different viruses, the
majority of these cells expressed high levels of KLRG1 (6)
and CD94/NKG2 (11), and 12–40% of these cells expressed
modest levels of NKR-P1 and CD49b (8, 12). The stimulatory
NKG2D receptor is also induced in all Ag-specific murine
CD8+ T cells following in vitro activation
(13) or viral infection (14). Whether
infection leads to up-regulation of Ly49 family members is not as
clear-cut. Only 
2% of lymphocytic choriomeningitis virus
(LCMV)3-specific
CD8+ T cells in infected mice express Ly49G2
(15), which is similar to the percentage of
Ly49G2+CD8+ T cells in
uninfected mice. However, a significantly larger population of
Ly49+CD8+ T cells was
reported in mice infected with influenza virus (12).
In this report, we document the expression of several murine NK cell receptors in Ag-specific CD8+ T cells responding to in vivo infections, and we explore the conditions under which receptor expression is initiated. We found that expression of the CD94/NKG2A receptor could be induced in CD8+ T cells under many circumstances, such as during viral and bacterial infections, during in vitro stimulation, in the absence of IL-15, and even during T cell proliferation in the absence of cognate Ag. Additional experiments addressed the effects of CD94/NKG2A receptor ligation on Ag-driven T cell activation and effector functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J (B6, H-2b), BALB/cJ (BALB/c, H-2d), and B6-Rag-1-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B6-Ly5.1 congenic mice (catalog name, B6-Ly5.2/Cr) were purchased from Charles River Laboratories (Frederick, MD). CD94 transgenic mice (16), OT-1 TCR-transgenic mice (17), Db-/- mice (18), and IL-15-/- mice (19) were all maintained on the B6 background.
NKG2A transgenic mice will be described in greater detail elsewhere
(A. M. Jamieson and D. H. Raulet, unpublished data). In
brief, NKG2AB6 cDNA was cloned into the class I
promoter/Igµ enhancer expression cassette (20).
Transgenic founders were backcrossed to B6 at least four times, and
mice were crossed to CD94 transgenic mice to yield CD94/NKG2A
double-transgenic mice. Double-transgenic mice expressed the CD94/NKG2A
heterodimer on the surface of all NK cells and T cells (see Fig. 8
A and data not shown); these cell populations appeared to
develop normally, although peripheral CD8+ T cell
numbers were reduced to 60% of normal.
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LCMV Armstrong CA1371 strain and clone 13 variant stocks were grown in BHK-21 cells and quantitated by plaque assay on Vero cell monolayers as previously described (21). For acute infections mice were injected i.p. with 2 x 105 PFU of LCMV Armstrong. Chronic LCMV infections were established by injecting mice i.v. with 2 x 106 PFU of LCMV clone 13. In some cases, mice were rechallenged i.v. with 3 x 106 PFU LCMV Armstrong at least 2 mo after clearance of the primary infection.
For primary Listeria infections, BALB/c mice were injected i.v. with 3 x 103 CFU of Listeria monocytogenes 10403S (kindly provided by D. Portnoy, University of California, Berkeley, CA). In some cases, mice were rechallenged i.v. with 105 CFU at least 1 mo after clearance of the primary infection.
Generation of 10D7 mAb and MHC class I tetramers
10D7 mAb (Syrian hamster IgG) was generated using protocols
described previously (22). Briefly, hamsters were
immunized five times in the footpad with activated NK (day 10 A-LAK)
cells from B6 mice. Popliteal lymph node cells were fused with the
P3 x 63-Ag.8.653 murine myeloma (TIB-9; American Type Culture
Collection, Manassas, VA), and
hypoxanthine/aminopterin/thymidine-resistant hybridomas were
screened for Abs specific for NK cell surface proteins. 10D7 mAb was
purified from supernatants by 45% ammonium sulfate precipitation
followed by size exclusion chromatography. The following tetrameric MHC
class I-peptide complexes were produced as described previously in
Escherichia coli in association with human
2-microglobulin (23, 24, 25):
Db/gp33–41 (LCMV),
Db/nucleo-protein
(NP)396–404 (LCMV), and
Kd/listeriolysin
O91–99 (Listeria).
Monomeric complexes were tetramerized by the addition of
PE-streptavidin (Molecular Probes, Eugene, OR).
