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Department of Pathology, University of Massachusetts Medical Center, Worcester, MA 01655;
Department of Pathology, University of Texas, Southwestern, Dallas, TX; and
Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent, perforin- and
Cmv-1-independent process, and each NK cell subset
mediated anti-viral activity. In adult C57BL/6 mice, the control of
MCMV in the spleen is mediated by a perforin-dependent mechanism,
regulated in part by the Cmv-1 gene, which maps closely
to the Ly49 family. In vivo depletions of either one or two of the Ly49
subsets in adult mice did not affect the ability of the residual NK
cells to regulate MCMV synthesis. These data provide evidence of NK
cell subset distribution and function in MCMV infection, but no
individual subset was required for the Cmv-1-like
regulation of MCMV synthesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is a non-MHC linked resistance gene to MCMV that maps very closely to the NK1.1 and Ly49 loci within the NK gene complex (8, 9, 10). This gene, designated Cmv-1, confers resistance to MCMV in the spleen but not in the liver (8). The effects of Cmv-1 are mediated through NK1.1+ cells, and mice that have the gene (Cmv-1r) have lower splenic MCMV titers than strains of mice that do not have the gene (Cmv-1s) (9). This organ-dependent genetic resistance is clearly linked to Cmv-1 in the NK gene complex, as Cmv-1s BALB/c mice, when made congenic with the Cmv-1r C57BL/6 NK gene complex, have their resistance to MCMV changed from a Cmv-1s phenotype to a Cmv-1rphenotype (11). Furthermore, this organ-dependent genetic susceptibility to MCMV may reflect the different NK cell-dependent control mechanisms in the two organs.
Three days post-MCMV infection, NK cells in C57BL/6 mice control MCMV
infections predominantly by a perforin-dependent, IFN--independent
mechanism in the spleen and by a perforin-independent,
IFN--dependent mechanism in the liver (12). Close linkage of the
Cmv-1 gene to the Ly49 receptor family suggests that
Cmv-1 may be an existing or undefined member of the Ly49
multigene family (13). The Ly49 NK cell receptor family belongs to a
group of transmembrane proteins that share common properties: dimeric
type II transmembrane proteins whose extracellular domains have
structural features of calcium-dependent (C-type) lectins. Most of the
Ly49 NK cell receptors have been shown to bind to MHC class I molecules
(14, 15, 16, 17). The interactions between Ly49A, C, or G2 with their
respective MHC class I ligands have an inhibitory effect on the NK
cells, whereas the Ly49D NK cell receptor activates the NK cells
(18, 19, 20).
Members of the Ly49 family have physiological roles in bone marrow transplants, especially in the phenomenon of F1 hybrid resistance (18, 21, 22), but the role of Ly49 NK cell receptors in the regulation of virus infections is not known. Recently, it was demonstrated that MCMV, like human CMV, encodes a MHC class I homologue that may interfere with the ability of NK cells to regulate the virus (23, 24). It is possible that MCMV may exploit the inhibitory effects on NK cells caused by the binding of Ly49 NK cell receptors to class I molecules by presenting pseudo class I molecules to the NK cells.
To address this possibility, we examined 1) the distribution of Ly49 NK cell receptors in the spleens and peritoneal cavities of uninfected and 3-day MCMV-infected C57BL/6 mice, 2) the mechanisms of the control of MCMV in C57BL/6 suckling mice adoptively reconstituted with NK cells, 3) the activity of NK cells of different Ly49 subsets to protect the suckling mice, and 4) the regulation of MCMV infections in adult C57BL/6 mice treated with mAbs to the different members of the Ly49 family.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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YAC-1, a highly NK-sensitive lymphoma line, was cultivated in suspension in RPMI 1640 (Sigma, St. Louis, MO), supplemented with 10% FBS, L-glutamine, and antibiotics. Mouse embryonic fibroblasts (MEF) from C57BL/6 mice and vero cells (monkey kidney cells) were cultivated as monolayers in MEM (Life Technologies, Grand Island, NY) with L-glutamine, antibiotics, and 20 or 10% FBS, respectively.
