?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
Absence of mouse 2B4 promotes
NK cell–mediated killing of activated
CD8+ T cells, leading to prolonged viral
persistence and altered pathogenesis
Stephen N. Waggoner,1 Ruth T. Taniguchi,2 Porunelloor A. Mathew,3
Vinay Kumar,2 and Raymond M. Welsh1
1Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. 2Department of Pathology, University of Chicago,
Chicago, Illinois, USA. 3Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, USA.
Tolerance of NK cells to self-tissue is predominately maintained
through inhibitory signals derived from interaction of certain
NK cell receptors (e.g., Ly49C) with self class I major histocom-
patibility complex (MHC) molecules (e.g., H-2Kb) (1, 2). How-
ever, MHC-independent inhibitory signals may also contribute
to tolerance, including inhibitory signals provided via inter-
action of CD244 (2B4) with its ligand on hematopoietic cells,
CD48 (3). 2B4 is a member of the signaling lymphocyte activa-
tion molecule (SLAM) receptor family (4–6). Expression of 2B4
is restricted to NK cells, γδ T cells, basophils, monocytes, and a
subset of CD8+ αβ T cells, where both activating and inhibitory
signals have been observed upon 2B4 engagement in vitro (7,
8). The recent generation of 2B4 (CD244)–deficient mice has
established an inhibitory function for this receptor on NK cells
both in vitro and in vivo (9, 10).
Expression of 2B4 on CD8+ T cells strongly parallels that of the T
cell exhaustion marker programmed death 1 (PD-1) and has been
postulated to contribute to the dysfunction of antiviral CD8+ T
cells during persistent viral infection of mice with the clone 13
strain of lymphocytic choriomeningitis virus (LCMV) (11, 12).
Although expression of 2B4 is limited on naive CD8+ T cells and
only transiently upregulated during acute virus infections, sus-
tained high-level expression of 2B4 on virus-specific CD8+ T cells
is characteristic of persistent viral infections in both humans and
mice (12–14). For example, 2B4 is upregulated on CD8+ T cells
from patients with persistent HIV infection (15).
In this study, we sought to determine the role of 2B4 in the
development and functionality of LCMV-specific CD8+ T cell
responses during LCMV infection of WT and 2B4-KO mice. Per-
sistent LCMV infection of 2B4-KO mice resulted in significantly
diminished LCMV-specific CD8+ T cell responses, prolonged
viral persistence, and altered tissue pathology. Surprisingly, this
abnormal phenotype of 2B4-KO mice was not directly related to
2B4 expression by CD8+ T cells but was instead mediated through
cytolytic targeting of activated CD8+ T cells by activated NK cells
in a 2B4-regulated and perforin-dependent manner. These results
identify an important role for 2B4 in maintaining tolerance of
highly activated NK cells during the early stages of persistent
infection that is nonredundant with the role of MHC in self-tol-
erance. Moreover, NK cell–mediated killing of highly activated
virus-specific CD8+ T cells in the absence of 2B4 hampers host
defenses during persistent viral infection.
Effects of 2B4 deficiency on antiviral T cell responses during persistent
LCMV infection. 2B4 deficiency had a pronounced effect on CD8+
T cell responses during a persistent, highly disseminated infection
induced by i.v. inoculation with 2 × 106 PFU of the clone 13 vari-
ant of LCMV (Figure 1). The proportion of LCMV-specific, IFN-γ–
producing CD8+ T cells was reduced in the spleen and peripheral
blood of 2B4-KO mice at all times analyzed (Figure 1, A and B).
Likewise, there were reduced total numbers of splenic GP33-41–spe-
cific (Figure 1C), NP396-404–specific (WT: 2.8 ± 0.3 × 105 vs. KO:
1.9 ± 0.1 × 105, n = 10, P = 0.015), and GP276-286–specific (WT: 2.5 ±
0.3 × 105 vs. KO: 1.4 ± 0.2 × 105, n = 10, P = 0.0058) IFN-γ+ CD8+
T cells at day 6 of infection in 2B4-KO mice.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2010;120(6):1925–1938. doi:10.1172/JCI41264.
1926?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
Despite a diminished magnitude, LCMV-specific CD8+ T cell
responses in 2B4-KO mice had a characteristic hierarchy (i.e.,
GP33 > NP205 > NP396) of partial exhaustion (Figure 1A), including
clonal deletion of NP396-404–specific T cells (Figure 1D), similar to
WT mice. Moreover, LCMV-specific IFN-γ–producing CD8+ T cells
in WT and 2B4-KO mice failed to coproduce either TNF or IL-2
(data not shown), a hallmark of virus-induced T cell exhaustion
(16). LCMV-specific CD8+ T cell responses were also reduced in the
lungs and inguinal lymph nodes (iLNs) of 2B4-KO mice (Figure 1E),
and the intense foci of infiltrating lymphocytes surrounding the
portal areas of WT livers were largely absent in 2B4-KO mice (Sup-
plemental Figure 1; supplemental material available online with
this article; doi:10.1172/JCI41264DS1). LCMV GP61-80–specific
and anti-CD3–induced CD4+ T cell IFN-γ responses did not differ
between WT and 2B4-deficient mice (Figure 1A).
As T cells can become dysfunctional during persistent LCMV infec-
tion and fail to produce cytokines (16, 17), we also determined antivi-
ral CD8+ T cell frequencies using LCMV peptide–loaded MHC class I
tetramer staining. The proportion and number of CD8+ T cells stain-
ing positive for H-2Db LCMV NP396-404 (Figure 1F) and H-2Db LCMV
GP33-41 (Figure 1G) tetramers were significantly reduced in 2B4-KO
mice at days 6 and 8 of infection, respectively. Despite differences
in frequency, LCMV-specific CD8+ T cells in WT and 2B4-KO mice
displayed similar functional avidities in regard to IFN-γ production
in response to a range of peptide doses (Supplemental Figure 2).
The cytolytic activity of virus-specific CTLs was also assessed
in LCMV clone 13–infected mice using a conventional in vivo
cytotoxicity assay (18). Consistent with the reduced frequency of
LCMV-specific CD8+ T cells in the absence of 2B4 (Figure 1), lysis
of LCMV GP33-41– (n = 5, P = 0.0025) and NP396-404–labeled (n = 5,
P = 0.0006) targets was significantly reduced in 2B4-KO mice
as compared with WT controls (Figure 2A) at postinfection day
4 (day 4 p.i.). Thus, 2B4 may play a role in regulating activation,
expansion, or acquisition of effector functions of CD8+ T cells in
the context of disseminated infection and high viral loads.
Elevated viral loads and prolonged viral persistence in 2B4-KO mice. At
early time points after infection (e.g., day 4 p.i.), viral loads were
comparably high in the spleen and liver of WT and 2B4-KO mice
(Figure 2B). However, an increased viral burden was evident in
2B4-KO mice, beginning 9 days after infection, when viral loads
began to slowly decline in WT mice. By day 63, most WT mice
(3 of 4) but not 2B4-KO mice (0 of 4) had cleared LCMV from these
tissues. By day 92, all 8 2B4-KO mice had detectable virus within
the spleen and liver, while 7 of 8 WT mice had cleared LCMV.
Accumulation of naive-phenotype CD8+ T cells and enhanced spleen size
in LCMV clone 13–infected 2B4-KO mice. In addition to a reduction in
the frequency of LCMV-specific CD8+ T cells, a large proportion
(Figure 3A) of 2B4-deficient splenic CD8+ T cells displayed a naive
phenotype (CD44loCD62Lhi CD127hiPD-1–KLRG1–CD43[1B11]–),
whereas WT CD8+ T cells had a predominately activated pheno-
type (CD44hiCD62LloCD127loPD-1+KLRG1+CD43[1B11]+) at day
8 of infection. This discrepancy in activation marker expression
resulted in a significant accumulation of splenic CD8+ T cells with
a naive phenotype (CD44lo) and a reduction in the total number
of activated-phenotype (CD44hi) CD8+ T cells in 2B4-KO mice in
comparison to WT control mice at day 8 of infection (Figure 3B).
An enhanced population of naive-phenotype CD8+ T cells was also
evident in peripheral blood and in iLNs (Figure 3C) and persisted
into late time points (e.g., day 100 p.i.) after infection (Figure 3D).
Changes in activation marker expression were not observed on
CD4+ T cells (Figure 3A).