Ab staining
Labeled mAbs specific for the following molecules were
purchased: Ly5.1, Ly5.2, NKR-P1c (NK1.1), CD49b (DX5), CD19,
Qa-1b, CD4, TCR V
2, CD3
, TCR, and
TNF- (BD PharMingen, San Diego, CA); and CD8 and CD44 (Caltag
Laboratories, Burlingame, CA). Additional anti-NK receptor mAbs
were prepared in our laboratory: NKG2A/C/E (20d5) (26),
NKG2AB6 (16a11) (16), KLRG1 (2F1)
(22), Ly49C/F/H/I (14B11) (27), Ly49A
(JR9-318) (28), and Ly49G2 (4D11) (HB-240; American Type
Culture Collection). These mAbs were used as direct conjugates to FITC,
except for the 20d5-biotin conjugate used in Fig. 4A. Before
staining with labeled mAbs, cells were preincubated for 20 min with
2.4G2 (HB-197; American Type Culture Collection) hybridoma supernatant
to block Fc
II/III receptors. Flow cytometry was performed on EPICS
XL-MCL machines (Coulter, Hialeah, FL) and data were analyzed on FlowJo
software (Tree Star, San Carlos, CA).
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Donor cells (B6 splenocytes) were enriched for naive
CD8+ T cells by passage over nylon wool followed
by mAb staining and removal of CD44high and
CD94/NKG2+ cells using an autoMACS magnetic cell
sorter (Miltenyi Biotec, Auburn, CA). Postsort
CD8+ T cells (40% of total) were >95%
CD44low and >99%
CD94/NKG2-. A total of 5 x
106 donor cells were injected i.v. into
lymphopenic hosts (B6-Rag-1-/- mice) or into
nonlymphopenic hosts (unirradiated B6-Ly5.1 mice). Splenocytes from at
least three mice of each group were analyzed by flow cytometry on day
21. Where applicable, anti-Ly5.2 mAb (B6 specific) or
anti-Ly5.1 mAb (B6-Ly5.1 specific) was used to distinguish donor
from host cells.
CTL lines and effector function assays
Derivation of the cloned CTL line 30NX/B10-1 has been described (29). A CTL line specific for OVA peptide OVAp (SIINFEKL) in the context of Kb was established by culturing splenocytes from OT-1 TCR-transgenic mice with irradiated B6 splenocytes pulsed with 1 µM OVAp peptide. In some experiments, nylon wool nonadherent OT-1 splenocytes were first labeled with the fluorescent intracellular dye CFSE (Molecular Probes) by culturing the cells (5 x 106/ml) for 10 min at 37°C in PBS containing 0.1 µM CFSE. CTL were restimulated weekly, supplementing the culture with 5% T-STIM supernatant (BD Biosciences, Franklin Lakes, NJ) during the third stimulation and thereafter. A CTL line specific for LCMV gp276 peptide (SGVENPGGYCL) in the context of Db was established in a similar manner from B6 mice infected 8 days before with LCMV Armstrong. CTL lines expressed uniformly high surface levels of CD94/NKG2A and were used as effectors 5–8 days after restimulation.
Con A blast targets were made by culturing B6 or
B6-Db-/- splenocytes for 24 h with 2
µg/ml Con A, followed by Con A neutralization with methyl
-mannopyranoside. Targets were radiolabeled with
51Cr for 1 h and were isolated with
Histopaque 1119 (Sigma-Aldrich, St. Louis, MO). Standard 4-h
51Cr release assays were performed to measure CTL
activity of the OT-1 T cell line, using an E:T ratio of 4:1 and the
indicated concentrations of peptides OVAp, N6 (SIINFNKL), or G4
(SIIGFEKL).
Direct ex vivo CTL activity was measured using splenocytes from B6 mice infected with LCMV (Armstrong) 8 days previously. Peptide-loaded EL-4 cells and EL-4/Qa-1 transductants were used as target cells at an E:T ratio of 12:1. Standard 4-h 51Cr release assays were performed in the presence of the indicated concentrations of LCMV gp33 peptide (KAVYNFATM).
EL-4/Qa-1 stimulators were generated by subcloning
Qa-1b cDNA into a MSCV-IRES-GFP retroviral
vector, transducing the EL-4 lymphoma (H-2b,
TIB-39; American Type Culture Collection) and using a cell sorter to
isolate clones with high surface expression of
Qa-1b, using methods described previously
(13). To measure CTL cytokine secretion, stimulator cells
were preincubated for 3 h in 96-well plates (5 x
105 cells/well) with the indicated concentrations
of gp276 peptide, followed by three washes to remove free peptide. In
some cases, stimulator cells were preincubated with 50 µM Qdm peptide
for 12 h before addition of gp276 peptide to maximize Qa-1/Qdm
presentation. A total of 2 x 105
gp276-specific CTL were added to each well of Ag-pulsed
stimulators. Cells were cultured for 5 h in the presence of
brefeldin A, followed by cell staining for CD8 and intracellular
TNF- using the Cytofix/Cytoperm Plus kit (BD PharMingen).
