IMMUNOLOGY
The CD8a–PILRainteraction maintains CD8
T cell quiescence
Linghua Zheng^1 ,XueHan^1 ,ShengYao^1 ,YuwenZhu^1 ,JohnKlement^2 ,ShirleyWu^2 ,LanJi^1 , Gefeng Zhu^1 ,
Xiaoxiao Cheng^1 ,ZuzanaTobiasova^1 ,WeiweiYu^1 ,BaozhuHuang^1 , Matthew D. Vesely^1 ,JunWang^1 ,
Jianping Zhang^1 , Edward Quinlan^1 , Lieping Chen^1 *
T cell quiescence is essential for maintaining a broad repertoire against a large pool of diverse
antigens from microbes and tumors, but the underlying molecular mechanisms remain largely
unknown. We show here that CD8ais critical for the maintenance of CD8+T cells in a physiologically
quiescent state in peripheral lymphoidorgans. Upon inducible deletion of CD8a,bothnaïve
and memory CD8+T cells spontaneously acquired activation phenotypes and subsequently died
without exposure to specific antigens. PILRawas identified as a ligand for CD8ain both
mice and humans, and disruption of this interaction was able to break CD8+T cell quiescence.
Thus, peripheral T cell pool size is actively maintained by the CD8a–PILRainteraction in the
absence of antigen exposure.
C
D8+T cells are major effector cells with-
in the immune system for eliminating
infected and malignant cells. Therefore,
it is important to maintain a large and
diverse repertoire of CD8+T cells within
lymphoid organs prior to their enlistment.
T cells are actively maintained in a metabol-
ically inactive or quiescent state to ensure
survival ( 1 ). Genetic deficiency of the intra-
cellular proteins FOXP1, TSC1, SLFN2, and
BTG1/BTG2, as well as inducible loss of regu-
latory T cells, has been shown to abrogate T cell
quiescence ( 1 – 3 ). However, the extracellular
signals that sustain CD8+T cell quiescence
remain unknown.
The transmembrane protein CD8 is a hetero-
dimer composed ofaandbsubunits linked
by disulfide bonds and serves as a lineage
marker to identify a major subset of T cells
with cytotoxic capacity ( 4 , 5 ). CD8ainteracts
with the major histocompatibility complex
class I (MHC I) via its extracellular domain and
stabilizes lymphocyte-specific protein tyrosine
kinase (Lck) via its intracellular domain ( 6 , 7 ).
CD8 is thought to primarily function by de-
livering Lck to the T cell receptor (TCR) to
strengthen TCR signaling upon antigen stim-
ulation ( 8 ). CD8ais also required for CD8+
T cell development in the thymus because
genetic deletion of CD8aleads to a nearly com-
plete elimination of cytotoxic T cells ( 9 ).
To study the potential functions of CD8ain
the periphery, we generatedCd8aloxp/loxpmice,
which were subsequently backcrossed with
tamoxifen-inducibleCre-ERT2transgenic mice
(fig. S1, A and B). CD8+T cells purified from
Cre+/+Cd8aloxp/loxpmice were adoptively trans-
ferred into wild-type (WT) mice followed by
tamoxifen treatment. This allowed the func-
tion of CD8aon peripheral CD8+T cells to be
studied while avoiding any effects on thymic
development. As expected, tamoxifen treat-
ment abolished the expression of both CD8a
and CD8bon adoptively transferred CD8+
T cells (fig. S1C). Thus, CD8bsurface expres-
sion depends on CD8aexpression, consistent
with a previous report ( 10 ). Both TCR and CD3
expression remained unchanged after CD8a
deletion (fig. S1C). Tamoxifen-induced dele-
tion of CD8aled to the progressive loss of CD8+
T cells, especially those proliferating cells in
the blood, lymph node, and spleen (fig. S2, A to
D). Thirty-four days after tamoxifen treatment,
memory CD8+T cell survival was decreased
by 30 to 40% (Fig. 1, A and B). Similarly, naïve
CD8+T cell survival was decreased by 30 to
40% (Fig. 1, C and D) and 80% (fig. S3), re-
spectively, 36 and 55 days after tamoxifen treat-
ment. By contrast, without tamoxifen treatment,
CD8+Tcells(Cre+/+Cd8a+/+or Cre+/+Cd8aloxp/loxp)
showed equivalent survival (fig. S4, A and B).
There was also increased expression of the
T cell activation markers CD69 and Fas (fig.