Spleen size is usually reduced during infection with the immu-
nosuppressive clone 13 strain of LCMV (19), but we observed strik-
ingly greater spleen size and leukocyte cellularity in LCMV clone
13–infected 2B4-KO mice compared with their infected WT coun-
terparts as early as day 9 after infection, and these were still evi-
dent at 32 weeks after infection (Figure 4). The spleens of infected
2B4-KO mice were slightly larger than spleens of either uninfected
WT or 2B4-KO mice. Although the increased numbers (Figure 3B)
of CD8+ T cells with a naive phenotype (CD44lo) present in 2B4-KO
mice contributed to the increased spleen size and leukocyte count
(Figure 4), the frequencies of erythrocytes (WT: 3.7 × 108 ± 0.7 × 108
vs. KO: 11.1 × 108 ± 3.6 × 108, n = 3), CD11c+ DCs (WT: 1.1 × 105 ±
0.7 × 105 vs. KO: 3.5 × 105 ± 1.4 × 105, n = 4, P = 0.02), CD11b+ mac-
rophages (WT: 5.4 × 106 ± 3.2 × 106 vs. KO: 17.0 × 106 ± 7.0 × 106,
n = 4, P = 0.03), CD3+ T cells (WT: 2.9 × 106 ± 1.6 × 106 vs. KO:
5.3 × 106 ± 2.1 × 106, n = 4, P = 0.07), and CD19+ B cells (WT:
1.2 × 107 ± 0.6 × 107 vs. KO: 2.3 × 107 ± 0.9 × 107, n = 4, P = 0.08) were
also increased at day 9 of infection in the spleens of 2B4-KO mice
relative to WT controls. In contrast, lymphocyte numbers were not
increased in the iLNs, liver, or lungs (data not shown) of 2B4-KO
mice relative to WT controls at any time point after infection.
Reduced activation of CD8+ T cells during LCMV infection of 2B4-KO
mice is not an intrinsic defect of 2B4-deficient CD8+ T cells. To test whether
a 2B4-associated signaling defect in T cells was responsible for the
weak T cell response and altered pathogenesis in 2B4-KO mice, we
isolated bulk splenocytes from WT congenic mice (Thy1.1+) and
transferred them into either Thy1.2+ WT or Thy1.2+ 2B4-KO mice
prior to infection of recipient mice with LCMV clone 13. In WT
recipients, both WT donor (Thy1.1+) and WT host (Thy1.2+) CD8+
T cells displayed a primarily activated phenotype (CD44hi) at day 6
of infection (Figure 5A). In contrast, WT donor CD8+ T cells trans-
ferred into a 2B4-deficient mouse maintained a principally naive
phenotype (CD44lo), similar to that observed among host 2B4-KO
CD8+ T cells after infection (Figure 5A). Reciprocal transfers of
2B4-deficient splenocytes (Ly5.2+) into congenic WT mice (Ly5.1+)
resulted in both donor 2B4-KO (Ly5.2+) and host WT (Ly5.1+) CD8+
T cells at day 6 of infection displaying a predominantly activated
(CD44hiCD43[1B11]+) phenotype similar to that of WT donor
Reduced magnitude of the LCMV-specific CD8+ T cell response dur-
ing persistent LCMV clone 13 infection of 2B4-KO mice. (A) Repre-
sentative viral peptide–induced IFN-γ expression by splenic T cells
9 days after infection with 2 × 106 PFU LCMV clone 13 i.v. Numbers
represent mean ± SD of the percentage of IFN-γ+ CD4+ or CD8+ T
cells from all the similarly treated mice in the experiment (n = 4). (B)
Proportions of LCMV GP33-41–stimulated IFN-γ+ splenic or peripheral
blood CD8+ T cells are plotted as mean ± SEM (n = 3–8/group) across
a range of time points during persistent LCMV clone 13 infection. (C)
Total numbers (mean ± SEM) of GP33-41– and NP396-404–specific IFN-γ+
splenic CD8+ T cells in WT and 2B4-KO mice (n = 10–15/group) at day
6 of infection. (D) Representative CD8+ T cell IFN-γ expression (mean
± SD) in the blood (n = 4) at day 35 of LCMV clone 13 infection. (E)
IFN-γ responses (mean ± SD) by GP33-41–specific CD8+ T cells in the
iLNs and lungs of WT and 2B4-KO mice (n = 3–4/group) at days 6 and
21 of LCMV clone 13 infection. (F and G) Frequencies (mean ± SEM)
of day 6 (n = 6–11/group) LCMV NP396/Db (F) and day 8 (n = 8/group)
GP33/Db (G) tetramer-binding CD8+ T cells in the spleen. *P < 0.05,
**P < 0.01 (2-tailed unpaired Student’s t test). Data are from 1 of 3–4
experiments with similar results.
1928? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
(Ly5.2+) CD8+ T cells transferred into WT (Ly5.1+) host mice (Supple-
mental Figure 3). Together these results indicated that the high fre-
quency of naive phenotype CD8+ T cells in 2B4-KO mice (Figure 3)
was not an intrinsic defect of 2B4-deficient CD8+ T cells.
In order to assess whether loss of 2B4 affects the activation and
expansion of virus-specific CTLs, WT Thy1.1+ CD8+ T cells were labeled
with CFSE and transferred into both WT (Thy1.2+) and 2B4-KO
(Thy1.2+) mice. Following infection with LCMV clone 13, the pro-
portion of WT donor CD8+ T cells that diluted CFSE was greater
in WT mice than in 2B4-KO mice at day 6 of infection (Figure 5B).
Moreover, the proportion of tetramer-defined LCMV NP205-212–spe-
cific WT donor CD8+ T cells was higher in WT hosts than in 2B4-KO
recipients (WT: 13.7% ± 0.2% vs. KO: 2.0% ± 0.5%, n = 3, P = 0.0001).
Consistent with the unaltered phenotype of endogenous CD4+ T cells
in 2B4-KO mice, WT donor CD4+ T cells transferred into 2B4-KO
mice upregulated CD44 and diluted CFSE in a similar fashion to
WT CD4+ T cells transferred into WT mice (data not shown).
Role for NK cells in regulating LCMV-specific CD8+ T cell responses.
As NK cells constitutively express 2B4 (7), we tested whether NK
cells may regulate CD8+ T cell activation in 2B4-KO mice. Specif-
ic depletion of NK cells was achieved through i.p. administration
at 1 day before LCMV infection of a single dose of 25 μg anti-
NK1.1, which, because of their lower expression of NK1.1 in com-
parison to NK cells, did not reduce numbers of γδ T cells (data
not shown) or NK T cells (Supplemental Figure 4). Depletion of
NK cells in 2B4-KO mice prior to transfer of WT donor spleno-
cytes and subsequent LCMV infection resulted in restoration of
both host (2B4-KO) and donor (WT) CD44 expression to levels
observed on CD8+ T cells in WT mice at day 6 p.i. (Figure 5A),
when most of the cells expressed an activated (CD44hi) pheno-
type. In addition, the dilution of CFSE by donor CD8+ T cells in
2B4-KO mice was similar to WT levels following depletion of NK
cells (Figure 5B). Of note, depletion of NK cells in either strain
of recipient mice appeared to slightly increase the proportion of
host and donor CD8+ T cells that display high CD44 or low CFSE
expression (Figure 5, A and B), suggesting that there is also a
role for WT NK cells in regulating CD8+ T cell responses during
LCMV clone 13 infection.