T cell activation assay
Stimulator splenocytes were irradiated (2500 cGy) and incubated in 96-well plates (106/well) with the indicated concentrations of staphylococcal enterotoxin B (SEB; Toxin Technology, Sarasota, FL) for 3 h at 37°C, followed by three washes to remove unbound SEB. Enriched T cells (nylon wool nonadherent spleen and lymph node cells) from CD94/NKG2A double-transgenic mice or nontransgenic littermates were labeled with CFSE (as above) and 3 x 105 responders were added to each well of stimulator cells. After culturing for 12 h, CD69 surface expression on responder CD4+ T cells was measured by flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Acute infection with LCMV (Armstrong strain) triggers a massive expansion of CD8+ T cells specific for viral epitopes, and these cells are crucial for clearance of the virus (30). Using class I tetramers folded with LCMV-derived peptides (24), we directly visualized LCMV-specific CD8+ T cells from B6 mice and assessed the expression of various NK cell receptors during and after viral infection.
Strikingly, LCMV infection induced the expression of the inhibitory
CD94/NKG2A receptor in essentially all CD8+ T
cells specific for an immunodominant viral epitope (Fig. 1). Most CD8+ T
cells not specific for this epitope also up-regulated CD94/NKG2A. It is
likely that these cells are also responding specifically to the virus,
as it has been shown that at the height of the immune response >50%
of peripheral CD8+ T cells are LCMV specific
(24). In addition, although cytokines induced by
infectious agents trigger the Ag-independent "bystander
proliferation" of memory CD8+ T cells
(31), we found that poly(I:C)-induced bystander
proliferation did not trigger up-regulation of any of the NK cell
receptors studied (data not shown).
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, A and B) and the
CD94-specific mAb 18d3 (data not shown). These results confirm that the
inhibitory CD94/NKG2A heterodimer is up-regulated by virus-specific CTL
but do not eliminate the possibility that stimulatory NKG2C or -E
receptors may also be expressed. To address this issue, a quantitative
assay taking advantage of gene-specific restriction enzymes
(26) revealed that NKG2A message represents >90% of the
total NKG2 mRNA in virus-specific CTL (data not shown).
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Surface expression peaked on day 9 postinfection, which corresponds to
the height of the CTL response, and then remained at this level for at
least a month (Fig. 2A). This level of surface expression
was similar to, but slightly lower than, that observed on the surface
of CD94/NKG2A+ NK cells (data not shown).
LCMV-specific memory T cells continued to express the CD94/NKG2A
receptor 6 mo after infection; however, the surface expression levels
gradually decreased on long-term memory cells (Fig. 2A).
Maximal surface expression on memory CD8+ T cells
was restored upon secondary LCMV infection (Fig. 2A).
A recent report demonstrated that many CD8+ T
cells during acute viral infections express the KLRG1 NK cell receptor
(6). Similarly, we observed that KLRG1 was expressed at
high levels on 86% of gp33-specific CD8+ T
cells on day 9 postinfection (Fig. 1). In addition, the percentage of
KLRG1-bearing memory cells gradually decreased over time (Fig. 1 and
data not shown), indicating a down-regulation of surface expression or
a selective loss of KLRG1+ memory cells.
Interestingly, KLRG1 expression on gp33-specific
CD8+ T cells was bimodal, with a distinct
population of cells remaining KLRG1 low or negative. This differed from
the expression of CD94/NKG2A (Fig. 1) and 10D7 (described below in this
section), which appeared to be homogeneously expressed by almost
all Ag-specific cells.
The expression of Ly49 receptors was also examined. Each of the
inhibitory Ly49 family members is expressed on 4–25% of memory
phenotype CD8+ T cells in uninfected B6 mice
(3). During LCMV infection, a small fraction of
virus-specific CD8 T cells was found to express the inhibitory
receptors Ly49G2 (15), -A, -C, -F, or -I (Fig. 1 and data
not shown). For each Ly49 molecule, this fraction is similar to (or
lower than) the fraction of memory CD8+ T cells
found in uninfected mice expressing the same receptor. Thus, only a
small percentage of LCMV-specific effector cells express Ly49
receptors, and this percentage does not change significantly in the
memory population (Fig. 2A).
