the human skin forms a barrier to the external environment that is constantly exposed to colonizing microbiota , invasive pathogens , and allergens ( belkaid and segre , 2014 , pasparakis et  al . ,
these encounters drive differentiation and colonization of tissue - resident memory t ( trm ) cells ( gaide et  al . , 2015 ,
2012 ) , originally characterized in barrier tissues such as skin , gut , lung , and the female genital tract ( gaide et  al . , 2015 ,
, 2014 , watanabe et  al . , 2015 ) . of the 20 billion t  cells contained in human skin ( clark et  al . , 2006 ) , 50%70% express the trm cell markers cd103 ( -subunit of the e7 integrin receptor ) and cd69 ( watanabe et  al . , 2015 ) , both implicated in lodging trm cells in peripheral tissues .
cd103 binds e - cadherin , which is highly expressed on epithelia , whereas cd69 antagonizes sphingosine 1-phosphate receptor 1 ( s1pr1)-mediated egress from tissues ( mackay et  al . , 2015 ,
trm cells provide strong defense against recurrent infections ( ariotti et  al . , 2014 ,
2014 ) . while such local immune responses contribute to immunity ( gebhardt et  al . , 2009 ,
2012 ) , aberrant activation might cause disease ( hondowicz et  al . , 2016 , jabri and abadie , 2015 , park and kupper , 2015 ) . in the skin , the patchy appearance of several t  cell - mediated diseases , such as psoriasis and vitiligo ( boehncke and schn , 2015 , ezzedine et  al . ,
2015 ) , suggests that tissue - resident rather than circulating cells drive immunopathology ( cheuk et  al . , 2014 ,
demarcated , inflamed and hyperproliferative plaques are maintained by interleukin-23 ( il-23 ) and il-17 in psoriasis ( hueber et  al . , 2010 ,
leonardi et  al . , 2008 ) , whereas vitiligo presents with persistent depigmentations attributed to local interferon- ( ifn- ) production and t  cell - mediated killing of melanocytes ( harris et  al .
thus , if trm cells drive these diseases , then vitiligo and psoriasis would be expected to involve the contribution of functionally different subsets .
trm cell subsets with distinct cytokine profiles have been reported in skin ( naik et  al . , 2015 ,
sanchez rodriguez et  al . , 2014 , watanabe et  al . , 2015 ) , possibly reflecting a diversity of  immune functionalities and responses to pathogenic and commensal microbiota
nonetheless , phenotypic markers of trm cell subsets with specialized effector functions have not been established .
in addition to cd103 and cd69 , murine trm cells that create local immunity to recurrent viral infection homogenously express cd49a ( gebhardt et  al . , 2009 , ray et  al . , 2004 ,
cd49a constitutes the -subunit of the 11 integrin receptor , also known as very late antigen 1 ( vla-1 ) , and binds collagen iv enriched in the basement membrane separating epidermis and dermis . as only 15% of human skin - derived t  cells express cd49a ( purwar et  al .
, 2011 ) , cd49a expression might distinguish trm cell subsets with distinct effector functions and putative roles in immunopathology .
here , we determined the anatomical localization , transcriptional profiles , and functional properties of trm cell subsets in human skin with respect to cd49a , cd69 , and cd103 . in healthy individuals , cd8cd103cd49a trm cells were present in both  the dermis and epidermis , whereas cd8cd103cd49a trm  cells specifically localized to the epidermis .
epidermal cd8cd103cd49a trm cells were the predominant il-17-producers ,  whereas cd8cd103cd49a trm cells excelled at ifn- production and rapidly gained a cytotoxic capacity following il-15 stimulation .
this functional dichotomy was evident in the comparison of distinct immune - mediated skin diseases , with skin biopsies from vitiligo patients showing a predominance of cytotoxic cd8cd103cd49a trm cells while skin biopsies from psoriasis patients featured the accumulation of the il-17 producing cd8cd103cd49a counterparts .
together , our results reveal a functional specialization of distinct skin trm cell subsets in health and disease .