Reduced LCMV-specific CTL activity and increased viral burden in the absence of 2B4 in vivo. Splenocytes from uninfected WT donor mice were
loaded with LCMV peptides (GP33-41 or NP396-404) or no peptide, labeled with various concentrations (2.5, 1, or 0.4 μM) of CFSE, mixed at equal
ratios, and injected (2 × 106 total targets) i.v. into LCMV clone 13–infected recipients (day 4 p.i.) or uninfected WT or 2B4-KO control mice. After
20 hours, spleens were harvested from WT and 2B4-KO recipient mice and analyzed for recovery of each CFSE-labeled target population. (A)
Representative histograms demonstrate recovery of NP396-404–labeled (low CFSE), GP33-41–labeled (middle CFSE), and unlabeled (high CFSE)
peaks. Numbers represent the percent specific lysis of LCMV peptide–coated targets relative to unlabeled control targets within each experi-
mental mouse. Specific lysis of LCMV peptide–coated targets within individual mice is plotted on the right. Each circle represents an individual
mouse, and horizontal lines denote the mean. (B) At various time points after infection, organs were harvested from LCMV clone 13–infected
mice, and infectious virus was quantified by standard plaque assay. Titers are plotted as the arithmetic mean ± SD of the log10 of PFU per organ
(n = 3–10 mice/group). The y axis lower limit is set at the limit of detection for the liver (log10 2.0) and spleen (log10 1.0) assays. *P < 0.05, **P < 0.01
(2-tailed unpaired Student’s t test). Data are from 1 of 3 experiments with similar results.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
The enhancement of spleen size and splenocyte number (Figure 6A),
reduction of LCMV GP33-41–specific IFN-γ responses (Figure 6B),
and increase in numbers of naive phenotype CD8+ T cells in 2B4-KO
mice (Figure 5A) were all abrogated by transient depletion of
NK cells at the time of LCMV clone 13 inoculation. The reduced
cytolysis of CTL targets in 2B4-KO mice during in vivo cytotoxicity
assays was also restored to WT levels by NK cell depletion (data
not shown). Moreover, the prolonged viral persistence evident at
day 90 of infection in 2B4-KO mice was completely prevented by
NK cell depletion just prior to infection (Figure 6C). In order to
confirm the role of NK cells in regulating CD8+ T cell responses
in 2B4-KO mice, NK cells were selectively depleted with a carefully
titrated dose of anti-asialo GM1 antibody, as described previously
(20). Similar to anti-NK1.1 antibody administration, the decreased
numbers of LCMV GP33-41–specific IFN-γ–producing splenic CD8+
T cells (WT: 3.9 × 105 ± 1.3 × 105 vs. KO: 0.7 × 105 ± 0.2 × 105, n = 3)
at day 6 of infection in 2B4-KO mice were restored to WT levels by
NK cell depletion using anti-asialo GM1 antibody (WT: 5.8 × 105 ±
0.4 × 105 vs. KO: 4.6 × 105 ± 0.5 × 105, n = 3).
By day 10 of LCMV clone 13 infection, the thymus underwent
dramatic involution, with a greater than 90% reduction in total
thymic cellularity in both WT and 2B4-KO mice (data not shown).
Elevated frequency of naive-phenotype (CD44lo) CD8+ T cells in LCMV clone 13–infected 2B4-KO mice. (A) Representative histograms demon-
strate the expression of various activation receptors by CD8+ and CD4+ T cells at day 8 of LCMV clone 13 infection in WT (shaded histograms)
and 2B4-KO (solid line) mice. The y axis represents percent of maximum. Numbers are mean proportion ± SD of WT (non-bold type) and 2B4-KO
(bold type) T cells (n = 4 mice/group) falling with the gated expression range of each activation marker. (B) The mean ± SEM total number of
naive (CD44lo) or activated (CD44hi) phenotype CD8+ T cells in the spleen of uninfected (n = 4/group) or day 8 clone 13–infected (n = 8/group)
is plotted. (C) Histograms demonstrating reduced expression of CD44 by CD8+ T cells in the iLNs and lungs of 2B4-KO mice (n = 3–4/group,
mean ± SD) at days 8 and 14 p.i. (D) Representative plots demonstrating a reduced frequency of CD44hi activated phenotype CD8+ T cells in
2B4-KO mice (n = 4/group, mean ± SD) at later time points of LCMV clone 13 infection. *P < 0.05, **P < 0.01 (2-tailed unpaired Student’s t test).
Data are from 1 of 3 experiments with similar results.
1930?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
Nevertheless, the frequency of CD4/CD8 double-positive (DP) thy-
mocytes was higher in 2B4-deficient mice (Figure 6D). The num-
bers of DP thymocytes were restored to infected WT levels after
NK cell depletion of 2B4-KO mice (Figure 6D). Thus, NK cells
appear to play a critical role in all of the phenotypes of 2B4-KO
mice described in this article.
Time requirements for NK cell depletion. Administration of anti-
NK1.1 depleting mAbs at 0, 1, or 2 days after infection converted
the day 10 phenotype of CD8+ T cells in 2B4-deficient hosts to
the WT infection phenotype (Figure 6E); however, this treatment
began to lose its efficacy when anti-NK1.1 antibody was adminis-
tered at 3 or 4 days after infection. Figure 6E shows the enhanced
naive CD44lo phenotype and reappearance of large spleens charac-
teristic of 2B4-KO mice when NK cells were depleted from these
mice at day 3 or 4. Taken together, these data suggest that 2B4-
deficient NK cells have an inhibitory effect on CD8+ T cell activa-
tion in the first few days of persistent infection.
2B4-deficient NK cells cytolytically target activated CD8+ T cells. In order
to determine whether reduced CD8+ T cell responses in 2B4-KO
mice were a result of NK cell targeting of CD8+ T cells directly or
through disruption of antigen presentation by NK cell targeting
of DCs, we determined the frequencies of various DC subpopula-
tions as well as the expression of T cell activation markers on CD8+
T cells in WT and 2B4-KO mice at early time points after infec-
tion. At very early stages of infection (day 4 p.i.), we began to see
accumulation of highly activated CD8+ T cells (CD3+Thy1.2+CD8
αβ+CD44hi) in the spleen (Figure 7, A and B) and iLN (Figure 7C)
of WT mice. These activated CD8+ T cells were characterized by
upregulated expression of both the activation-associated isoform
(1B11) of CD43 (21) and the DC marker CD11c (22) (Figure 7A).
The frequency of these highly activated CD8+ T cells (CD3+Thy1.2+
CD8αβ+CD44hiCD43[1B11]+CD11c+) was significantly reduced in
both the spleen (Figure 7, A and B) and iLNs (Figure 7C) of 2B4-KO
mice. Depletion of NK cells prior to infection restored the frequen-
cy of these highly activated CD8+ T cells in 2B4-KO mice to WT
levels (Figure 7, B and C). In addition, the frequencies of several DC
subsets were decreased in the spleens of both WT and 2B4-KO mice
after LCMV infection (Supplemental Figure 5), but in contrast to
frequencies of activated CD8+ T cells, DC loss at this time point was
not prevented by NK cell depletion (data not shown).
We previously demonstrated that 2B4-deficient NK cells kill other
NK cells upon activation with IL-2 in vitro and CpG DNA in vivo,
resulting in reduced peripheral NK cell numbers (23). However,
splenic NK cell frequencies (WT: 1.8 × 106 ± 0.9 × 106 [3.3% ± 0.2%]
vs. KO: 1.4 × 106 ± 0.8 × 106 [3.2% ± 0.3%], n = 15, P = 0.3) were
not significantly reduced in the spleen of 2B4-KO mice at day 4
of LCMV clone 13 infection. NK cell frequencies were also similar
in the spleens of WT and 2B4-KO mice at day 3 (WT: 9.0 × 105 ±
1.9 × 105 [1.5% ± 0.3%] vs. KO: 8.6 × 105 ± 1.8 × 105 [1.4% ± 0.3%],
n = 11, P = 0.9) and day 6 (WT: 1.3 × 106 ± 0.4 × 106 [2.0% ± 0.3%] vs.
KO: 1.0 × 106 ± 0.1 × 106 [2.4% ± 0.1%], n = 3) of infection. Thus, the
importance of NK cell fratricide in the absence of 2B4 (23) may be
dependent upon the context of NK cell activation.
In order to determine whether NK cells may directly target activat-
ed CD8+ T cells for cytolysis, perforin-deficient (Prf1-KO) and 2B4/
perforin double-KO (2B4/Prf1-KO) mice were infected with LCMV
clone 13. At day 4 p.i., the two strains of mice displayed similar num-
bers (Figure 7D) and proportions (Supplemental Figure 6) of highly
activated (CD44hiCD11c+) CD8+ T cells. Importantly, depletion of
NK cells in Prf1-KO and of 2B4/Prf1-KO mice had minimal impact
on the frequency of activated CD8+ T cells at day 4 (Figure 7D).
In order to determine whether NK cell killing accounts for the
decrease of the virus-specific T cell responses, we first assessed
the activity of WT and 2B4-KO NK cells against activated CD8+
T cells in vitro. WT lymphokine-activated killer (LAK) cells dis-
played low levels of cytolytic activity against WT concanavalin A–
Splenomegaly during persistent
LCMV clone 13 infection of 2B4-KO
mice. At various time points after
LCMV clone 13 infection, spleens
were photographed and the total
number of splenic leukocytes
determined after red blood cell
lysis. Mean splenocyte counts
(±SEM) are plotted for WT and KO
mice (n = 4–10/group) throughout
the course of infection. *P < 0.05,
**P < 0.01 (2-tailed unpaired Stu-
dent’s t test).