We have generated a novel mAb, 10D7, that recognizes a cell surface
molecule expressed by essentially all NK cells (Fig. 3A) and memory
CD8+ T cells (Fig. 3B). Low or
negligible levels of 10D7 were found on the cell surface of naive
CD8+ T cells, CD4+ T cells,
and B cells (Fig. 3B). We found that LCMV infection induced
a striking up-regulation of the 10D7 receptor on all gp33-specific CD8
T cells (Fig. 1). 10D7 surface expression peaked slightly earlier than
that observed for the CD94/NKG2A receptor (day 7 vs day 9) and began to
gradually decrease immediately thereafter (Fig. 2A). The
identity of the protein recognized by the 10D7 mAb is as yet unknown;
however, we have excluded many NK cell receptors and markers of T cell
activation (see Discussion).
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B). Notably, cell
surface expression of these receptors appeared to remain high on
Ag-specific T cells 101 days after LCMV clone 13 infection. The same NK
cell receptors were up-regulated in CD8+ T cells
responding to Listeria infection (Fig. 2C), with
kinetics similar to what was observed during LCMV Armstrong infection.
The surface expression levels of each of these receptors during
Listeria infection were significantly lower than during LCMV
Armstrong infection; however, high-level surface expression did occur
following secondary infection (Fig. 2C). Thus, both viral
and bacterial infections induce up-regulation of these NK cell
receptors on Ag-specific CD8+ T cells. Naive CD8+ T cells initiate CD94/NKG2A expression in vitro
We investigated whether stimulation of naive CD8+ T cells with cognate Ag was sufficient to initiate NK cell receptor expression. CD8+ T cells specific for an OVA peptide (OVAp) in the context of Kb were isolated from B6-OT-1 TCR-transgenic mice (17) and were cultured with OVAp-pulsed stimulator cells. Before culturing, the OT-1 CD8+ T cells were labeled with the intracellular dye CFSE to allow the visualization of cell divisions.
As seen in Fig. 4A, expression
of the CD94/NKG2A receptor was initiated early, at approximately day 2
poststimulation. Cell surface levels of CD94/NKG2A gradually increased
over time, and they increased further following restimulation. Uniform,
high levels of receptor expression were observed after a third in vitro
stimulation (Fig. 4B, left panel). A similar
up-regulation of the 10D7 receptor was observed (data not shown).
Conversely, it has been documented that KLRG1 is not up-regulated in T
cells stimulated in vitro (6, 32).
Interestingly, all OT-1 T cells initiated CD94/NKG2A expression to a
similar degree, independent of the number of cell divisions (Fig. 4A, see day 2 or 3 postinfection). This was true even for
those V2+ cells on day 2 or 3 with the highest
level of intracellular CFSE, which were cells that had not yet divided
(determined by equating to nondividing
CFSE+V2- cells in the
same cultures). Therefore, the onset of CD94/NKG2A expression preceded
cell division. Taken together, these data demonstrate that naive
CD8+ T cells initiate de novo expression of the
CD94/NKG2A receptor following antigenic stimulation. It is very likely
that up-regulation of NK cell receptors occurs on most or all murine
CD8+ T cells restimulated in vitro, as we have
observed analogous expression of CD94/NKG2A and 10D7 on 11 independent
CD8+ T cell lines and clones (Fig. 4B
and data not shown).
NK cell receptor up-regulation occurs in the absence of IL-15
Mice lacking IL-15 or the high-affinity IL-15R have reduced
numbers of NK cells, CD1-restricted NKT cells, and memory phenotype
CD8+ T cells (19, 33), demonstrating
the importance of IL-15 for the normal development or maintenance of
these cell populations. In addition, human CD8+ T
cells up-regulate the CD94/NKG2A receptor upon antigenic stimulation in
vitro, but only upon the addition of exogenous IL-15 or TGF-
(34, 35). Therefore, we addressed the role of IL-15 in the
expression of NK cell receptors on murine CD8+ T
cells.