to gain insights into the diversity among trm cells with respect to  spatial niches , we determined the relative expression of tissue - residency markers cd69 , cd103 , and cd49a on cd4 and cd8 t  cells from healthy human skin ( figure  s1a ) . compared to the dermis , an increased proportion of epidermal cd4 and cd8 t  cells co - expressed cd69 and cd103 ( figures 1a and 1b ) as previously reported ( watanabe et  al . , 2015 ) .
in these healthy skin samples , cd49a expression was restricted to cd8cd69cd103 t  cells that were located in the epidermis ( figures 1a and 1c ) .
considerable variation in the frequencies of  cd8cd69cd103cd49a t  cells was apparent among ( figure  1c ) and within different donors ( figures s1b and s1c ) , indicating high inter- and intra - individual variation of trm cell compositions within the epidermal compartment .
skin cd4 trm cells were predominately cd62lcd45racd28 , whereas cd8 trm cells were cd62l but heterogeneous with respect to expression of cd27 , cd28 , and cd45ra ( figures s1d and s1e ) . to delineate the microanatomical localization of skin cd8 t  cell subsets , we performed confocal imaging of healthy skin .
regardless of cd49a expression , epidermal cd8 t  cells were juxtaposed to the collagen iv - rich basement membrane , embedded among basal keratinocytes ( figures 1d and 1e ) .
perivascular dermal cd8 t  cells were  predominately detected in papillary dermis well separated from epidermis ( figure  1d ) . in total , 24% ( 6/33 ) of epidermal and  6% ( 3/53 ) of dermal cd8 t  cells expressed cd49a in direct  contact with the basement membrane ( figure  1e ; data not shown ) .
bulk t  cells in full thickness human skin display a diverse t  cell receptor ( tcr ) repertoire that is in equilibrium with that of circulating t  cells ( clark et  al . , 2006 ,
to determine whether epidermal cd8cd103cd49a trm cells shared a clonal origin with circulating effector memory cd8 t  cells or skin t  cell subsets , cd8 t  cell subsets from five healthy donors were sorted ( figures s1f and s1 g ) and  analyzed by targeted , deep , high - throughput genomic dna sequencing of the tcr complementarity - determining region 3 ( cdr3 ) .
a single v family dominated the epidermal cd8cd103cd49a trm cell population in three donors , with cdr3 diversity more restricted in cd8cd103cd49a versus cd8cd103cd49a trm cells ( figures 1f and 1 g ) .
although only a small proportion of the detected clonotypes overlapped between epidermal cd8cd103cd49a and cd8cd103cd49a trm cells ( figure  1h ) , a few dominant clones were shared and comprised the majority of total reads in two donors ( figures 1h and 1i ) . furthermore , whereas the most prevalent cd8cd103cd49a trm clonotypes showed some overlap with other epidermal and dermal cd8 t  cell subsets ( figure  1j ) , v family distribution differed in epidermal and dermal cd8cd103cd49a trm cells ( figure  1f ) .
the ten largest cd8cd103cd49a trm cell clones were highly enriched in the epidermis ( figure  1j ) .
although the mechanical procedure of  dermal and epidermal preparations might result in some degree of cellular cross - contamination , these results together demonstrate the epidermal localization of expanded , oligoclonal cd8cd103cd49a trm cells in human skin . to further interrogate the functional properties of epidermal cd8cd103cd49a and cd8cd103cd49a trm cell populations
, we determined their transcriptional profiles , along with those of dermal cd8cd103cd49a and cd8cd103cd49a t  cells as well as peripheral blood cd8cd62lcd45ra t  cell populations expressing or lacking the cutaneous leukocyte antigen ( cla ) .
cells were sorted from healthy donors ( figures s1f and s1 g ) and analyzed by rna - sequencing ( ramskld et  al . , 2012 ) .
principal component analysis on all sorted t  cells subsets from six donors distinguished circulating and skin t  cell populations ( figure  2a ) . among skin t  cells , principal component analysis discriminated epidermal cd8cd103cd49a and cd8cd103cd49a trm cells from dermal populations ( figure  2b ) . focusing on the epidermal trm cells , 92 genes were differentially expressed between cd8cd103cd49a and cd8cd103cd49a trm cells ( figure  2c , tables s1 and s2 ) . gene enrichment analysis ( david ) indicated acquisition of cytotoxicity - related function as the most compelling difference between the  populations ( figure  2d ) .