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activated (ConA-activated) syngeneic CD8+ T cells, but 2B4-defi-
cient LAK cells readily lysed these activated CD8+ T cell targets
(Figure 8A). Likewise, WT LAK cells mediated killing of activated
CD8+ T cells when these targets were derived from Cd48-KO mice,
which lack the ligand for 2B4 (Figure 8B). The killing of Cd48-KO
target cells was abrogated by perforin deficiency within the
effector cell population (Figure 8B). Importantly, the ability of
2B4-deficient NK cells to kill CD8+ T cells was dependent upon
activation of these T cells, as very little killing of nonactivated
(0 μg/ml ConA) T cells occurred (Figure 8A). These experiments
directly demonstrate a perforin-dependent killing of activated T
cells by activated 2B4-KO NK cells in vitro.
We next asked whether a similar NK cell–mediated elimination
of activated CD8+ T cells would occur in vivo. In a modified in vivo
cytotoxicity assay, mixed populations of naive and activated CD8+
T cells from LCMV-infected congenic (Ly5.1+) WT mice (day 4
p.i.) were labeled with CFSE and transferred into WT or 2B4-KO
mice at day 4 of LCMV clone 13 infection. The survival of donor
(Ly5.1+CFSE+) CD8+ T cells with an activated (CD44hiCD43+) or
naive (CD44loCD43–) phenotype was examined 5 hours after trans-
fer (Figure 8, C and D). Over the course of this 5-hour assay, there
was no detectable target cell division (i.e., dilution of CFSE) among
the donor CD8+ T cell populations (data not shown). Importantly,
the frequencies of activated but not naive phenotype donor CD8+
Reduced activation of CD8+ T cells in 2B4-KO mice is caused by NK cells rather than defects in CD8-intrinsic 2B4 signaling. WT congenic
(Thy1.1+) splenocytes (3 × 107) were i.v. transferred into WT and 2B4-KO mice (Thy1.2+ host) 1 day before infection with LCMV clone 13. Two
days before transfer, some groups of recipient mice received 25 μg of either isotype (IgG2a) control or anti-NK1.1 mAb. (A) Representative
gating of Thy1.1+ (donor, red) and Thy1.1– (host, blue) CD8+ T cells in WT and 2B4-KO recipient mice at day 6 p.i. is shown at left. Expression
of CD44 on donor (red histograms) and host (blue histograms) CD8+ T cells is shown, with numbers representing mean ± SD of CD44hi CD8+ T
cells. (B) CFSE dilution (mean ± SD CFSElo) is shown for Thy1.1+ (donor) CD8+ T cells in the spleen, iLN, and blood of recipient mice. *P < 0.05,
**P < 0.01 (2-tailed unpaired Student’s t test). Data are from 1 of 3 experiments with similar results.
1932?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
Depletion of NK cells restores LCMV-specific CD8+ T cell responses and viral clearance in 2B4-KO mice to WT levels. (A–D) One day prior
to infection, WT and 2B4-KO mice (n = 4–8/group) were treated with 25 μg isotype (IgG2a) or anti-NK1.1 mAb i.p. (A) Splenic leukocytes
(mean ± SD) were enumerated on day 9 p.i. in WT and 2B4-KO mice. (B) Mean proportion (±SD) of IFN-γ+ CD8+ T cells in spleen after GP33-41
stimulation. (C) Viral titers at day 90 p.i. (n = 7–8/group) are displayed as log10 PFU/liver. The horizontal line represents the limit of detection
for the plaque assay (log10 2.0). (D) Mean (±SD) fraction of DP thymocytes within thymus of uninfected or day 9 p.i. WT and 2B4-KO mice.
(E) A single injection of anti-NK1.1 mAb was administered at various time points (day 0 to day 4) relative to the time of infection with LCMV
clone 13. Representative spleen size (photo) and CD44 expression (y axis represents percent of maximum) on splenic CD8+ T cells was
determined at day 10 of infection (n = 2 mice/group, mean ± SD). *P < 0.05, **P < 0.01 (2-tailed unpaired Student’s t test). Data are from
1 of 3 experiments with similar results.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
T cells were reduced in 2B4-KO host mice (Figure 8, C and D),
and this loss was abrogated by NK cell depletion of 2B4-KO
recipient mice prior to infection (Figure 8D). Similar transfers of
activated CD8+ T cell–containing populations were conducted in
Prf1-KO and 2B4/Prf1-KO mice, with comparable survival of acti-
vated donor CD8+ T cells in both strains (Figure 8D). T cell targets
from LCMV-infected Cd48-KO mice were not tested in this system
because Cd48-KO mice have a defect in T cell activation (24). Taken
together, these results suggest that activated 2B4-deficient NK
cells kill activated CD8+ T cells in a perforin-dependent manner
in vitro and in vivo.
Here we demonstrate that a long-term persistent viral infection
associated with altered T cell activation may actually be regulated
by NK cells acting during the first few days of infection. This NK
cell–mediated regulation of T cell responses is itself regulated by
2B4 on the NK cells, not the T cells, which also can express 2B4.
In the absence of 2B4-mediated inhibition, activated NK cells
cytolytically targeted highly activated CD8+ T cells, resulting in a
significant culling of these LCMV-specific effectors. This loss of
virus-specific CD8+ T cells impaired control of virus replication,
causing delayed viral clearance and altered immune pathologies.
Therefore, our results suggest that 2B4-mediated regulation of NK
cell activity within a highly inflammatory lymphoid environment
is both crucial for antiviral defense and nonredundant with the
role of MHC in maintaining self-tolerance of NK cells.
Recently, much emphasis has been placed on the role of inhibi-
tory receptors such as PD-1 in CD8+ T cell exhaustion during
chronic virus infections in mice (LCMV clone 13) (25) and humans
(HIV, HCV) (26–28). Blockade of PD-1 in vitro and in vivo results
in “rescue” of the proliferation and effector functions of exhausted
virus-specific CD8+ T cells, thereby leading to enhanced control
of virus (25, 26, 29–31). However, exhausted CD8+ T cells express
a number of different inhibitory receptors, including 2B4, which
have been suggested to synergistically contribute to the severity
of T cell dysfunction (11, 12). We demonstrate here that 2B4-defi-
cient LCMV-specific CD8+ T cells underwent clonal exhaustion
and were deleted (e.g., NP396-404) similarly to 2B4-sufficient T cells
in WT mice. However, the magnitude of the total LCMV-specific
CTL response was reduced by NK cells lacking 2B4. Although 2B4
expressed on CD8+ T cells has been suggested to both augment
NK cells mediate early loss of highly activated CD8+ T cells in 2B4-KO mice in a perforin-dependent manner. (A) At day 4 of LCMV clone 13 infec-
tion, splenocytes (n = 4/group) were gated on Thy 1.2-expressing cells, and the proportion of CD8α+ T cells expressing CD11c was determined.
The Thy1.2+CD8α+CD11c+ events were uniformly CD8β+, CD3ε-expressing, and CD44hi. Right: Proportion of CD44hiCD43(1B11)+ events among
gated CD8αβ+ T cells. Numbers represent mean (±SD) proportion of gated events. Total numbers (mean ± SEM) of Thy1.2+CD11c+CD8α+ cells
were determined in the spleens (B) as well as iLNs (C) of isotype-treated or anti-NK1.1–treated WT and 2B4-KO mice (n = 5/group), as well as
in the spleens (D) of similarly treated Prf1-KO and 2B4/Prf1-KO (n = 3–4/group) mice. **P < 0.01 (2-tailed unpaired Student’s t test). Data are
from 1 of 3 experiments with similar results.
1934? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
(13) and inhibit (11) T cell activation, our adoptive transfers of WT
as well as 2B4-KO lymphocytes into 2B4-deficient and 2B4-suffi-
cient environments demonstrated that deficiencies in CD8+ T cell
activation associated with genetic ablation of 2B4 were CD8+ T cell
extrinsic and regulated by NK cells. In fact, WT and 2B4-KO CD8+
T cells from infected mice were phenotypically indistinguishable
in the absence of NK cells. These experiments demonstrate that
despite similar expression patterns of 2B4 and PD-1 on LCMV-
specific T cells, it is the inhibition of NK cell cytolytic activity by
2B4 expressed on NK cells that regulates antiviral T cell responses
during persistent infection. Therefore, the effect of 2B4 blockade
on NK cell activity should be considered when designing therapies
for persistent virus infection that are based on antibody blockade
of 2B4 expressed on exhausted CD8+ T cells.