Examination of uninfected IL-15-deficient mice revealed an almost
complete absence of memory phenotype CD8+ T cells
that express Ly49 family members (Fig. 5A); this population normally
makes up over one-quarter of
CD44highCD8+ T cells in B6
mice (3). The 30-fold decrease in
Ly49+CD8+ T cells far
exceeds the 4-fold decreases in
CD44highCD8+ T cells (Fig. 5A) and CD1-restricted NKT cells (19). We next
infected IL-15-deficient mice with LCMV Armstrong and examined the
expression of NK cell receptors on LCMV-specific
CD8+ T cells on day 9 postinfection. Although the
overall CD8+ T cell expansion in IL-15-deficient
mice was reduced (13.6 ± 7% of total splenocytes compared with
37.8 ± 7% in wild type), the fractions of
CD8+ cells specific for LCMV epitopes gp33 and
NP396 were very similar between IL-15-deficient
and wild-type mice. Unexpectedly, the up-regulation of CD94/NKG2A,
KLRG1, and 10D7 on virus-specific CD8+ T cells
was not significantly affected by the absence of IL-15 (Fig. 5B). These results demonstrate that IL-15 plays a critical
role in the appearance of
Ly49+CD8+ T cells but is
unnecessary for the expression of other NK cell receptors in
CD8+ T cells responding to viral infection.
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It has been demonstrated that naive CD8+ T cells transferred to hosts deficient in lymphocytes, such as sublethally irradiated mice or mice lacking lymphocyte compartments due to depletion or genetic manipulation, will proliferate in response to low-affinity self peptides (36, 37, 38, 39). This T cell activation and expansion in the absence of cognate peptide is hypothesized to be a means of maintaining homeostasis of the T cell compartment. We investigated whether homeostatic proliferation of CD8+ T cells was sufficient to induce the expression of NK cell receptors.
Naive phenotype CD8+ T cells were transferred to
B6-Rag-1-/- hosts, which are lymphopenic due to
a complete absence of B and T lymphocytes. To serve as a negative
control in which T cells do not proliferate or become activated, cells
were also transferred to normal unirradiated mice. At an early time
point (day 6 posttransfer), many donor-derived
CD8+ T cells isolated from lymphopenic recipients
had acquired a CD44high phenotype (37, 38, 40), but NK cell receptor expression on these cells was not
observed (data not shown). However, by day 21 posttransfer a sizable
fraction of donor CD8+ T cells transferred to
B6-Rag-1-/- hosts expressed high levels of NK
cell receptors CD94/NKG2A (an average of 49%) and KLRG1 (an average of
40%) (Fig. 6). It is unlikely that these
cells expanded from preexisting CD94/NKG2A+ or
KLRG1+ T cells, as CD44high
and CD94/NKG2+ cells were selectively removed
from the donor cell population before transfer. 10D7 and Ly49 receptors
did not appear to be significantly up-regulated in
B6-Rag-1-/- hosts, as the modest shifts in
fluorescence seen in these cases (compared with cells from unirradiated
hosts) were similar to the shift seen using an isotype control mAb
(Fig. 6). Thus, homeostasis-driven proliferation can induce CD94/NKG2A
and KLRG1 expression on CD8+ T cells, even
without high-affinity TCR binding.
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Binding of Qa-1 ligand by CD94/NKG2A mediates potent inhibition of
NK cell killing (41). Therefore, we tested whether this
interaction could inhibit peptide-specific CTL cytokine secretion and
target lysis. After repeated in vitro stimulations, OVAp-specific and
gp276-specific CTL lines express uniformly high levels of CD94/NKG2A
(see Fig. 4B). Target cells derived from
Db-deficient mice (Db-/-)
lack the Qdm peptide presented by Qa-1 that is required for CD94/NKG2A
recognition and are therefore expected to not inhibit
CD94/NKG2A+ effector cells (42).
However, CTL specific for OVAp in the context of
Kb lysed OVAp-coated B6 and
Db-deficient lymphoblasts equivalently (Fig. 7A, left panel).
Furthermore, the presence of Qa-1/Qdm ligand did not inhibit lysis of
targets presenting OVAp variants N6 and G4, which are weak agonists of
OVAp-specific CTL (43).
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B,
right panel). Nonetheless, EL-4/Qa-1 transductants
presenting gp276 peptide did not inhibit TNF- production by a
gp276-specific CTL line (Fig. 7B, left panel). In
addition, EL-4 cells and EL-4/Qa-1 transductants were lysed
equivalently by CTL isolated directly from LCMV-infected mice
(Fig. 7C), and similar results were obtained in cytokine
secretion (TNF-, IFN-) and cytolysis assays using the
OVAp-specific CTL line (data not shown). In all assays, preincubating
the Qa-1-bearing cells (B6 lymphoblast targets or Qa-1-transduced
stimulators) overnight with Qdm peptide, which enhances functional
inhibition of NK cells (42), did not result in detectable
CTL inhibition (Fig. 7, B and C, and data not
shown). CD94/NKG2A heterodimers expressed as transgenes can inhibit T cell activation
A transgenic mouse line expressing a NKG2A cDNA construct was
generated and crossed to CD94 transgenic mice (16),
resulting in double-transgenic mice with high-level surface expression
of the CD94/NKG2A heterodimer on all peripheral NK cells and T cells
(Fig. 8A and data not shown).