specifically , expression of prf1 , gzmb , gzmh , gzmk , gnly , and nkg7 transcripts , all encoding cytotoxic granule components , were elevated in epidermal cd8cd103cd49a trm cells ( figure  2e ) , indicating that this subset might mediate cellular cytotoxicity .
moreover , genes mediating response to viruses , lymphocyte activation , and chemotaxis were also upregulated in cd8cd103cd49a trm cells ( figure  2d ) .
conversely , gene enrichment analyses did not identify any significantly downregulated gene programs ( figure  2d ) .
nonetheless , transcripts of genes associated with il-17 production , such as il17f , rorc , il23r , and ccr6 , were significantly decreased in cd8cd103cd49a relative to cd8cd103cd49a trm cells , whereas transcripts for ifn- were elevated ( figures 2d - e ) .
transcription of itga1 , encoding cd49a , was not significantly different following correction for  multiple comparisons ( uncorrected p  = 0.0007 , adjusted p  =  0.12 ) .
quantitative pcr ( qpcr ) analysis demonstrated significantly higher itga1 transcription in cd8cd103cd49a versus cd8cd103cd49a trm cell subsets ( figure  2f ; p  =  0.002 ) .
moreover , regulation of gzmb , ifng , and ccr6 was confirmed by qpcr , whereas no difference in itgae , encoding cd103 , was found ( figure  2f ) . further validating transcriptional data ,
cxcr3 expression was higher on cd8cd103cd49a trm cells , whereas il-23r and ccr6 were preferentially expressed by cd8cd103cd49a trm cells ( figure  2 g ) .
as such , transcriptional profiles as well as surface receptor expression suggested functional differences between cd8cd103cd49a and cd8cd103cd49a trm cells . despite enrichment of transcripts for cytotoxic granule constituents in epidermal cd8cd103cd49a trm cells , perforin , and  granzyme
b protein expression was virtually undetectable when examined ex  vivo ( figures 3a and s2a and s2b ) .
we reasoned that in  situ inflammatory stimuli might be required to elicit expression of cytotoxic granule constituents in trm cell subsets , as recently proposed for il-15 ( jabri and abadie , 2015 ) .
following incubation of epidermal cell suspensions with a variety of proinflammatory cytokines relevant to skin inflammation , intracellular protein expression of perforin and granzyme b was detected in cd8cd103cd49a trm cells following il-2 and il-15 stimulation ( figures 3a3c ) .
granzyme b expression was upregulated as early as 1  hr after il-15 stimulation , whereas perforin expression was detected at later time points ( figure  3d ) .
antibody - mediated blockade of mhc class i did not diminish il-15-mediated granzyme b upregulation ( data not shown ) and other inflammatory cytokines , including il-1 , il-6 , il-7 , il-12 , il-23 , and ifn- , did not elicit expression of cytotoxic granule constituents ( figure  3 g ) . in an inflammatory milieu , several cytokines might interact for induction of cytotoxic granule constituent expression . accordingly ,
il-15-dependent expression of perforin and granzyme b was augmented by il-6 , but not other cytokine combinations tested ( figures s2c  s2e ) .
differential expression of il-2 and il-15 receptor components could have explained the preferential responses of epidermal cd8cd103cd49a trm cells .
however , the il-2 receptor  and  chains were expressed in both epidermal subsets ( figures s2f and s2 g ) .
thus , epidermal cd8cd103cd49a trm cells appeared quiescent in healthy skin , but il-2 from activated immune cells or il-15 from keratinocytes ( loser et  al . , 2004 )
besides the skin , trm cells are widely distributed in other barrier tissues . to determine whether cd49a expression more broadly defined trm cells with a cytotoxic potential , we examined two mucosal barriers , gut and cervix ( figure  s3a ) .