Several recent studies have also suggested that NK cells indirect-
ly regulate antiviral T cell responses during murine cytomegalo-
virus (MCMV) infection through interactions with DCs (32–34).
However, interpretation of these results is hampered because NK
cells are vital to control of MCMV replication (35, 36), and high
viral loads in the absence of NK cells may adversely affect virus-
specific CD8+ T cell responses (37). In the case of LCMV, NK cells
do not play a role in direct control of early viral loads (38, 39).
In our study, the NK cell–independent reduction of DC numbers
in both WT and 2B4-KO mice suggested that NK cell–mediated
regulation of CD8+ T cells was not a result of NK cell–DC interac-
tions. Moreover, CD4+ T cell frequencies were not altered after NK
cell depletion, indicating that they were getting sufficient stimu-
lation from antigen-presenting cells. These data indicate that NK
cells did not control activated CD8+ T cell responses through a
DC intermediary in this system, though they do not rule out NK
cell–mediated regulation of immune responses via interaction
with DCs in other circumstances.
NK cells mediate specific lysis of activated CD8+ T cells in the absence of 2B4 in vitro and in vivo. (A) WT (black bars) or 2B4-KO (white bars)
LAK cell killing (mean ± SD) of WT CD8+ T cell targets, activated in vitro with various doses of ConA. (B) Specific lysis (mean ± SD) of WT or
CD48-KO ConA-activated CD8+ T cells by WT (left) or by Prf1-KO (right) LAK cells is shown at various effector to target (E/T) ratios. (C and D) A
modified in vivo cytotoxicity assay was done by injecting CFSE-labeled splenocytes (3 × 107) from LCMV-infected (day 4 p.i.) congenic (Ly5.1+)
WT mice into uninfected or LCMV-infected (day 4 p.i.) WT and 2B4-KO mice (Ly5.2+, n = 5–6/group), some of which were depleted of NK cells
1 day prior to infection. Five hours after transfer, the proportion of activated (CD44hiCD43[1B11]+) donor (Ly5.1+CFSE+) cells was determined
in each mouse. (C) Representative CD44 and CD43(1B11) expression by donor Ly5.1+CFSE+ CD8+ T cells. Numbers are the percentage of
donor (Ly5.1+CFSE+) CD8+ T cells that are CD44hiCD43(1B11)+ for the representative sample shown. (D) Top graphs depict the percentage of
activated CD44hiCD43(1B11)+ donor (Ly5.1+CFSE+) CD8+ T cells in isotype- (left) or anti-NK1.1–treated (right) WT and 2B4-KO mice. Lower left
plot demonstrates the proportion of naive phenotype (CD44loCD43[1B11]–) donor (Ly5.1+CFSE+) CD8+ T cells in isotype-treated WT and 2B4-KO
mice. Lower right plot depicts the percentage of activated CD44hiCD43(1B11)+ donor (Ly5.1+CFSE+) CD8+ T cells in Prf1-KO and 2B4/Prf1-KO
mice. Results are presented as mean ± SD. **P < 0.01 (2-tailed unpaired Student’s t test). Data are from 1 of 3 similar experiments.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
Activated CD8+ T cells transferred into LCMV-infected 2B4-KO
mice were rapidly (within 5 hours) lost in an NK cell– and perfo-
rin-dependent manner. This rapid loss of CD8+ T cells without evi-
dence of cellular division (e.g., CFSE dilution) argues for a direct
mechanism whereby NK cells eliminate activated CD8+ T cells. In
mice with a combined genetic deficiency of perforin and 2B4, NK
cells did not mediate a reduction of highly activated CD8+ T cells
within lymphoid tissues. NK cell depletion did not further augment
the number of activated CD8+ T cells present in lymphoid organs of
2B4/Prf1-KO mice, suggesting that perforin is a crucial component
of the NK cell–mediated regulation of CD8+ T cell activation. Of
note, the frequencies of activated CD8+ T cells were also marginally
increased in perforin-deficient and NK cell–depleted WT mice rela-
tive to nondepleted WT controls. Indeed, we have found that under
certain conditions of infection, NK cells may shape the kinetics and
functionality of antiviral CD8+ T cell responses in WT mice (S.N.
Waggoner and R.M. Welsh, unpublished observations).
In contrast to the reduced spleen size typically observed dur-
ing infection of WT C57BL/6 mice with the immunosuppressive
clone 13 strain of LCMV (19), we observed an increase in both the
size and leukocyte cellularity of the spleen during LCMV clone
13 infection of 2B4-deficient mice. Increased frequencies of mul-
tiple lymphocyte lineages contributed to the enhanced splenic
leukocyte counts in 2B4-KO mice, including T cells, B cells, mac-
rophages, and DCs. Acute infection of WT mice with the Arm-
strong strain of LCMV characteristically results in splenomegaly
associated with the accumulation of large numbers of activated,
LCMV-specific T cells (40, 41). In contrast, the majority of CD8+ T
cells in the enlarged spleens of LCMV clone 13–infected 2B4-KO
mice displayed a naive phenotype (CD44lo), and these naive cells
accumulated in 2B4-KO mice to 3-fold-higher levels than in their
infected WT counterparts. The enhanced frequency of CD4/CD8-
DP thymocytes in 2B4-KO mice may be indicative of increased
thymic output of naive CD8-single-positive lymphocytes dur-
ing LCMV infection of 2B4-KO mice, which could contribute to
increased numbers of peripheral CD8+ T cells with a naive pheno-
type. However, the frequency of naive-phenotype 2B4-KO CD4+ T
cells was not increased in a similar fashion. Through depletion of
NK cells, we established that NK cells play an important role in
regulating both the altered splenic and thymic compositions of
2B4-KO mice, although the exact mechanism driving augmented
naive CD8+ T cell as well as total lymphocyte accumulation in the
spleen remains to be determined.
According to the missing self hypothesis, loss or reduction of
MHC class I molecule expression renders cells susceptible to NK
cell–mediated killing (42). We found that the highly activated
CD8+ T cells susceptible to NK cell–mediated killing in LCMV-
infected 2B4-KO mice actually expressed increased levels of class I
MHC molecules (S.N. Waggoner and R.M. Welsh, unpublished
observations), indicating that the protective effect of MHC class
I molecule expression is insufficient for self-tolerance to activated
NK cells in the absence of 2B4. Thus, as described elsewhere (3),
the protective effect of MHC class I and CD48 on control of self-
killing are nonredundant. Of interest is that the numbers and
phenotype of LCMV-specific CD4+ T cells were not altered in 2B4-
deficient mice. This suggests that the features of activated CD8+
T cells that target them for NK cell–mediated cytolysis may not
be shared by activated CD4+ T cells, or that the kinetic delay of
LCMV-specific CD4+ T cells responses previously described (43,
44) may temporally separate activated NK and CD4+ T cells.
We demonstrate that, in the absence of 2B4, NK cells cytolytical-
ly targeted activated (CD44hi) but not naive (CD44lo) CD8+ T cells
during persistent LCMV infection. This suggests that although
2B4 can both enhance and suppress NK cell activation (7, 8), 2B4-
mediated inhibitory signals are of greater significance in the reg-
ulation of antiviral immune responses during virus infection in
vivo. Furthermore, the specificity of NK cell–dependent killing of
activated CD8+ T cells but not other 2B4 ligand (CD48)–express-
ing lymphocytes in 2B4-KO mice suggested that additional NK
cell receptor ligands must be present on activated CD8+ T cells
that distinguish these cells for killing. A previous report suggested
that ligands of the activating NK cell receptor NKG2D are tran-
siently upregulated during in vitro activation of CD8+ T cells (45).
To date, our investigation of CD8+ T cells (WT and 2B4-KO) acti-
vated during LCMV infection in vivo has failed to detect expres-
sion of NKG2D ligands by these activated lymphocytes, regardless
of the presence or absence of NK cells (S.N. Waggoner and R.M.
Welsh, unpublished observations). In addition, our vivo blockade
of NKG2D by i.p. anti-NKG2D antibody (CX5) administration did
not restore CD8+ T cell activation in 2B4-KO mice to WT levels
(data not shown). Further study is required to identify the features
of activated CD8+ T cells that distinguish these lymphocytes from
naive lymphocytes as targets for NK cell killing.