We tested whether the expression of the CD94/NKG2A transgenes in naive
T cells could inhibit the induction of the early activation marker CD69
following TCR-mediated stimulation. T cells from CD94/NKG2A
double-transgenic mice or nontransgenic littermates were cultured with
stimulator cells coated with SEB. SEB, when presented by class
II+ APCs, polyclonally stimulates
CD4+ T cells that express reactive TCR V
elements (25% of peripheral CD4+ T cells in
B6 mice). However, we found significantly decreased CD69 induction in
double-transgenic T cells responding to B6 APC that express the
Qa-1/Qdm ligand (Fig. 8B). This inhibition of activation
could be attributed to Qa-1 ligand binding by the CD94/NKG2A receptor,
as double-transgenic T cells up-regulated CD69 as well as
nontransgenic T cells in response to APCs that lack Qa-1/Qdm
(Db-/-). Using similar assay conditions, the
CD94/NKG2A transgenes also partially inhibited T cell proliferation,
but this effect was not consistently reproducible (data not shown). It
is notable that the inhibition of T cell activation and proliferation
were relatively modest, especially when considering that the surface
expression of transgenic CD94/NKG2A on T cells is even higher than that
of the endogenous receptor on NK cells (Fig. 8A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is interesting that up-regulation of CD94/NKG2A and 10D7 differs in
several respects from that of the KLRG1 receptor. First, CD94/NKG2A and
10D7 are rapidly induced on naive CD8+ T cells
upon in vitro stimulation (Fig. 4 and data not shown), whereas KLRG1 is
not induced in vitro (32) and more than 10 cell divisions
are required before KLRG1 surface expression is seen in vivo
(6). This demonstrates that unique conditions are required
for KLRG1 up-regulation; conversely, CD94/NKG2A and 10D7 expression
appear to be general features of CD8+ T cell
activation. Second, KLRG1 is not uniformly up-regulated on Ag-specific
CD8+ T cells during infection, as 20% of
activated cells remained KLRG1 negative or low (see Fig. 1). A recent
report demonstrated that sorted KLRG1+ memory T
cells proliferated poorly compared with KLRG1-negative memory cells,
thus correlating KLRG1 expression with senescence (6).
Because the expression patterns of CD94/NKG2A and 10D7 are not bimodal,
these receptors do not likewise segregate functional T cell subsets
during LCMV infection.
Surprisingly, we found that the up-regulation of CD94/NKG2A and KLRG1 receptors can occur on some CD8+ T cells in the absence of stimulation by cognate Ag, during homeostasis-driven proliferation in B6-Rag-1-/- hosts. These data suggest the possibility that NK cell receptors play a role in T cell homeostasis, perhaps by helping to inhibit proliferation once homeostasis is achieved. This hypothesis is currently difficult to test experimentally. It is important to note that we did not observe expression of CD94/NKG2A or KLRG1 receptors on T cells transferred to sublethally irradiated hosts (data not shown). The discrepant results from the two types of lymphopenic mice may be related to the observation that T cells transferred to B6-Rag-1-/- hosts, unlike those transferred to irradiated hosts, continue to proliferate indefinitely (40). Thus, in lymphopenic model systems expression of NK cell receptors may only occur in T cells that have undergone prolonged proliferation or are undergoing rapid proliferation—situations that may not occur in irradiated lymphopenic mice, at least at the time points examined.