Mice
C57BL/6 mice were purchased from The Jackson Laboratory (Bar
Harbor, ME). These mice were conventionally housed, bred in our animal
facility, and used at 6–12 wk of age. C57BL/6 suckling mice were used
at 4–6 days of age in the adoptive transfer experiments. Breeder pairs
of 129 wild-type control and 129 mice homozygous for a targeted
mutation disrupting the mouse IFN- gene (IFN-R0/0)
were originally derived and kindly supplied by Dr. M. Aguet (University
of Zurich, Switzerland) (25). Breeder pairs of 129 x C57BL/6 mice
heterozygous for a targeted mutation disrupting the gene for perforin
were originally derived and kindly provided by Drs. C. M. Walsh
and W. R. Clark (University of California, Los Angeles, CA) (26).
F1 offspring of the above heterozygous breeding were typed
using PCR (12), and perforin-deficient (perforin 0/0) mice were then
bred homozygously. Both perforin 0/0 and IFN-R0/0 mice
and their respective wild-type controls were housed in a specific
pathogen-free environment and were used at 6–12 wk of age.
Virus
The Smith strain of MCMV was a salivary gland-passaged virus stock prepared in BALB/c mice. MCMV was titrated by plaque assay on MEF and was given to adult and suckling mice i.p. at a dose of 1–2 x 104 PFU/mouse or 2 x 103 PFU/mouse, respectively, 3 days before use. The Armstrong strain of lymphocytic choriomeningitis virus (LCMV) was propagated in BHK21 cells (1). LCMV was titrated by plaque assay on vero cells and was given i.p. at a dose of 5 x 104 PFU/mouse 3 days before use.
Cytotoxicity assay
Standard 4–6 h 51Cr release microcytotoxicity assays were used to determine NK cell activity on YAC-1 targets (1). YAC-1 cells were used at 5 or 10 x 103 targets/well and at a variety of E:T ratios. All microcytotoxicity assays were performed using U-bottom 96-well plates (Falcon, Lincoln Park, NJ).
Immune reagents
The anti-NK1.1 mAb, PK136 (provided by Dr. G.C. Koo, Merck Sharpe and Dohme Research Laboratories, Rahway, NJ) (27), was produced in ascites, NH4SO4-cut and affinity-purified before use. PK136 was inoculated into mice i.v. at a dose of 200 µl of a 1:40 dilution/mouse, 24 h before the day of infection. NH4SO4-cut anti-Ly49G2 ascites, 4D11 (Hazelton Technologies, Vienna, VA), was given i.v. at a dose of 200 µg/mouse 1 day before virus infection. NH4SO4-cut anti-Ly49D ascites, 12A8, was given i.v. at a dose of 300 µg/mouse at day 2 and day 1. NH4SO4-cut anti-Ly49C ascites, 5E6, and anti-Ly49A (A1) ascites were given i.v. at a dose of 2 mg/mouse and in 100 µl of a 1:2 dilution of ascites/mouse, respectively, 1 day before virus infection. (We have recently found that the 5E6 anti-Ly49C Ab also detects a determinant on Ly49I, and results should be interpreted accordingly.) These anti-Ly49C and anti-Ly49G2 Abs were titrated in vivo by measuring the rejection of parental bone marrow cells by Ly49C+ and Ly49G2+ cells in F1 (b x d) mice (18, 19). The mAb A1 directed against Ly49A was titrated by assessing the depletion of Ly49A+ cells by flow cytometry (28). Anti-Ly49D Abs were also previously titrated in vivo by our collaborators to ensure that the correct doses were used to deplete in vivo the respective Ly49 NK cell subsets (20).
Immunofluorescence
Phycoerythrin-labeled anti-NK1.1 mAb, FITC-labeled anti-Ly49A mAb, and FITC-labeled Ly49C mAb were purchased from PharMingen (San Diego, CA). These Abs were used according to manufacturer’s specifications. FITC-labeled anti-Ly49D (12A8) and FITC-labeled anti-Ly49G2 (4D11) were used at 50 µl of 1:80/106 cells and 50 µl of 1:40/106 cells, respectively. The anti-Ly49D (12A8) Ab used in the in vivo depletion studies and the adoptive transfer studies was cross-reactive with the Ly49A NK cell receptor (20). However, a different FITC-labeled anti-Ly49D Ab (4E5), which was not cross-reactive with Ly49A receptor, was used in the study of NK cell distribution. This Ab was used at a concentration of 50 µl of a 1:1000 dilution/106 cells. The significance of differences in proportions of Ly49 subpopulations within NK1.1+ cells was evaluated by the ANOVA test.