cd8cd103cd69cd49a trm cells were abundant in these tissues , with an overall higher frequency of cd49a t  cells ( figure  4a ) . unlike skin , substantial proportions of cd8cd49a t  cells lacking cd69 or cd103 expression were present in both gut and cervix and a population of cd4cd103cd69cd49a trm cells existed in cervical epithelium ( figure  4a ) .
with respect to expression of cytotoxic granule constituents , gut , and particularly cervical trm cells displayed higher basal expression of perforin and granzyme b relative to skin ( figures 3a3c , 4b and s3b ) .
perforin and granzyme b were more highly expressed in cd8cd103cd69cd49a compared to cd8cd103cd69cd49a gut and cervical trm cells , being further augmented by il-15 stimulation ( figure  4b ) .
these results indicate that cd49a expression defines trm cell subsets with cytotoxic potential in a variety of epithelial tissues .
having found that cd49a generally identifies trm cells with cytotoxic potential and having elucidated inflammatory signals for their priming , we set out to determine their cytotoxic capacity
. sorted t  cell subsets from blood and skin collected from healthy donors ( figure  s4 ) were incubated with cr - labeled target cells in 4  hr redirected antibody - dependent cellular cytotoxicity assays .
the cytotoxic activity of epidermal cd8cd103cd49a and cd8cd103cd49a trm cell subsets was compared to that of peripheral blood cd8cd57 t  cells , a highly cytotoxic effector memory subset ( chiang et  al . , 2013 ) , as well as
cd8cd57 t  cells displayed strong cytotoxic activity , whereas skin - derived cd8cd103cd49a trm cells mediated very poor activity following anti - cd3 antibody stimulation ( figure  5a ) .
however , following 48  hr of il-15 pre - stimulation , epidermal cd8cd103cd49a trm cells manifested cytotoxic activity comparable to that of peripheral blood cd8cd57 t  cells , whereas cd8cd103cd49a trm cells and cd8cd57 t  cells remained poor cytotoxic effectors ( figure  5a ) .
stimulation of epidermal t  cells with anti - cd3 antibodies for 24  hr induced similar levels of cd8cd103cd49a trm cell granzyme b and perforin expression as il-15 , whereas these stimulations in combination displayed an additive effect ( figure  5b ) . in summary ,
skin - derived cd8cd103cd49a trm cells lack perforin expression , with short - term tcr stimulation alone being insufficient to induce cytotoxicity .
t cell - derived cytokines are crucial for defense against invading  pathogens at barrier sites . generally , ifn- contributes to immunity toward intracellular infections while il-17 provides anti - fungal defense and both of these cytokines initiate inflammatory keratinocyte responses .
prompted by differences in transcription of ifng as well as rorc , a key transcriptional regulator of il-17 production , we determined whether cd49a expression also defined epidermal skin trm cell subsets in regards to cytokine production .
corroborating transcriptional profiles , cd8cd103cd49a trm cells produced il-17 while cd8cd103cd49a trm cells excelled in ifn- production upon stimulation with phorbol 12-myristate 13-acetate and ionomycin ( figures 6a6c ) .
tnf and il-2 were abundantly produced by dermal and epidermal trm cell subsets ( figures 6b and 6c ) .
tcr engagement using anti - cd3 antibodies also preferentially induced ifn- by epidermal cd8cd103cd49a trm cells ( figure  6d ) . moreover , il-15 stimulation potentiated tcr - dependent expression of il-17 and ifn- by epidermal cd8cd103cd49a and ifn- by cd8cd103cd49a trm cells , respectively ( figure  6d ) , substantiating effectual  chain receptor signaling in both subsets .
coating of wells with collagen iv in addition to anti - cd3 antibodies specifically augmented production of ifn- by epidermal cd8cd103cd49a trm cells ( figure  6e ) , indicating vla-1-mediated regulation of cytokine production .
unconventional mucosal - associated invariant t ( mait ) cells represent a fraction of peripheral blood il-17-producing t  cells ( dusseaux et  al . , 2011 ) .