S5, A and B, and Fig. 2, A and B) and decreased
expression of CD127, CD122, and CD5 (fig. S5,
C to E, and Fig. 2, C to E) onCd8a−/−memory
T cells. Similarly, inducible CD8adeletion re-
sulted in the up-regulation of CD69 and Fas
(fig. S6, A and B) and the down-regulation of
CD127, CD122, and CD5 (fig. S6, C to E) on
naïve CD8+T cells. Moreover, the inducible
deletion of CD8aincreased the proportion of
naïve T cells that exited the G 0 phase (fig. S7),
indicative of T cell activation. Thus, upon in-
ducible CD8adeletion, CD8+T cells acquire a
loss-of-quiescence phenotype without specific
antigen stimulation. Because cell surface CD5
transmits a tuning signal for TCR ( 11 ), decreased
CD5 expression onCd8a−/−T cells suggested
dampened TCR signaling. Similarly, Nur77, an
immediate early protein downstream of TCRpathway ( 12 ), was also decreased in naïve and
memoryCd8a−/−Tcells(fig.S8,AandB).Thus,
the activation or loss of quiescence induced by
CD8adeletion is not caused by TCR triggering.
ThedecreaseofCd8a−/−T cells observed in
lymphoid organs was not due to their migra-
tion or redistribution to other tissues because
there was no detectable increase ofCd8a−/−
T cells in nonlymphoid tissues (fig. S9, A and
B). Cleaved caspase-3 expression was increased
in Cd8a−/−T cells (fig. S10, A and B), indicating
activation of caspase-3. Furthermore, deletion
of CD8aresulted in elevated Fas expression
by T cells (Fig. 2B and fig. S6B), and anti-Fas
antibody treatment ex vivo induced significantly
more apoptosis ofCd8a−/−naïve or memory
T cells (fig. S11, A and B). Thus, the decrease
of CD8+T cells upon CD8adeletion may be
caused by programmed cell death.
To validate these findings, we generated an
anti-mouse CD8amonoclonal antibody (mAb,
clone 3D9). Mice were thymectomized before
3D9 inoculation to specifically study the effect
of 3D9 on mature peripheral T cells. 3D9 de-
creased the survival and increased the activa-
tion of CD8+T cells in thymectomized mice
(fig. S12, A to C). 3D9 also enhanced the cell
cycle transition from G 0 to G 1 by transferred
naïve CD8+T cells (fig. S13). Thus, 3D9 in-
duces a loss-of-quiescence phenotype similar
to that caused by CD8adeletion. To exclude
an agonistic or depletion effect of 3D9, the
genes encoding the Fab fragment of 3D9 were
cloned and expressed in vivo by intravenous
hydrodynamic gene delivery ( 13 ). 3D9 Fab
treatment reduced the survival of transferred
memory (fig. S14, A and B) and naïve (fig. S14,
A and C) CD8+T cells and increased the acti-
vation of memory (fig. S15, A and B) and naïve
(fig.S16,AandB)CD8+Tcells.
The loss of quiescence by genetic deletion or
antibody blockade suggested that CD8amay
interact with an unknown ligand to mediate this
function. We used a high-throughput genome-
scale receptor array ( 14 – 16 )tosearchthislig-
andbytestingthebindingofhumanCD8ato
around 6000 human transmembrane proteins
that were individually displayed on the surface
of 293T cells. The paired immunoglobulin-like
type 2 receptor alpha (PILRa) was identified
as a binding partner (Fig. 3A), and this inter-
action was confirmed by flow cytometry and
enzyme-linked immunosorbent assay (ELISA)
(Fig. 3, B to D). This CD8a–PILRainteraction
was also observed in mice (Fig. 3, E to G). The
CD8a–PILRainteraction appeared to be spe-
cific because CD8adid not bind PILRb,aclose
homolog of PILRa( 17 ), in either mice or hu-
mans (fig. S17, A and B). Both 3D9 mAb and
3D9 Fab could compete with CD8abinding to
PILRa(Fig. 3G and fig. S17C), suggesting that
the effect of 3D9 or its Fab fragment in vivo
may have been due to a blockade of the CD8a–
PILRainteraction.RESEARCH
Zhenget al., Science 376 , 996–1001 (2022) 27 May 2022 1of6
(^1) Department of Immunobiology, Yale University School of
Medicine, New Haven, CT, USA.^2 Yale College, Yale
University, New Haven, CT, USA.
*Corresponding author. Email: [email protected]