Generation of LAK cells
Six- to eight-week-old C57BL/6 spleens were asceptically excised, and a single-cell suspension was prepared using RPMI 1640 (Sigma) supplemented with 10% FBS, L-glutamine, antibiotics, nonessential amino acids, sodium pyruvate, and 2-ME (MLC media). Splenic leukocytes were washed and incubated on a nylon wool column for 45 min at 37°C. Nylon wool nonadherent cells were then cultured in MLC media supplemented with 800 U/ml of recombinant human IL-2 (Cetus Corporation, Emeryville, CA) at a density of 2 x 106 cells/ml in 60 x 15 mm tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ). After the initial 3 days of culture, the adherent cells were expanded for another 4–6 days before use.
Adoptive transfers
Adoptive transfer experiments were done by using a modification of a method previously described (6). Briefly, 4- to 6-day suckling mice were pooled and randomly assigned to lactating females. Groups of four to nine mice were given 5 x 105 LAK cells/mouse or 5 x 107 mouse spleen cells/mouse i.p. in 0.1 ml of media using a 1-ml syringe and a 30-gauge needle. The following day, the suckling mice were challenged with MCMV i.p. Three days postinfection, the mice were sacrificed, and the spleens were removed for virus titration.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine if there were any differences in the distribution of
the NK cell subsets before and after MCMV infection, spleen cells from
uninfected and 3-day MCMV-infected C57BL/6 mice were stained for NK1.1
and for Ly49A, C, D, and G2 NK cell receptors. The virus dose used in
all the experiments was shown previously to be sufficient to activate
the NK cells (12). Compared with uninfected mice, MCMV infection did
not alter the number of spleen leukocytes (data not shown). However,
FACS analyses based on 104 gated lymphocytes showed that
there were fewer NK1.1+ cells (two- to threefold decrease)
in the spleens of MCMV-infected mice compared with that of uninfected
mice (Fig. 1
and Table I). Analyses of the Ly49 NK cell subset
distribution within the splenic NK1.1+ population per
104 lymphocytes before and after MCMV infection revealed
that there were increases in the percentages of Ly49A+
(Expt. 1, 15–17%; Expt. 2, 19–25%; Expt. 3, 17–51%; Expt. 4,
20–27%; p = 0.2) and Ly49G2+ (Expt. 1,
39–54%; Expt. 2, 43–55%; Expt. 3, 57–70%; Expt. 4, 44–58%;
p = 0.001) NK cell subsets and decreases in the
percentages of Ly49C+ (Expt. 1, 45–23%; Expt. 2,
40–16%; Expt. 3, 48–30%; Expt. 4, 41–31%; p =
0.008) and Ly49D+ (Expt. 1, 45–40%; Expt. 2, 42–29%;
Expt. 3, 61–45%; Expt. 4, 45–39%; p = 0.03) NK cell
subsets (see Fig. 3). This pattern of the Ly49 subsets was not observed
at 8 days post-MCMV infection (data not shown), and control mice
injected with uninfected salivary gland extract also did not produce
any of the changes seen in 3-day MCMV-infected mice (data not shown).
These results suggest that, compared with uninfected mice, 3-day MCMV
infection of C57BL/6 mice resulted in a decrease in the total number of
NK1.1+ cells and changes in the percentages of
Ly49+ NK cell subsets within the spleen.
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Analyses of the NK1.1+-Ly49+ NK cell
subsets in the spleen indicated that even though the absolute number of
splenocytes was similar between uninfected and infected mice, there
were decreases in the number of NK1.1+ cells and changes in
the proportion of the NK1.1+-Ly49+ subsets
after MCMV infection. To examine if there were any changes in the NK
cell population in the peritoneal cavity, peritoneal exudate cells
(PEC) from uninfected and 3 day MCMV-infected mice were stained with
Abs to NK1.1, Ly49A, C, D, and G2 NK cell receptors. Compared with the
uninfected controls, there was usually about a twofold increase in the
absolute number of PEC in MCMV-infected mice (data not shown). FACS
analyses showed that there were significantly more NK1.1+
cells per 104 lymphocytes (two- to fourfold increase) in
the peritoneal cavity after infection (Table I
and Fig. 2). These data suggest that either there
is an influx of NK1.1+ cells into the peritoneal cavity
during MCMV infection or else the resident NK cells are proliferating.