We further sought to examine whether NK cell regulation of
antiviral CD8+ T cell responses in the absence of 2B4 is restricted
to persistent LCMV clone 13 infection or is a characteristic of mul-
tiple virus infections. Our preliminary data demonstrated an NK
cell–dependent 50% reduction in the frequency of LCMV-specific
splenic CD8+ T cells in 2B4-KO mice relative to WT controls at day
6 of acute LCMV Armstrong infection (S.N. Waggoner and R.M.
Welsh, unpublished observations). However, both strains of mice
mediated clearance of replicating virus by day 9 of infection, and
2B4-KO mice established LCMV-specific T cell memory popula-
tions that were similar to those present in immune WT mice. Our
preliminary experiments also revealed that 2B4 deficiency altered
T cell responses to Pichinde virus, MCMV, and mouse hepatitis
virus infections. Therefore, although NK cell regulation of T cell
responses in the absence of 2B4 is characteristic of numerous virus
infections, the impact of this regulation on antiviral immunity
may be virus strain–dependent.
Signaling in NK or CD8+ T cells following 2B4 engagement
depends in part upon association of 2B4 with the SLAM-asso-
ciated protein (SAP/SH2D1A) (46, 47). In patients with the rare
genetic immunodeficiency X-linked lymphoproliferative syn-
drome (XLP), the SH2D1A (SAP) gene is altered or absent (48),
resulting in a cytolytic defect of NK cells and CTLs. Frequent
childhood fatality in XLP patients is associated with uncontrolled
virus infections and activated CD8+ T cell–mediated hepatic
necrosis (49). Persistent, but not acute, LCMV or herpesvirus
infections of SAP-deficient (Sh2d1a–/–) mice recapitulate many
facets of XLP disease (50, 51). Recently, we demonstrated that
low expression levels of SAP result in strong suppression of NK
cell activation following 2B4 engagement (52). Although 2B4 has
not been directly implicated in the phenotypes of XLP patients or
SAP-deficient mice, 2B4-induced inhibitory signals in the absence
of SAP may contribute to the cytolytic defects of NK and CD8+
T cells. Moreover, 2B4-mediated inhibitory signals may prevent
NK cells from restraining the activated antiviral CTLs responsible
for tissue destruction. Of note, polymorphisms associated with
increased expression of 2B4 correlate with increased incidence of
1936?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
autoimmune diseases (53). The importance of 2B4 in restricting
NK cell killing of activated lymphocytes during pathogenesis of
human disease remains to be determined.
Our results therefore demonstrate that NK cells acting early in
infection can alter the magnitude and duration of viral persis-
tence through regulation of developing antiviral T cell responses.
The two persistent human infections with greatest similarity to
that of LCMV in the mouse are HIV, whose infections are char-
acterized by distinct antigen load set points per individual (54),
and HCV, whose infections are characterized by wide variations in
timing of clearance. We suggest that NK cells acting early in these
infections should be considered as possible contributors to these
T cell–regulated events.
Mice. WT C57BL/6, B6.PL Thy-1a/Cy (Thy1.1), and perforin-deficient mice
(10) were obtained from The Jackson Laboratory. Ly5.1 congenic mice on
a C57BL/6 background (B6.SJL-Ptprc<a>) were purchased from Taconic
Farms. 2B4-deficient mice were generated in C57BL/6-derived embryonic
stem cells as previously described (10). 2B4-deficient mice were bred and
maintained at the University of Massachusetts Medical School (UMMS).
Perforin- and 2B4-deficient (2B4/Prf1-KO) mice were bred and maintained
at the University of Chicago. CD48-deficient mice were provided by Arlene
Sharpe (Harvard University, Cambridge, Massachusetts, USA) (24). Experi-
ments were routinely done using male mice at 6–12 weeks of age. All mice
were maintained under specific pathogen–free conditions within either the
Department of Animal Medicine at UMMS or at the University of Chicago.
All experiments were performed according to a protocol approved by the
Institutional Animal Care and Use Committee of UMMS.
Virus infections and in vivo NK cell depletion. The highly disseminating clone
13 variant of LCMV was propagated in baby hamster kidney BHK21 cells
(55, 56). Virus was titrated by plaque assay on Vero cells. Persistent infec-
tions were initiated by infecting mice i.v. with 2 × 106 PFU of the clone
13 strain of LCMV. In order to specifically deplete NK cells, mice received
a single i.p. injection of 25 μg anti-NK1.1 (PK136) or control rat IgG2a
produced by Bio-X-Cell, 1 day before virus infection. Alternatively, mice
received 10 μl of anti-asialo GM1 antibody (Wako Pure Chemical) diluted
in 200 μl PBS i.p. 1 day prior to virus infection.
Antibodies and FACS analysis. Fluorescently labeled mAbs purchased from
BD Biosciences were the following: CD3 (145-2C11), CD4 (RM4-5), CD8α
(clone 53-6.7), CD8β (clone 53-5.8), CD48 (HM48-1), H-2Kb (AF6-88.5),
CD90.2 (clone 53-2.1), CD45.2 (1D4), I-Ab (AF6-120.1), CD11c (HL3),
CD11b (M1/70), NK1.1 (PK136), CD44 (IM7), CD62L (MEL-14), CD69
(H1.2F3), IL-2 (3C7), IFN-γ (XMG1.2), TNF (MP6-XT22), CD107a (1D4B),
CD107b (ABL-93), B220 (RA3-6B2), CD19 (1D3), and CD49b (DX5). mAbs
purchased from eBioscience were the following: CD90.1 (OX-7), PD-1
(J43), CD127 (A7R34), CD45.1 (A20), KLRG1 (2F1), PDCA-1 (eBio927),
2B4 (eBio244F4), and granzyme B (16G6). Antibodies purchased from Bio-
Legend include F4/80 (BM8), CD43 (1B11), and CD44 (IM7). Antibodies
purchased from R&D Systems included anti-NKp46. For flow cytometric
analysis, cells were analyzed on an LSR II cytometer (BD Biosciences), and
data were analyzed using FlowJo software (Tree Star).
Tetramers and peptides. Several previously defined T cell epitopes encoded
by LCMV were used in this study (57, 58). LCMV-specific epitopes includ-
ed NP396-404 (FQPQNGQFI), GP33-41 (KAVYNFATC), GP276-286 (SGVEN-
PGGYCL), NP205-212 (YTVKYPNL), GP118-125 (ISHNFCNL), and GP61-80
(GLKGPDIYKGVYQFKSVEFD). All peptides listed were purchased from
21st Century Biochemicals and were purified with reverse-phase HPLC to
90% purity. MHC class I peptide tetramers specific for LCMV NP205/Kb,
LCMV NP396/Db, and LCMV GP33/Db were generated as described previ-
ously (59). CD1d-PBS57-allophycocyanin tetramers were provided by the
National Institute of Allergy and Infectious Diseases Tetramer Facility
and were a gift of Leslie J. Berg (UMMS).
CFSE labeling and adoptive transfer. Single-cell suspensions were prepared
from spleens of congenic (Thy1.1+ or Ly5.1+) WT mice or 2B4-KO and WT
mice (Thy1.2+Ly5.2+), and erythrocytes were removed by lysis using a 0.84%
NH4Cl solution. Splenocytes were labeled with the 2 μM fluorescent dye
CFSE (CFDA-SE, Molecular Probes, Invitrogen) for 15 minutes at 37°C,
washed, and transferred i.v. (107 donor cells) to recipient mice.
In vitro cytotoxicity assay. NK LAK cells were prepared by culturing sple-
nocytes for 4 days in 1,000 U/ml recombinant human IL-2 (rhIL-2) (60).
CD8+ T cells used as targets were enriched from spleen by coating sple-
nocytes with FITC anti-CD8α mAb and using the EasySep FITC Selec-
tion Kit from StemCell Technologies. Enriched CD8+ T cells (75%–90%
CD8+) were incubated for 24–48 hours in 4 μg/ml ConA from Sigma-
Aldrich, unless otherwise described.
Target cells were labeled with 100 μC of sodium chromate (51Cr) for
1 hour at 37°C, washed, and then plated at 2,000 cells per well. Effector
NK LAK cells were added at the indicated ratios to triplicate wells. After
5 hours of incubation at 37°C, supernatants were collected for analysis,
and percent specific lysis was calculated using standard methods.