although up to 30% of skin cd8 t  cells expressed cd161 , cd8tcr - v7.2cd161 mait cells comprised less than 25% of dermal or epidermal skin cd8 trm cell populations ( figures s5a and s5b ) and made up less than 25% of the il-17-producing  cd8cd103 t  cells
thus , cd49a expression delineated a dichotomy in trm cell cytokine production , augmented by il-15 , with cd8cd103cd49a and cd8cd103cd49a trm cells preferentially producing il-17 and ifn- , respectively .
a population of epithelial trm cells that upon controlled activation perform cytotoxic killing and ifn- production would be perfectly placed to safeguard against infections and malignant transformation in  situ .
hypothetically , such specialized trm cells might also participate in focal immune pathology in vitiligo ( figure  s6a ) , a disease where cytotoxic t  cells have been implicated in eradication of melanocytes .
the proportion of epidermal cd8cd103cd49a trm cells was increased in lesional vitiligo but not in psoriasis ( figures 7a and 7b ) .
cd49a was expressed on almost half of dermal cd8cd103 trm cells in lesional vitiligo ( figures 7a and 7b ) , adjacent to collagen iv - expressing vessels ( figure  s6b ) , while healthy skin , non - lesional vitiligo and psoriasis was devoid of this population ( figures 7a and 7b , s6c and s6d ) .
in contrast , cd4cd103 t  cells in lesional vitiligo did not express cd49a ( figure  s6e ) .
in vitiligo , a substantial proportion of lesional cd8cd103cd49a trm cells recognized melanocyte - derived antigens ( figures s6f and s6 g ) .
perforin and granzyme b - expressing trm cells were detected in vitiligo , but not in  healthy skin or psoriasis ( figures 7c and 7d ) .
moreover , cd8cd103cd49a trm cells co - expressed perforin and granzyme b in lesional vitiligo ex  vivo ( figures 7e and 7f ) . in line with  increased cd49a frequencies , ifn- producing trm cells were enriched in vitiligo lesions ( figure  7 g ) .
conversely , il-17-producing trm cells were enriched in psoriasis plaques ( figure 7 g ) . as in skin from healthy volunteers ,
cd8cd103cd49a trm cells thus preferentially expressed ifn- and cd8cd103cd49a trm cells il-17 in both vitiligo and psoriasis ( figures 7h7j ) .
similar to healthy skin and in agreement with a previous study ( teunissen et  al . , 2014 ) ,
less than 5% of skin il-17 producing cells were cd161v7.2 mait cells ( figures s6h  s6j ) .
trm cells with the capacity to co - express ifn- and il-17 were present in psoriasis plaques ( figure  7h and 7j ) , indicating a degree of functional plasticity in the context of chronic inflammation . however , constrained plasticity of healthy skin - derived trm cells was indicated , as sorted cd8cd103cd49a trm cells from healthy skin failed to produce il-17 following 72  hr of stimulation with il-1 , il-6 , and il-23 ( figures s6l and s6 m ) . in resolved psoriasis , cd8 trm cells poised for il-17 production accumulate ( cheuk et  al . , 2014 ) , and il-17 expression
thus , functional dichotomies associated with cd49a expression were generally preserved in disease - associated trm cells arguing that cd49a marks subsets of trm cells with imprinted effector profiles and distinct functions in inflammatory skin diseases .
at epithelial boundaries , trm cells differentiate and form a first line of adaptive defense against multiple pathogens , tailored to effectively control recurrent infections .
consequently , it would be expected that distinct subsets of trm cells respond to activation with heterogeneous cytokine responses ( naik et  al . , 2015 , sanchez rodriguez et  al . , 2014 , schlapbach et  al . , 2014 , watanabe et  al . , 2015 ) despite their protective role in recurrent viral infections , direct proof of trm cell - mediated cytotoxicity is lacking ( mueller and mackay , 2016 ) .
in addition to anti - microbial defense , pathogenic trm cells are implicated in several inflammatory diseases ( clark , 2015 ) .
here , we identify cd49a expression as a marker delineating a subpopulation of  cd8 trm cells in human skin that specifically localize to the  basal layer of epidermis , preferentially produce ifn- , and display high cytotoxic capacity upon stimulation .
we also find a high proportion of cd49a trm cells poised for cellular cytotoxicity in other epithelial tissues .