FACS analyses of the Ly49 NK cell subsets within the NK1.1+
population showed that there was a decrease in the percentage of
Ly49A+ (Expt. 1, 34–26%; Expt. 2, 36–32%; Expt. 3,
45–21%; Expt. 4, 37–23%; p = 0.06) cells while
there were increases in the percentages of Ly49C+ (Expt. 1,
34–46%; Expt. 2, 44–52%; Expt. 3, 30–46%; Expt. 4, 51–52%;
p = 0.06), Ly49D+ (Expt. 1, 24–50%; Expt.
2, 37–52%; Expt. 3, 26–53%; Expt. 4, 41–52%; p =
0.02), and Ly49G2+ (Expt. 1, 46–61%; Expt. 2, 47–57%;
Expt. 3, 46–60%; Expt. 4, 53–62%; p = 0.006) cells
(Fig. 3). Again, this specific
distribution of the Ly49 subsets 3 days postinfection was not observed
at 8 days after MCMV infection (data not shown), nor was it seen in
control mice injected with salivary gland extract from uninfected mice
(data not shown).
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, which indicates that 3 days
post-MCMV infection there was within the NK1.1+ population
an increase in the percentage of Ly49A+ cells, while there
were decreases in the percentages of Ly49C+ and
Ly49D+ cells, in the spleen. This was accompanied by the
concomitant decrease in the percentage of Ly49A+ PECs and
increase in the percentage of Ly49C+ and Ly49D+
PECs. In both the spleen and the peritoneal cavity, the percentage of
Ly49G2+ cells increased after MCMV infection. Distribution of NK1.1+-Ly49+ NK cells in the spleen and peritoneal cavity in uninfected and 3-day LCMV-infected C57BL/6 mice
To determine whether the above mentioned pattern of NK cell
accumulation only occurred during MCMV infection, C57BL/6 mice were
infected with a different virus, LCMV, and spleen cells and PECs were
then stained with the various NK cell receptor Abs. There were few
differences in the absolute number of splenic leukocytes and PECs from
3-day LCMV-infected mice compared with uninfected mice (data not
shown). FACS analyses also showed that there were few differences in
the number of NK1.1+ cells in the spleen (Table I), and in
only one of three experiments was there an increase in
NK1.1+ cells per 104 lymphocytes in the
peritoneal cavity (Table I). The distribution of the various Ly49 NK
cell receptors in the spleen and the peritoneal cavity did not follow a
reproducible pattern after 3 days of LCMV infection (Fig. 4). There were few to no changes in the
percentages of Ly49A+, Ly49C+, and
Ly49D+ cells in the spleen, while the changes in the
percentage of these three (A, C, and D) Ly49 NK cell subsets were
erratic in the peritoneal cavity. However, there was an increase in the
percentage of Ly49G2+ NK cell subsets in both the spleen
and the peritoneal cavity (spleen: Expt. 1, 39–62%; Expt. 2,
43–58%; Expt. 3, 36–58%; p = 0.02; and peritoneal
cavity: Expt. 1, 46–60%; Expt. 2, 47–48%; Expt. 3, 45–58%;
p = 0.2) (Fig. 4). Thus, the distribution of Ly49 NK
cell subsets after MCMV infection is distinct for that particular
NK-sensitive virus infection, thereby suggesting that the Ly49 NK cell
subsets may play a role in the regulation of MCMV in C57BL/6 mice.