We found that the surface expression levels of CD94/NKG2A, KLRG1, and
10D7 were lower during primary Listeria infection, as
compared with acute LCMV infection. This probably does not reflect a
difference between viral and bacterial infections (or B6 vs BALB/c
strain differences), as Listeria rechallenge resulted in
high surface levels of all three receptors. Rather, we favor the idea
that receptor up-regulation is governed by the potency of the
CD8+ T cell response. At the height of the immune
response to LCMV Armstrong, the spleen contains 3 x
107 LCMV-specific CD8+ T
cells (24). In comparison, spleens from
Listeria-infected mice contain 5 x
105 Ag-specific CD8+ T
cells but significantly higher numbers (8 x
106) after rechallenge (25). Thus,
the cell surface levels of induced NK cell receptors may be subject to
the extent and/or speed of T cell proliferation. The gradual increase
of CD94/NKG2A expression in T cells stimulated and restimulated in
vitro (Fig. 4) is consistent with this model. Along these lines, it is
perhaps significant that receptor surface expression remains relatively
high long after infection with the persistent LCMV variant clone 13
(Fig. 2B); this suggests that chronic stimulation may
prevent the gradual receptor down-regulation observed following acute
viral and bacterial infections.
The cell surface receptor recognized by the 10D7 mAb is expressed on
essentially all NK cells in all mouse strains tested (B6, B10, BALB,
SJL, and 129) and is also expressed at slightly lower levels on all
activated and memory (CD44high)
CD8+ T cells (Fig. 3). Tests suggest that the
10D7 epitope is not any of the NK cell-specific Ags previously
characterized. The variegated expression of many NK cell receptors
eliminates them as candidates, such as KLRG1, CD94/NKG2 heterodimers,
and individual Ly49 family members. Cells transfected with other
receptors (NKG2D, NKR-P1a, -b, -c and -d) failed to stain with 10D7,
eliminating them as 10D7 epitopes (data not shown). In addition,
counterstaining NK cells with 10D7 vs individual mAbs to other
candidate receptors (DX5, CD44, IL-2R, FcR, Ly6C, 2B4, etc.) did
not result in the staining pattern observed when a pair of labeled Abs
bind to the same cell surface molecule, i.e., a double-positive
population that clusters along the x = y
diagonal of a two-color flow cytometry plot (Fig. 3A and
data not shown). Thus, the 10D7 epitope may represent a novel NK cell
receptor, and the 10D7high phenotype is a useful
marker for all NK cells and activated CD8+ T
cells in many mouse strains.
We found that IL-15 was dispensable for LCMV-induced up-regulation of
CD94/NKG2A, 10D7, and KLRG1 receptors in T cells (Fig. 5B).
This result was somewhat unexpected, because IL-15 is thought to be
important for NKG2A expression during NK cell development
(46). In addition, it was shown that in vitro stimulation
of human T cells induced CD94/NKG2A expression, but only when exogenous
IL-15 or TGF- was added to the culture (34, 35). These
studies suggest that there are cytokine requirements for CD94/NKG2A
up-regulation, but that there may be some redundancy with respect to
the particular cytokine(s) required. In contrast, we found that in
vitro CD94/NKG2A induction on murine T cells did not require the
addition of any exogenous cytokines (Fig. 4). Although this implies
that murine and human T cells require different conditions for
CD94/NKG2A induction, it is also possible that sufficient levels of the
required cytokine(s) were generated in our cultures to mediate receptor
up-regulation.
It is unknown what circumstances are necessary for the appearance of
the population of Ly49+CD8+
T cells found in normal mice. We found that the large majority of
memory CTLs specific for different viral and bacterial Ags did not
express Ly49 molecules (Figs. 1 and 2). The fact that we did always
observe a small subpopulation of Ag-specific
Ly49+ T cells suggests that Ly49 expression is
not directly linked to TCR specificity, unless the
Ly49+ cells cross-react with undefined Ags. It is
noteworthy that Ly49 receptors were also not significantly up-regulated
in CD8+ T cells during LCMV clone 13 infection
(Fig. 2B). This result argues against the hypothesis that
Ly49+CD8+ T cells may arise
as a result of chronic infections (9). It still may be the
case that Ly49 induction depends upon the nature of the infectious
agent, such as a requirement for a type of Ag presentation that occurs
only with certain routes of infection or infected cell types.
Interestingly, Ly49+CD8+ T cells are completely absent in IL-15-deficient mice. It is possible that IL-15 plays a direct role in Ly49 receptor up-regulation, but this is difficult to test without knowing the conditions that induce Ly49 receptor expression in T cells in vivo. Notably, IL-15 is not sufficient to trigger Ly49 expression in NK cells during in vitro development, but it may be required (47, 48). Alternatively, Ly49+CD8+ T cells may represent a discrete lymphocyte subpopulation that requires IL-15 for development or survival. Finally, it is plausible that the absence of Ly49+CD8+ T cells is due to the apparent defect in memory cell maintenance in IL-15-deficient mice. However, we feel this is unlikely, as IL-15-deficient mice have a reduced but significant number of memory phenotype CD8+ T cells (19), and some LCMV-specific memory cells persist in IL-15-deficient mice following infection (data not shown).