In vivo cytotoxicity assays. Assays to measure T cell cytolytic activity in vivo
were done as described previously (18). Briefly, spleens were harvested from
uninfected WT mice, and single-cell suspensions were prepared. Separate
populations of splenocytes were then loaded with LCMV peptides (1 μM)
for 10 minutes at 37°C before labeling with different concentrations of
CFSE (2.5, 1, or 0.4 μM, Molecular Probes, Invitrogen) for 15 minutes at
37°C. These populations were then washed and combined at equal ratios
and adoptively transferred i.v. into naive or infected recipients. Spleens
from recipient mice were harvested 4–20 hours later, and the survival
of each transferred population was assessed by flow cytometry. Specific
lysis was calculated using the following equation: 100 – ([% LCMV target
population in infected experimental/% unlabeled population in infected
experimental) ÷ (% LCMV target population in naive control/% unlabeled
population in naive control)] × 100).
An unconventional in vivo cytotoxicity assay was adapted to determine
NK cell killing of lymphocyte populations in vivo as follows. Congenic
(Ly5.1) WT mice were infected with 2 × 106 PFU LCMV clone 13 i.v., and
at day 4 p.i., single-cell suspensions were made from the spleens of these
infected donor mice. These cells were then labeled, for ease of detection,
with CFSE, and 2 × 107 bulk splenocytes containing both activated- and
naive-phenotype populations of CD8+ T cells were transferred into experi-
mental recipient mice on day 4 of LCMV clone 13 infection. The recipient
mice were WT (Ly5.2), 2B4-KO (Ly5.2), Prf1-KO (Ly5.2), or 2B4/Prf1-KO
(Ly5.2) mice that were administered anti-NK1.1 or isotype control antibod-
ies 1 day prior to infection with 2 × 106 PFU LCMV clone 13 i.v. Some recip-
ient mice were uninfected and served as controls. CFSE-labeled target cells
were transferred at day 4 of infection, and after 5 hours, spleens of recipient
mice were harvested for analysis of survival of donor CD8+ T cells.
Lymphocyte preparation and intracellular cytokine assay. Single-cell leukocyte
suspensions were prepared from spleens, iLNs, and peripheral blood by
lysing erythrocytes using a 0.84% NH4Cl solution. Lung and liver lympho-
cytes were harvested by mechanical and enzymatic digestion of tissues, as
described previously (61, 62). Briefly, livers were homogenized and then
digested for 10 minutes at 37°C in HBSS containing 10 U/ml DNase I and
0.5 mg/ml collagenase type II (Sigma-Aldrich), and 10% fetal bovine serum.
These homogenates were washed and leukocytes isolated by centrifugation
on a Lympholyte M (Cedarlane Laboratories) gradient. Prepared single-cell
suspensions from various tissues were plated at 2 × 106 cells per well in
96-well plates and stimulated for 5 hours at 37°C with either 1 μM viral
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
peptide or 2.5 μg/ml anti-CD3 mAb in the presence of brefeldin A and
0.2 U/ml rhIL-2. Following stimulation, cells were preincubated with a
1:200 dilution of Fc Block (2.4G2) in FACS buffer (HBBS, 2% FCS, 0.1%
NaN3) and then stained for 20 minutes at 4°C with various combinations
of fluorescently tagged mAbs. After washing, cells were permeabilized
using BD Cytofix/Cytoperm solution and then stained in BD Perm/Wash
using mAbs specific for various cytokines.
Statistics. Results are displayed as mean ± SD (for variation within indi-
vidual experiments) or SEM (for variation between experiments), with
statistical differences between experimental groups determined using
a 2-tailed unpaired Student’s t test, where a P value less than 0.05 was
We thank Michael A. Brehm and Liisa K. Selin for helpful discussions
and Keisha Mathurin and Keith Daniels for technical support. We
also thank Arlene Sharpe (Harvard University) for CD48-deficient
mice and Leslie Berg (UMMS) for NK T cell tetramer. We thank Lewis
Lanier (UCSF) for providing us with anti-NKG2D blocking antibody
(CX5). We appreciate the role of Susan E. Stepp in initiating these
studies. This work was supported by NIH training grant AI07349
(to S.N. Waggoner) and research grants AI-17672, AR-35506,
and CA34461 (to R.M. Welsh). The views expressed are those of the
authors and do not necessarily express the views of the NIH.
Received for publication September 28, 2009, and accepted in
revised form February 24, 2010.
Address correspondence to: Raymond M. Welsh, Department of
Pathology, University of Massachusetts Medical School, 55 Lake
Avenue North, Worcester, Massachusetts 01655, USA. Phone:
508.856.5819; Fax: 508.856.5780. E-mail: Raymond.Welsh@
Ruth T. Taniguchi’s present address is: Diabetes Center, UCSF,
San Francisco, California, USA.
1. Yokoyama WM, Kim S. How do natural killer
cells find self to achieve tolerance? Immunity.
2. Raulet DH, Vance RE. Self-tolerance of natural
killer cells. Nat Rev Immunol. 2006;6(7):520–531.
3. McNerney ME, Guzior D, Kumar V. 2B4 (CD244)-
CD48 interactions provide a novel MHC class I-
independent system for NK-cell self-tolerance in
mice. Blood. 2005;106(4):1337–1340.
4. Schwartzberg PL, Mueller KL, Qi H, Cannons JL.
SLAM receptors and SAP influence lymphocyte
interactions, development and function. Nat Rev
5. Ma CS, Nichols KE, Tangye SG. Regulation of cel-
lular and humoral immune responses by the SLAM
and SAP families of molecules. Annu Rev Immunol.
6. Veillette A. Immune regulation by SLAM family
receptors and SAP-related adaptors. Nat Rev Immu-
7. McNerney ME, Lee KM, Kumar V. 2B4 (CD244) is
a non-MHC binding receptor with multiple func-
tions on natural killer cells and CD8+ T cells. Mol
8. Assarsson E, Kambayashi T, Persson CM, Chambers
BJ, Ljunggren HG. 2B4/CD48-mediated regulation
of lymphocyte activation and function. J Immunol.
9. Lee KM, et al. 2B4 acts as a non-major histo-
compatibility complex binding inhibitory recep-
tor on mouse natural killer cells. J Exp Med.
10. Vaidya SV, et al. Targeted disruption of the 2B4
gene in mice reveals an in vivo role of 2B4 (CD244)
in the rejection of B16 melanoma cells. J Immunol.
11. Blackburn SD, et al. Coregulation of CD8+ T
cell exhaustion by multiple inhibitory recep-
tors during chronic viral infection. Nat Immunol.
12. Wherry EJ, et al. Molecular signature of CD8+
T cell exhaustion during chronic viral infection.
13. Kambayashi T, Assarsson E, Chambers BJ, Ljung-
gren HG. Cutting edge: regulation of CD8(+) T cell
proliferation by 2B4/CD48 interactions. J Immunol.
14. Sayos J, et al. Potential pathways for regulation
of NK and T cell responses: differential X-linked
lymphoproliferative syndrome gene product SAP
interactions with SLAM and 2B4. Int Immunol.
15. Ostrowski SR, Ullum H, Pedersen BK, Gerstoft J,
Katzenstein TL. 2B4 expression on natural killer
cells increases in HIV-1 infected patients followed
prospectively during highly active antiretroviral
therapy. Clin Exp Immunol. 2005;141(3):526–533.
16. Wherry EJ, Blattman JN, Murali-Krishna K, van der
MR, Ahmed R. Viral persistence alters CD8 T-cell
immunodominance and tissue distribution and
results in distinct stages of functional impairment.
J Virol. 2003;77(8):4911–4927.
17. Zajac AJ, et al. Viral immune evasion due to persis-
tence of activated T cells without effector function.
J Exp Med. 1998;188(12):2205–2213.
18. Oehen S, Brduscha-Riem K, Oxenius A, Odermatt
B. A simple method for evaluating the rejection of
grafted spleen cells by flow cytometry and tracing
adoptively transferred cells by light microscopy.
J Immunol Methods. 1997;207(1):33–42.
19. Moskophidis D, Lechner F, Pircher H, Zinkernagel
RM. Virus persistence in acutely infected immuno-
competent mice by exhaustion of antiviral cytotoxic
effector T cells. Nature. 1993;362(6422):758–761.
20. Yang H, Yogeeswaran G, Bukowski JF, Welsh RM.
Expression of asialo GM1 and other antigens and
glycolipids on natural killer cells and spleen leuko-
cytes in virus-infected mice. Nat Immun Cell Growth
21. Jones AT, et al. Characterization of the activation-asso-
ciated isoform of CD43 on murine T lymphocytes.