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Three-week-old or younger suckling mice are very susceptible
to MCMV infections, because NK cells take about 3 wk from birth to
reach maturity (4, 5). Bukowski et al. have shown that the adoptive
transfer of syngeneic adult spleen cells or LAK cells into suckling
mice can protect them from a subsequent normally lethal MCMV infection
(6). Analyses of splenic MCMV titers indicated that suckling mice
adoptively reconstituted with adult spleen cells have lower MCMV titers
compared with control suckling mice. This protection is mediated by NK
cells, as the depletion of NK cells from the adoptively transferred
splenic population abrogated this protection, and splenocytes depleted
of T cells do confer protection. Bukowski et al. also showed that much
lower cell numbers of NK1.1+ LAK cell populations could
protect suckling mice from MCMV and that neither adult splenocytes nor
LAK cells protected suckling mice from the NK cell-resistant virus,
LCMV (6). To examine the importance of the different Ly49 subsets in
the early regulation of MCMV, we tested the ability of
NK1.1+, Ly49+ LAK cells to protect suckling
mice from an MCMV infection. LAK cells were generated using 6- to
12-wk-old C57BL/6 spleen cells. Fig. 5
shows the typical distribution of the different Ly49 subsets within the
NK1.1+ LAK cell population. The percentages of the
different Ly49 NK cell subsets in the LAK cell cultures were similar to
the percentages seen in spleen NK cells taken from naive C57BL/6 mice
as shown here and previously by others (18, 19, 20, 29). Before adoptive
transfers, the LAK cells were sorted into
NK1.1+-Ly49- or
NK1.1+-Ly49+ for all the four Ly49 subsets (A,
C, D, and G2). These sorted LAK cells were adoptively transferred into
4- to 6-day-old C57BL/6 suckling mice, which were subsequently infected
with MCMV, and splenic MCMV titers were measured 3 days later. Suckling
mice adoptively reconstituted with any of the different combinations of
NK1.1+ LAK cells had lower splenic MCMV titers compared
with the media control (Table II). For
example, both NK1.1+-Ly49A- LAK cells and
NK1.1+-Ly49A+ LAK cells significantly decreased
the amount of MCMV in the spleen of the suckling mice compared with the
media control (Table II, Expt. 2). It should be noted that a hundred
times more naive adult splenocytes is required to see the same level of
protection afforded by LAK cells (6). These results indicate that
subpopulations of NK cells expressing any of the Ly49 NK cell receptors
could protect the suckling mice from MCMV.
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The mechanisms by which adoptively transferred NK cells protect
suckling mice from MCMV had not been previously determined. Experiments
were performed to determine the mechanisms of MCMV control in the
suckling mice and also to see if it was related to the Cmv-1
resistance gene. As shown previously, C57BL/6 leukocytes or C57BL/6 LAK
cells when transferred into the suckling mice significantly lowered
MCMV titers in the spleen compared with the media controls (Table III), and it required 5 x
107 splenic leukocytes but only 3 x 105
LAK cells to mediate protection (6). Interestingly, 129 spleen
cells and LAK cells when adoptively transferred into C57BL/6 suckling
mice also had a protective effect in the spleen compared with the media
controls (Table III, Expts. 1 and 2). These results are surprising in
view of the fact that the 129 strain is Cmv-1s,
and the regulation of spleen MCMV titers in adult C57BL/6 mice is
Cmv-1-dependent (12). Therefore, these data suggest that at
3 days post-MCMV infection, the protection afforded by the Ly49-bearing
LAK cells in the spleens of C57BL/6 suckling mice is not
Cmv-1-dependent.
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Cmv-1-dependent, NK cell regulation of MCMV in the
spleen of adult mice is mediated by a perforin-dependent mechanism
(12). The results presented above suggested that the regulation of
spleen titers of MCMV in the suckling mouse was not
Cmv-1-dependent, but the role of perforin was not evaluated.
Therefore, the abilities of perforin 0/0 spleen cells and perforin 0/0
LAK cells to control MCMV synthesis in the spleens of C57BL/6 suckling
mice were tested. Perforin 0/0 spleen cells or LAK cells when
adoptively transferred into 4- to 6-day-old C57BL/6 suckling mice
significantly lowered MCMV titers in the spleens compared with control
spleens (Table III, Expts. 3–5). The levels of protection afforded by
perforin 0/0 spleen cells and LAK cells were similar to that of C57BL/6
spleen cells and C57BL/6 LAK cells, respectively. Therefore, results
presented in Table IV suggest that
adoptively transferred spleen cells or LAK cells protect C57BL/6
suckling mice from MCMV via a Cmv-1-independent,
perforin-independent mechanism.
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R0/0 mice
NK cells can regulate MCMV in adult mice livers via the production
of IFN- and it was possible that the transferred NK cells controlled
splenic MCMV titers in the suckling mice via the production of IFN-
by the NK cells introduced into the peritoneal cavity. To test for
this, adult C57BL/6 spleen cells were adoptively transferred into 4- to
6-day-old 129 or IFN-R0/0 suckling mice, and their
abilities to reduce MCMV synthesis in the spleens were measured. In
C57BL/6 suckling recipients, adult C57BL/6 spleen cells significantly
reduced splenic MCMV titers compared with infected control spleens
(Table IV, Expts. 1 and 2), but they had no effect on MCMV splenic
titers in MCMV-infected IFN-R0/0 suckling mice. These
data suggest that the control of MCMV synthesis in suckling mice by
adoptively transferred cells is IFN--dependent. Thus, the numerous
experiments listed in Tables III and IV collectively suggest that the
control of spleen MCMV titers in suckling mice by adoptively
transferred NK cells is Cmv-1- and perforin-independent but
dependent on IFN-.
