decreased 2 days after 9B12 treatment (fig.
S18D). In a complementary approach, treat-
ment with 9B12 reduced the absolute number
of carboxyfluorescein diacetate succinimidyl
ester (CFSE)–labeled and transferred CD8+
T cells, but not CD4+T cells, in both lymph
nodes and the spleen 32 days after treatment
(fig. S19). To determine the effect of 9B12 on
individual CD8+T cell subsets, we transferred
naïve or memory CD8+T cells into mice. 9B12
treatment reduced the survival and broke the
quiescence of both naïve (fig. S20, A and B)
and memory (fig. S21, A and B) CD8+T cells.
9B12 had minimal effects on Fas, CD127, and
CD122 expression (fig. S20, C to E, and fig.
S21, C to E). Moreover, 9B12 enhanced G 0
phase exit in transferred naïve CD8+T cells
(fig. S22). Soluble PILRacould not be de-
tected in mouse sera (fig. S23), suggesting
that cell–cell interactions were important
in maintaining CD8+T cell quiescence. We
cannot explain the rapid effect of 9B12 on
thelossofquiescenceofCD8+T cells in the
lymph node compared with its delayed effect
in the spleen, but the contribution of tissue-
specific signals in these organs cannot pre-
sently be excluded.
We next induced bona fide monoclonal
memory CD8+T cells from OT-I TCR trans-
genic mice using ovalbumin peptide (OVA)
antigen. As described above for polyclonal
memory CD8+T cells, the quiescence of antigen-
specific memory OT-I CD8+T cells was also
broken after 9B12 (fig. S24, A and B) or 3D9
(fig.S25,AandB)antibodytreatment.Tostudy
naïve OT-I T cell quiescence, we backcrossed
OT-I transgenic mice to theCre+/+Cd8aloxp/loxp
strain. The inducible deletion of CD8adis-
rupted naïve OT-I CD8+T cell quiescence and
significantly reduced OT-I CD8+T cell survival
in the absence of OVA antigen stimulation (fig.
S26, A to D). Similarly, treatment with anti-PILRa
9B12 resulted in up-regulated CD69 expression
on naïve OT-I T-cells, which was associated
with decreased survival (fig. S27, A and B).
The ability of 9B12 to alter CD8+Tcellqui-
escence was dependent on PILRa,asthiseffect
was abolished inPilra−/−mice (figs. S28, A
and B, and S29D). Consistent with a previous
report ( 19 ), we did not observe any changesin the frequency of memory or naïve CD8+or
CD4+T cells in peripheral lymphoid organs
in 3-month-oldPilra−/−mice (fig. S29, A to C).
CD8+T cell quiescence inPilra−/−mice also
remained unchanged (fig. S29D). Moreover,
purified memory and naïve CD8+T cells from
WT mice did not show decreased survival after
transfer intoPilra−/−mice (fig. S30, A and B).
Although the differences were not pronounced
in 6-month-old mice (fig. S31A), the frequency
of memory CD8+T cells was decreased sig-
nificantly by 20 to 30% in both lymph nodes
and spleens ofPilra−/−mice in comparison with
controlsat1yearofage(fig.S31B).Bycontrast,
the frequency of naïve CD8+T cells or CD4+
T cells remained unchanged in these mice
(fig. S31B). Thus, the genetic ablation of PILRa
in mice results in a partial phenotype as com-
pared to the mAb against CD8aor PILRa.One
possible interpretation for this discrepancy is
that while interacting with CD8ato main-
tain T cell quiescence, PILRamay also engage
other receptors promoting T cell activation,
which may partially or completely counter
the effect of the PILRa–CD8ainteraction.Zhenget al., Science 376 , 996–1001 (2022) 27 May 2022 3of6
**
05101520*
A
**120014001600180020002200**
12001400160018002000*
6008001000120014001600****
6008001000120014002500300035004000(^4500) *
2500
3000
3500
(^4000) **
BCD
3500
4000
4500
5000
5500
6000
6500
E
3500
4000
4500
5000
5500
6000
Cd8a+/+ Cd8aloxp/loxp
lymph node
CD69
- %CD69
- %
spleen
0
5
10
15
20
FAS MFI
lymph node
FAS MFI
spleen
CD122 MFI
spleen
lymph node
CD122 MFI CD127 MFI
lymph node
CD127 MFI
spleen
CD5 MFI
lymph node
CD5 MFI
spleen
Cd8a+/+ Cd8aloxp/loxp
Cd8a+/+ Cd8aloxp/loxp
Cd8a+/+ Cd8aloxp/loxp
Cd8a+/+ Cd8aloxp/loxp
Cd8a+/+ Cd8aloxp/loxp
Cd8a+/+ Cd8aloxp/loxp
Cd8a+/+ Cd8aloxp/loxp
Cd8a+/+ Cd8aloxp/loxp Cd8a+/+ Cd8aloxp/loxp
Fig. 2. CD8amaintains memory CD8+T cell quiescence and CD127 and CD122 expression in the periphery.The lymph node and spleen cells analyzed here were from
the same populations as those used in Fig. 1, A and B. As described in Fig. 1, A and B, memory CD45.2+CFSE+T cells were gated and analyzed for (A)CD69+percentage, (B)FAS
MFI, (C)CD122MFI,(D)CD127MFI,and(E)CD5MFI.Seealsofig.S5forrepresentativeflow cytometry plots and histograms. MFI:mean fluorescence intensity. Mean ± SD is
shown. P <0.05,P < 0.01, P < 0.001, ****P <0.0001byunpairedStudent’s t test. n = 5 mice per group. Data are representative of three independent experiments.
RESEARCH | REPORT