exposure 5 weeks after birth is insufficient to
alter the methylation ofCxcl16and minimize
the accumulation of iNKT cells ( 13 ). Thus, epi-
thelial cells are permissive to epigenetic modi-
fications in early life that consequently have
long-lasting effects on the abundance of iNKT
cells in barrier tissues and predispose animals
to immune pathologies.
In addition to imprinting the frequency of
iNKT cells through the regulation of chemo-
kines, the microbiota also provides antigens
that can directly modulate the development
of iNKT cells during the first 2 weeks of life.
The intestinal symbiontBacteroides fragilis
expresses sphingolipids that inhibit iNKT ac-
tivation by preventing CD1d-mediated pre-
sentation of agonistic lipids ( 3 ), including
endogenous ligands that mediate iNKT selec-
tion in the absence of commensals. As such,
early-life exposure toB. fragilisprevents the
accumulation of iNKT cells within the colon,
but not the lungs, of the progeny, indicating
that the inhibitory effects of the sphingoli-
pids are localized to the predominant site of
B. fragiliscolonization ( 3 ). Although these
animals are protected from experimentally
induced colitis once they become adults, col-
onization of GF mice at 2 weeks of age is in-
sufficient to inhibit iNKT cell proliferation,
resulting in a higher frequency of these pro-
inflammatory T cells and rendering the adult
animals more susceptible to inflammation ( 3 ).
As well as inhibiting the accumulation of
iNKT cells in the colon via sphingolipids,
B. fragilisalso regulates the abundance of
other unconventional T cells through another
microbial molecule, polysaccharide A (PSA).
Using the transcription factor PLZF to identify
innate and innate-like lymphocytes ( 14 , 15 ),
B. fragiliscolonization was found to increase
the frequency and number of PLZF+innate-
likeabT cells and PLZF+innate lymphocytes
(TCRb–TCRgd–) within the neonatal thymus
in a PSA-dependent manner ( 16 ). Although
antibiotic treatment of adult mice did not
alter the abundance of PLZF+innate-like cells
within the thymus, mice treated with anti-
biotics until weaning exhibited a reduced
number of these thymic lymphocytes as adults
and were more susceptible to experimentallyinduced colitis ( 16 ). Thus, microbial coloni-
zation during the neonatal period imprints
the abundance of iNKT cells in barrier tis-
sues, which can have enduring effects on host
physiology.
In contrast to mice, where iNKT cells are
abundant, the predominant unconventional
abT cells in humans are MAIT cells, which
recognize intermediates of vitamin B2 (ribo-
flavin) synthesis presented by MR1 ( 17 ). Be-
cause riboflavin synthesis is broadly conserved
among bacteria and fungi, MAIT cells respond
to a diverse array of microbes, including known
commensals ( 4 , 18 ). Genetically identical mice
housed in distinct cages exhibit a striking var-
iability in MAIT cell abundance ( 4 ), which is
suggestive of a dependence on the microbiota
and parallels the variability in MAIT cell fre-
quency between human individuals ( 19 , 20 ).
In further support of a role for the microbiota,
mice devoid of live microbes have fewer thymic
CD24+CD44–MAIT cells ( 21 ). Additionally,
these immature MAIT cells express reduced
levels of Nur77, a transcription factor that cor-
relates with TCR signal strength, indicating
that microbial antigens promote thymic selec-
tion of MAIT cells ( 21 ). Consequently, MAIT
cellsarenearlyabsentfromperipheraltissues
in animals that are either GF or colonized with
commensals that do not synthesize riboflavin
( 4 , 21 – 23 ). Among all lymphocyte subsets,
MAIT cells are the most affected by the ab-
sence of the microbiota ( 4 ). In convention-
ally housed mice, MAIT cells accumulate in
barrier tissues before weaning, and neonatal
colonization of GF animals with riboflavin-
synthesizing intestinal commensals results
in a comparable number of MAIT cells at these
sites ( 4 , 21 ). However, although colonization
of adult GF mice promotes the maturation of
MAIT cells in the thymus, microbial expo-
sure later in life is insufficient to induce MAIT
cells in peripheral tissues ( 4 , 21 ), indicating
that MAIT cell abundance in tissues is im-
printed by the microbiota in early life. Topical
application of purified riboflavin intermedi-
ates on neonatal, but not adult, GF mice also
results in the maturation of MAIT cells in the
thymus and their accumulation in tissues
( 4 , 21 ). Thus, these ligands are sufficient to
induce MAIT cells in the absence of additional
microbial antigens and can derive from com-
mensals at other barrier surfaces, including
the skin. The systemic effects of riboflavin de-
rivatives on MAIT cell development parallel
the distal impact of other microbial metab-
olites, including short-chain fatty acids, aryl
hydrocarbon receptor ligands, polyamines, and
PSA ( 24 ). In utero transfer of these metabolites
from the maternal microbiota would explain
the seemingly paradoxical presence of func-
tionally mature MAIT cells in human fetal
tissue before microbial colonization ( 5 ). Entero-
bacteriaceae, a family of Gram-negative bacteriaConstantinides and Belkaid,Science 374 , eabf0095 (2021) 10 December 2021 2of6
Intestine17%Liver38%Lungs6%Skin11%Blood6%Intestine8%Liver41%Lungs12%12%Skin91%Spleen / Blood5%5%Fig. 2. Frequency of unconventional T cells.Unconventional T cells are enriched in barrier tissues and
organs that drain these sites, such as the liver. The cumulative frequency of MAIT, iNKT, andgdT cells
among all T cells is shown for the indicated tissues of humans and mice.
Fig. 1. Emergence of unconventional T cells.The emergence of unconventional T cells during murine
development correlates with the evolutionary age of the MHC molecules they recognize.
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