Effects of in vivo anti-Ly49 mAb treatment on the regulation of MCMV in C57BL/6 mice
As the role of the innate resistance gene Cmv-1 has
been best described in the adult mouse/MCMV model (12), adult C57BL/6
mice were used to test the hypothesis that the regulation of MCMV was
dependent exclusively on any one of the Ly49 subsets defined by the
four available Ly49 Abs. Individual Ly49 NK subsets or a combination of
Ly49 subsets were depleted with mAbs in vivo, and NK cell activity and
MCMV titers in the spleen and the liver were then measured in the adult
mice 3 days postinfection. As mentioned in Materials and
Methods, the anti-Ly49C and anti-Ly49G2 Ab stocks were
titrated in vivo by measuring the rejection of parental bone marrow
cells by Ly49C+ and Ly49G2+ cells in
F1 (b x d) mice (18, 19). The anti-Ly49A and
anti-Ly49D Abs were also previously titrated in vivo, and the
depletions of the respective NK cell subsets were checked by FACS
analysis. Depletion of any single Ly49 NK cell subset (Ly49A, Ly49C,
Ly49D, or Ly49G2 alone) had very little effect on NK cell cytotoxicity,
in contrast to the near complete NK cell depletion in MCMV-infected
mice treated with anti-NK1.1 mAb (Fig. 6, A–C). This
suggests that the residual NK cells can rapidly compensate for the
depletion in any NK cell subset after virus infection. When compared
with the infected controls, MCMV-infected C57BL/6 mice treated with
either anti-Ly49A, C, D, or G2 mAbs alone exhibited no increase in
virus titers in the spleen and the liver (Table V, Expts. 1–3). Depletion of two subsets
of Ly49+ NK cells (Ly49C and Ly49D, Ly49C and Ly49G2, Ly49D
and Ly49G2) caused some reduction in NK cell cytotoxicity (Fig. 6, D and E), but these depletions had no effect on
the regulation of MCMV in the spleen and the liver (Table V, Expts. 4
and 5). In two separate experiments, depletion of three
Ly49+ NK cell subsets (Ly49C, Ly49D, and Ly49G2) resulted
in diminished NK cell activity (Fig. 6), but in only one experiment did
such a depletion result in an increase of MCMV titers in the spleen and
in one other experiment it caused an increase in liver titers (Table V,
Expts. 5 and 6). As a positive control in all the experiments, C57BL/6
mice treated with anti-NK1.1 mAb to deplete the NK cells exhibited
the expected increase in MCMV titers in both the spleen and the liver.
These results indicated that in adult C57BL/6 mice the residual NK
cells can compensate for the deletion of any one of the defined subsets
for which Abs are currently available.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), but these changes in NK1.1+ cell
numbers in the spleen and peritoneal cavity were not apparent during
LCMV infection. As i.p. MCMV infection resulted in increases in both
the absolute number of PEC and NK1.1+ cells in the
peritoneal cavity, these results indicate that there either is
proliferation of NK cells at the sites of infection or that there is
trafficking of NK cells to those sites.
There are not only changes in NK cell numbers but also changes in the
Ly49 subsets within the NK population. Even though MCMV did not cause
any apparent change in the absolute number of splenocytes, the virus
infection caused an increase in the percentage of Ly49A and Ly49G2 NK
cell subsets but a decrease in the percentage of Ly49C and Ly49D NK
cell subsets. Interestingly, these changes in the proportion of NK cell
subsets within the spleen are accompanied by decreases in the
percentages of the Ly49A NK cell subset and increases in the
percentages of Ly49C, D, and G2 NK cell subsets in the peritoneal
cavity. As this change of Ly49 subsets only occurs during MCMV
infection but not during LCMV infection, the different Ly49 NK cell
subsets may have different roles in the regulation of MCMV in vivo. The
differences in the Ly49 distribution after MCMV or LCMV infection may
be due to the ability of the virus infection to stimulate different
cytokine profiles. MCMV but not LCMV has been shown to stimulate the
production of IL-12 and IFN- 3 days postinfection (30, 31), and
these cytokines may stimulate the production of chemokines that would
attract the NK cells to the sites of infection. Another possibility is
that the different Ly49 NK cell subsets may recognize MCMV itself or
MCMV peptide/s-modified MHC molecules, such that the NK cells
specifically home to the sites of infection.