The human CD94/NKG2A receptor can inhibit T cell clones (49, 50). However, how the CD94/NKG2A receptor affects murine T cell
responses in vivo remains more of an open question. We were unable to
inhibit CTL effector functions using presenting cells expressing normal
(or above-normal) levels of the Qa-1 ligand, even at low doses of
antigenic peptide (Fig. 7). We hypothesized that inhibition might only
occur in T cells responding to low-affinity peptides, thus focusing the
T cell response upon high-affinity Ags. However, CD94/NKG2A ligation
did not inhibit the cytolytic response to weak agonists (Fig. 7A). The only situation where we reproducibly observed
CD94/NKG2A-mediated inhibition was in the initial activation of naive T
cells overexpressing CD94 and NKG2A transgenes (Fig. 8).
However, two recent reports have documented CD94/NKG2A-mediated
inhibition of killing by murine CTLs. In one case, noncytotoxic
NKG2+CD8+ T cells isolated
from mice infected with polyoma virus could be induced to lyse target
cells by first blocking and internalizing the Qa-1 ligand
(11). This discrepancy from our results cannot be
explained by differences in CD94/NKG2A levels, as our CTL lines and
CTLs isolated from LCMV-infected mice all expressed uniformly high
levels of CD94/NKG2A on the cell surface (Figs. 1 and 4B).
An alternative possibility is suggested by the finding that polyoma
virus-specific CTLs become cytolytic following in vitro restimulation
(51), which may indicate that the in vivo environment is
necessary for maintaining nonresponsiveness. However, we found that
CTLs isolated from LCMV-infected mice were not inhibited by CD94/NKG2A
(Fig. 7C). Therefore, reconciling the differing results
requires arguing that there is something unique about the
CD8+ T cells in polyoma virus-infected mice that
renders them sensitive to CD94/NKG2A signaling. A second report found
that human target cells expressing very high levels of Qa-1 and very
low levels of class I were killed poorly by an allospecific CTL line
(45). Thus, in this case it appears that significant
inhibition requires a combination of conditions, including low levels
of Ag, high levels of inhibitory receptor ligand, and perhaps a target
cell that does not express murine ligands for costimulatory receptors
(e.g., human cells).
There have been similar difficulties in demonstrating the inhibition of murine CTL functions by other endogenously expressed NK cell receptors, such as KLRG1 (6) and Ly49 molecules (3, 15). However, Ly49A expressed as a transgene by all T cells is a potent inhibitor of CD8+ T cell killing (52, 53). Similarly, a modest inhibition of CTL killing was observed upon cross-linking of transgenically expressed KLRG1 (7). Thus, these receptors clearly have the potential to dampen TCR-mediated responses, but the results taken together suggest that significant inhibition in vivo may occur only under favorable circumstances, such as high receptor and ligand levels combined with weak antigenic stimulus. Indeed, perhaps simultaneous signaling from multiple inhibitory receptors is necessary to overcome TCR-mediated activation. Because we have found that CD94/NKG2A induction is a general consequence of CD8+ T cell activation, it seems likely that the state of CTL nonresponsiveness found in polyoma virus-infected mice is maintained by a variety of mechanisms acting in concert, of which CD94/NKG2A-mediated inhibition may be only one. It also is possible that CD94/NKG2A signaling may play a more significant role in modifying non-effector functions such as CD8+ T cell activation, proliferation, or survival. Infectious model systems will prove useful in better describing how NK cell receptors may influence the waxing and waning of the CD8+ T cell immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David H. Raulet, Department of Molecular and Cell Biology, 489 Life Sciences Addition, University of California, Berkeley, CA 94720. E-mail address: raulet{at}uclink4.berkeley.edu
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; SEB, staphylococcal enterotoxin B; MFI, mean fluorescence intensity; NP, nucleoprotein.
Received for publication February 28, 2002. Accepted for publication June 3, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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8.1 transgenic mice. EMBO J. 8:719.[Medline]
TCR population in i-IEL with an extrathymic maturation. Int. Immunol. 4:699.-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. Eur. J. Immunol. 29:23.[Medline]
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and 
) or Db-/- mice
(Qa-1/Qdm-, 
and •) were preincubated with the
indicated concentrations of SEB. Responder T cells from
CD94/NKG2A double-transgenic mice (• and 
