Science - USA (2022-05-27)

This is reminiscent of B7’s interactions with
both CD28 (promoting T cell activation) and
CTLA-4 (suppressing T cell activation) ( 20 ).
PILRahas been shown to interact with sev-
eral transmembrane proteins such as NPDC1,
COLEC12 ( 21 ), and PANP ( 22 ), although these
proteins are not thought to be expressed by
T cells. PILRamay also bind CD99 ( 17 ), which
is highly expressed on B cells, T cells, and mac-
rophages ( 23 ) and which has been shown to
act as a costimulatory receptor on T cells ( 24 ).
Future studies characterizing PILRa’s possi-
ble interactions with other non-CD8arecep-
tors will be necessary to test this hypothesis.
The effect of CD8adeletion on CD8+T cell
development may be partial because the
quantityofCd8a−/−cytotoxic lymphocytes (CTLs)
in the spleen was decreased by only half in the
absence of MHC II ( 25 ). We established a
role for the PILRa–CD8ainteraction in mature

CD8+T cell quiescence, but its function in thymic

CD8+T cell development was unclear. To eval-

uate this, we treated pregnant mice and sub-

sequent neonates with anti-PILRamAb 9B12.

Thymic CD8+T cell development was normal

at postnatal day 14 (fig. S32A), whereas CD8+

T cells in the spleens of 9B12-treated neonates

were decreased significantly as expected (fig.

S32B). We also tested whether the PILRa–CD8a

interaction could affect thymic negative selec-

tion. BALB/c mice have been widely used as

a model of T cell negative selection owing to

this strain’s integration of mouse mammary

tumorvirustypes6,8,and9,resultinginthe

deletion of thymocytes bearing TCR Vb3, V b5,

Vb11, and Vb12 ( 26 – 28 ). CD8+Tcellnegative

selectioninthethymuswasunaffectedby9B12

mAb treatment of BALB/c mice (fig. S32C).

Thus, the PILRa–CD8ainteraction functions in

peripheral lymphoid organs but not in the thymus.

Our results support the notion that the qui-

escent state of T cells, especially CD8+T cells,

is actively maintained by constant receptor–

ligand interactions on the cell surface even

in the absence of antigen stimulation. PILRa

is broadly expressed on various types of mye-

loid cells ( 18 , 19 )andCD8ais highly expressed

on naïve and memory CD8+T cells, provid-

ing ample opportunity for this interaction to

occur in peripheral lymphoid organs. Sim-

ilarly, a recent study shows that cell surface

PD-1H/VISTA on naïve CD4+T cells may reg-

ulate T cell quiescence ( 29 ). Memory CD8+

T cells require the PILRa–CD8ainteraction

to maintain this quiescent status, suggesting

that this interaction may contribute to de-

tuning memory CD8+T cells during antigen-

induced activation and help them return to

a normal state after the resolution of an

immune response. Our findings therefore

Zhenget al., Science 376 , 996–1001 (2022) 27 May 2022 4of6

PILRα

FcR

A negative PILRα FcR

B

293T/mock

293T/h-CD8A

293T/mock

293T/h-PILRA

C

E G

flag-hIg

h-PILRα-hIg

0

0.5

1.0

1.5

2.0

0 5 10

flag-mIg

m-PILRα-mIg

0

1

2

3

4

5

0 5 10

D

F

293T/mock 293T/mock+ctrl Ig

293T/m-Cd8a+ctrl Ig

293T/m-Cd8a+9B12

293T/m-Cd8a+3D9

293T/m-Pilra

OD (450)

biotin h-CD8α-hIg (μg/ml)

OD (450)

biotin m-CD8α-his (μg/ml)

Fig. 3. Identification of PILRaas a ligand for CD8ain both mice and humans.
(A) Screening for the binding partner of human CD8a-hIg fusion protein against
a human transmembrane protein library on the Mirrorball Fluorescence Cytometer.
Representative images of 384-well plates (left) and single wells (right) are shown.
(B) Flow cytometry analysis of human CD8a-hIg fusion protein binding to mock or
humanPILRA-transfected 293T cells. (C) Flow cytometry analysis of human PILRa-
mIg binding to mock or humanCD8A-transfected 293T cells. (D)ELISAofbiotin-
labeled human CD8a-hIg binding to immobilized flag-hIg or human PILRa-hIg.

(E) ELISA of biotin-labeled mouse CD8a-his binding to immobilized flag-mIg or mouse

PILRa-mIg. (F) Flow cytometry analysis of mouse CD8a-hIg fusion protein binding

to mock or mousePilra-transfected 293T cells. (G) Flow cytometry analysis of

allophycocyanin (APC)-labeled mouse PILRa-hIg protein binding to mock or mouse

Cd8a-transfected 293T cells in the presence of anti-mouse CD8amAb 3D9 or anti-

mouse PILRamAb 9B12. In (D) and (E), values are mean ± SD, and three replicates

are included in each concentration. Representative data of two (D), three (E),

and at least four [(B), (C), (F), and (G)] independent experiments are shown.

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