Suckling mice less than 2 wk of age can be used as a model to study the role of NK cells in the regulation of MCMV because NK cells take about 3 wk from birth to reach maturity. In this model, spleen cells or culture-purified LAK cells when transferred into the suckling mice protect the mice from a lethal MCMV infection (6). These adoptive transfer studies cannot be performed in adult mice, as these mice would have to be irradiated to destroy the recipient’s immune system before the transfer. This is because resident NK cells in the adult mice have been shown to eliminate adoptively transferred LAK cells, preventing such adoptive transfer experiments to be performed in adult mice (32).
The regulation of MCMV in the spleen of adult mice is Cmv-1-
and perforin-dependent, but in suckling mice, results indicate that
this innate resistance gene and perforin do not play a role in the
regulation of MCMV by adoptively transferred NK cells. In suckling
mice, adoptive transfers of spleen cells or LAK cells derived from
either Cmv-1r (C57BL/6) or
Cmv-1s (129) mice significantly lowered splenic
MCMV titers in infected C57BL/6 suckling mice. Furthermore, perforin
0/0 spleen cells or LAK cells regulated MCMV in the spleens of the
suckling mice, indicating that the regulation of MCMV by the
transferred cells is perforin-independent (Table III). We and other
groups have previously shown that the regulation of MCMV in the liver
by NK cells in adult mice requires IFN- (12, 30). This mechanism is
also used by the transferred cells in suckling mice, as normal C57BL/6
spleen cells, when transferred into IFN-R0/0 suckling
mice, failed to regulate MCMV synthesis in the spleen. These results
suggest that in the suckling mouse model the regulation of MCMV does
not follow the convention seen in adult mice. Adoptively transferred
cells, unlike the NK cells in the adult mouse model, used a
perforin-independent, IFN--dependent mechanism to control MCMV in
the spleens of suckling mice. The adoptively transferred Ly49 NK cell
subsets may have controlled MCMV via an IFN--dependent mechanism.
It may not be surprising that NK cells use different mechanisms to
control MCMV in the spleen of different mouse models. In the suckling
mouse, both the effector cells and the pathogen are introduced into the
peritoneal cavity. As the effector cells are present in the suckling
mice before the introduction of the virus, it is possible that the
control of MCMV may have occurred within the peritoneal cavity, thereby
blocking the migration of the virus into the spleen. Early work by
Bukowski et al. had shown that cultured purified Lyt2+
(CD8+) LAK cells, as well as a T cell clone with NK
cell-like activity, when adoptively transferred into the suckling mice
protected the mice from MCMV infection (6, 7). These non-NK cells are
likely to have secreted IFN- and protected mice by that mechanism.
Although there was a change of the tested Ly49 NK cell subsets in adult
mice during MCMV infection and although individual Ly49 NK cell subsets
could control MCMV synthesis in suckling mice (probably by an IFN-
mechanism), in vivo depletion of any one of the four tested Ly49
subsets did not affect the ability of the NK cells to control MCMV in
adult spleen or liver. To date, there are nine cloned Ly49 NK cell
receptors, but there are only Abs available to five of the receptors.
The anti-Ly49D Ab (12A8) used in the above in vivo depletion and
adoptive transfer studies is cross-reactive with the Ly49A NK cell
receptor such that the depletion of Ly49D-bearing cells will also cause
the depletion of Ly49A+ cells (20). Depleting cells with
anti-Ly49G2 (4D11) Abs will not only deplete the
Ly49G2+ cells but will also inadvertently deplete some
Ly49A+ NK cells (33). This is because 
11% of all NK
cells express both Ly49A and Ly49G2 NK cell receptors (33). However,
the cross-reactivity of anti-Ly49D Ab and the coexpression of Ly49A
and Ly49G2 on some NK cells should not affect our understanding of the
function of Ly49A subset. Depletion of the Ly49A NK cell subset or the
adoptive transfer of the Ly49A+ or Ly49A- LAK
cells alone did not affect the ability of the NK cells to control MCMV
in vivo.
Recently, the anti-Ly49C Ab (SW5E6) used in our analyses was found
to bind not only with Ly49C but also with the Ly49I NK cell receptor.