J Immunol. 1994;153(8):3426–3439.
22. Lin Y, Roberts TJ, Sriram V, Cho S, Brutkiewicz RR.
Myeloid marker expression on antiviral CD8+ T cells
following an acute virus infection. Eur J Immunol.
23. Taniguchi RT, Guzior D, Kumar V. 2B4 inhibits
NK-cell fratricide. Blood. 2007;110(6):2020–2023.
24. Gonzalez-Cabrero J, Wise CJ, Latchman Y, Freeman
GJ, Sharpe AH, Reiser H. CD48-deficient mice have
a pronounced defect in CD4(+) T cell activation.
Proc Natl Acad Sci U S A. 1999;96(3):1019–1023.
25. Barber DL, et al. Restoring function in exhausted
CD8 T cells during chronic viral infection. Nature.
26. Day CL, et al. PD-1 expression on HIV-specific T
cells is associated with T-cell exhaustion and disease
progression. Nature. 2006;443(7109):350–354.
27. Urbani S, et al. PD-1 expression in acute hepatitis C
virus (HCV) infection is associated with HCV-specific
CD8 exhaustion. J Virol. 2006;80(22):11398–11403.
28. Boni C, et al. Characterization of hepatitis B virus
(HBV)-specific T-cell dysfunction in chronic HBV
infection. J Virol. 2007;81(8):4215–4225.
29. Ha SJ, et al. Enhancing therapeutic vaccination by
blocking PD-1-mediated inhibitory signals during
chronic infection. J Exp Med. 2008;205(3):543–555.
30. Nakamoto N, et al. Functional restoration of HCV-
specific CD8 T cells by PD-1 blockade is defined by
PD-1 expression and compartmentalization. Gas-
troenterology. 2008;134(7):1927–1937, 1937e1–e2.
31. Velu V, et al. Enhancing SIV-specific immunity in vivo
by PD-1 blockade. Nature. 2009;458(7235):206–210.
32. Andrews DM, Scalzo AA, Yokoyama WM, Smyth
MJ, gli-Esposti MA. Functional interactions
between dendritic cells and NK cells during viral
infection. Nat Immunol. 2003;4(2):175–181.
33. Andoniou CE, et al. Interaction between conven-
tional dendritic cells and natural killer cells is inte-
gral to the activation of effective antiviral immunity.
Nat Immunol. 2005;6(10):1011–1019.
34. Robbins SH, et al. Natural killer cells promote early
CD8 T cell responses against cytomegalovirus. PLoS
35. Bukowski JF, Warner JF, Dennert G, Welsh RM.
Adoptive transfer studies demonstrating the anti-
viral effect of natural killer cells in vivo. J Exp Med.
36. Bukowski JF, Woda BA, Habu S, Okumura K,
Welsh RM. Natural killer cell depletion enhances
virus synthesis and virus-induced hepatitis in vivo.
J Immunol. 1983;131(3):1531–1538.
37. Bukowski JF, Woda BA, Welsh RM. Pathogenesis of
murine cytomegalovirus infection in natural killer
cell-depleted mice. J Virol. 1984;52(1):119–128.
38. Tay CH, Szomolanyi-Tsuda E, Welsh RM. Control
of infections by NK cells. Curr Top Microbiol Immunol.
39. Biron CA, Nguyen KB, Pien GC, Cousens LP,
Salazar-Mather TP. Natural killer cells in anti-
viral defense: function and regulation by innate
cytokines. Annu Rev Immunol. 1999;17:189–220.
40. Masopust D, Murali-Krishna K, Ahmed R. Quantitat-
ing the magnitude of the lymphocytic choriomenin-
gitis virus-specific CD8 T-cell response: it is even big-
ger than we thought. J Virol. 2007;81(4):2002–2011.
41. Lau LL, Jamieson BD, Somasundaram T, Ahmed R.
Cytotoxic T-cell memory without antigen. Nature.
42. Karre K. NK cells, MHC class I molecules and the
missing self. Scand J Immunol. 2002;55(3):221–228.
43. de Boer RJ, Homann D, Perelson AS. Different
dynamics of CD4+ and CD8+ T cell responses dur-
ing and after acute lymphocytic choriomeningitis
virus infection. J Immunol. 2003;171(8):3928–3935.
44. Whitmire JK, Benning N, Whitton JL. Precursor
frequency, nonlinear proliferation, and functional
maturation of virus-specific CD4+ T cells. J Immunol.
45. Rabinovich BA, et al. Activated, but not resting, T
cells can be recognized and killed by syngeneic NK
cells. J Immunol. 2003;170(7):3572–3576.
research article Download full-text
1938?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 6 June 2010
46. Tangye SG, Lazetic S, Woollatt E, Sutherland
GR, Lanier LL, Phillips JH. Cutting edge: human
2B4, an activating NK cell receptor, recruits
the protein tyrosine phosphatase SHP-2 and
the adaptor signaling protein SAP. J Immunol.
47. Sayos J, et al. The X-linked lymphoprolifera-
tive-disease gene product SAP regulates signals
induced through the co-receptor SLAM. Nature.
48. Engel P, Eck MJ, Terhorst C. The SAP and SLAM
families in immune responses and X-linked
lymphoproliferative disease. Nat Rev Immunol.
49. Morra M, et al. X-linked lymphoproliferative dis-
ease: a progressive immunodeficiency. Annu Rev
50. Crotty S, McCausland MM, Aubert RD, Wherry
EJ, Ahmed R. Hypogammaglobulinemia and
exacerbated CD8 T-cell-mediated immunopathol-
ogy in SAP-deficient mice with chronic LCMV
infection mimics human XLP disease. Blood.
51. Yin L, et al. Mice deficient in the X-linked lympho-
proliferative disease gene sap exhibit increased
susceptibility to murine gammaherpesvirus-
68 and hypo-gammaglobulinemia. J Med Virol.
52. Chlewicki LK, Velikovsky CA, Balakrishnan
V, Mariuzza RA, Kumar V. Molecular basis of
the dual functions of 2B4 (CD244). J Immunol.
53. Suzuki A, et al. Functional SNPs in CD244 increase
the risk of rheumatoid arthritis in a Japanese popu-
lation. Nat Genet. 2008;40(10):1224–1229.
54. Mellors JW, et al. Plasma viral load and CD4+ lym-
phocytes as prognostic markers of HIV-1 infection.
Ann Intern Med. 1997;126(12):946–954.
55. Welsh RM Jr, Lampert PW, Burner PA, Oldstone
MB. Antibody-complement interactions with puri-
fied lymphocytic choriomeningitis virus. Virology.
56. Yang HY, Dundon PL, Nahill SR, Welsh RM. Virus-
induced polyclonal cytotoxic T lymphocyte stimu-
lation. J Immunol. 1989;142(5):1710–1718.
57. Oxenius A, Bachmann MF, Ashton-Rickardt PG,
Tonegawa S, Zinkernagel RM, Hengartner H. Pre-
sentation of endogenous viral proteins in associa-
tion with major histocompatibility complex class II:
on the role of intracellular compartmentalization,
invariant chain and the TAP transporter system.
Eur J Immunol. 1995;25(12):3402–3411.
58. van der Most RG, et al. Identification of Db-
and Kb-restricted subdominant cytotoxic T-cell
responses in lymphocytic choriomeningitis virus-
infected mice. Virology. 1998;240(1):158–167.
59. Mylin LM, et al. Quantitation of CD8(+) T-lympho-
cyte responses to multiple epitopes from simian
virus 40 (SV40) large T antigen in C57BL/6 mice
immunized with SV40, SV40 T-antigen-trans-
formed cells, or vaccinia virus recombinants express-
ing full-length T antigen or epitope minigenes.
J Virol. 2000;74(15):6922–6934.
60. Assarsson E, et al. NK cells stimulate proliferation
of T and NK cells through 2B4/CD48 interactions.
J Immunol. 2004;173(1):174–180.
61. Chen HD, Fraire AE, Joris I, Welsh RM, Selin LK. Spe-
cific history of heterologous virus infections deter-
mines anti-viral immunity and immunopathology in
the lung. Am J Pathol. 2003;163(4):1341–1355.
62. Daniels KA, Devora G, Lai WC, O’Donnell CL,
Bennett M, Welsh RM. Murine cytomegalovirus is
regulated by a discrete subset of natural killer cells
reactive with monoclonal antibody to Ly49H. J Exp