These data also suggest that in hybrid resistance, it is the
Ly49C+/I+ NK cells and not the
Ly49C+ NK cells that mediate the rejection of
H-2d bone marrow cells (34). Nonetheless, results
from the in vivo depletion of Ly49C- and Ly49I-bearing cells (using
SW5E6, Table II), or the adoptive transfer of Ly49C+,
Ly49I+ or Ly49C-, Ly49I- LAK
cells (Table II) again strongly indicate that the absence or the
presence of a particular Ly49 NK cell subset does not affect the
ability of NK cells to control MCMV.
The Ly49D and Ly49H NK cell receptors are the only members of the Ly49
multigene family to date that do not contain the immunoreceptor
tyrosine-based inhibitory motif (ITIM) motif in its cytoplasmic
tail (20). Ly49D is a putative NK cell receptor, and Ly49D-bearing NK
cells have been shown to have the ability to lyse tumor cells and ConA
blasts of different H-2 haplotypes and mediate reverse
antibody-dependent cell-mediated cytotoxicity (ADCC) through
anti-Ly49D Ab on FcR+ target cells (20). However,
depleting the Ly49D+ NK cell subset or transferring
Ly49D- NK1.1+ LAK cells into MCMV-infected
suckling mice did not affect the NK cells’ ability to control MCMV
(Table II).
At first glance, these results suggest that the interaction of Ly49 molecules with their class I MHC ligands expressed on infected cells are probably not involved in the anti-viral activity of NK cells. In this regard, the results with adoptive transfer of various Ly49+ subset are of particular relevance. For example, the 5E6+ subset receives negative signals from H2Kb; therefore, in uninfected C57BL/6 mice this subset would be prevented from reacting against H2Kb-expressing (self) cells. Following viral infection, MCMV-associated peptides presumably displace the H2Kb-associated self-peptides, and such cells might be expected to be killed because they fail to deliver a negative signal to 5E6+ NK cell subset. This would explain the ability of 5E6+ subset to offer partial protection against MCMV infection. However, in the reported experiments, every subset, including Ly49A, D, and G2, which do not express known H2b receptors, protected as well as the 5E6+ subset. These data are not easily reconciled with the role for Ly49 molecules in the antiviral activity of NK subsets. However, it is conceivable that each of the Ly49 subsets expresses other unknown Ly49 or non-Ly49 receptors that normally inhibit lysis of unmodified cells, but allow the lysis of virus-infected cells due to alterations in class I molecules. Such receptors would have to be expressed on all NK subsets, and may be far less class I-specific than Ly49 molecules. The recently described CD94 molecules would be candidates for such non-Ly49 class I MHC receptors. This receptor is expressed on all human NK cells and seems to have a broad reactivity with MHC molecules, and it is possible that mouse NK cells may also express such molecules that are used to detect virus-infected cells.
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Footnotes |
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2 Current address: Department of Pathology, Tufts University, 136 Harrison Avenue, Boston, MA 02111.
3 Address correspondence and reprint requests to Dr. Raymond M. Welsh, Department of Pathology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655. E-mail address:
4 Abbreviations used in this paper: aGM1, asialo GM1; IFN-0/0, IFN- receptor-deficient; LCMV, lymphocytic choriomeningitis virus; MCMV, murine CMV; MEF, mouse embryonic fibroblast; perforin 0/0, perforin deficient; LAK, lymphokine-activated killer; PEC, peritoneal exudate cell.
Received for publication January 16, 1998. Accepted for publication September 28, 1998.
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receptor. Science 259:1742.
ß TCR population of IEL with an extrathymic maturation. Int. Immunol. 4:699. in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J. Exp. Med. 182:1045.ß, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. J. Immunol. 156:4746.[Abstract]
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), Expt. 2 (
), Expt. 3
(
), and Expt. 4 (
) (1 x 104 PFU MCMV/mouse).
For Expts. 1, 2, and 4, 10,000 events were collected for flow
cytometric analysis, while in Expt. 3, 70,000 events were
collected.
), anti-Ly49D-treated (C, 
),
anti-Ly49G2-treated (C, 
), anti-Ly49C-,
-Ly49D-treated (D, 
), anti-Ly49D-,
-Ly49G2-treated (E, 
) C57BL/6
mice were used as effectors against YAC-1 targets in a standard 5-h
51Cr release assay.