lastly , we determined that this functional dichotomy among trm cell subsets was preserved in the inflammatory skin diseases psoriasis or vitiligo . in healthy skin , we found cd49a trm cells confined to basal epidermis in contact with collagen iv expressed in the basement membrane .
mouse epidermis displays analogous spatial restrictions to the number of trm cells during homeostasis ( zaid et  al . , 2014 ) .
interactions with collagen iv might anchor cd49a trm cells in the epidermis . in murine models ,
protective cd8cd49a trm cells develop in barrier tissues ( gebhardt et  al . , 2009 , ray et  al . , 2004 , zhang and bevan , 2013 ) following viral infections .
in line with our study of human skin , granzyme b - expressing trm cells are retained in mouse epithelium following epidermotropic viral infections ( gebhardt et  al .
epithelial tissues represent first sites of viral entry and with high cellular turnover of keratinocytes , offer an attractive niche for viral propagation and shedding .
thus , by expressing cd49a , trm cells seem well - positioned for immunosurveillance of infected epithelial cells via production of ifn- as well as  cellular cytotoxicity .
collagen iv - mediated engagement of cd49a enhanced ifn- production by cd8cd103cd49a trm cells , possibly through stabilizing ifng transcripts ( wang et  al . ,
similar to mouse cd8 trm cells in the context of viral infection ( mackay et  al . , 2013 ,
, 2013 ) , the transcriptional profiles of human cd8cd103cd49a trm cells suggested that anti - viral defense and target cell killing represent key functions of this subset .
such cells might also contribute to local surveillance and defense against malignancy ( jameson et  al .
freshly isolated human skin cd8cd103cd49a trm cells displayed a transcriptional profile indicative of cytotoxic function , but did not express key mediators of cellular cytotoxicity .
rather , their cytotoxic capacity was primed through il-2 and il-15-mediated induction of perforin and granzyme b expression .
, 2013 ) and entry into the epidermis ( adachi et  al . , 2015 ) . here
we find  that il-15 additionally acts both to potentiate cytokine responses and as a key mediator in cytotoxic licensing of cd8cd103cd49a trm cell .
our results suggest an important role for bystander keratinocytes or t  cells in activation of trm cell - mediated effector functions within the skin .
these observations validate the hypothesis that il-15 might generally act as a central danger signal regulating trm cell responses ( jabri and abadie , 2015 ) and extend knowledge by providing a cellular marker of  specific trm cell subsets capable of mediating cellular cytotoxicity . in the gut , il-15 is implicated in driving cytotoxic lymphocyte responses that turn pathological in celiac disease ( meresse et  al . , 2004 ) .
in our analyses , cd49a was abundantly expressed on cd8 trm cells in gut and cervix , representing mucosal barrier tissues . in these tissues ,
a greater proportion of trm cells expressed cd49a than in skin and displayed a more activated phenotype , with constitutive expression of perforin and granzyme b. it is possible that mucosa is a milieu more commonly challenged by pathogens , requiring activated , cytotoxic cd49a trm cells that provide continuous surveillance .
nonetheless , il-15-mediated potentiation of cd49a trm cells might represent a mechanism to safeguard tissues against immunopathology .
revealing functional specialization among epidermal trm cells with respect to cd49a expression , cd8cd103cd49a trm cells preferentially produced il-17 , a cytokine required for control of bacterial and fungal infections .
epidermal cd8cd103cd49a trm cells excelled in il-17 production relative to dermal  counterparts as well as dermal cd8cd103 t  cells .
this observation , combined with different gene - expression profiles , indicates distinct subsets of cd8cd103cd49a trm in dermis versus epidermis . to explore potential origins of epidermal cd8cd103cd49a trm cells , we sorted dermal cd8cd103 trm cells and stimulated them with il-15 and tgf- , cytokines implicated in trm cell differentiation ( mackay et  al . , 2013 , watanabe et  al . , 2015 ) .
a fraction of dermal cd8cd103 trm cells upregulated both cd103 and cd49a following stimulation ( s.c . and l.e .
, unpublished observations ) , indicating that inflammatory responses might differentiate dermal t  cells to functionally distinct trm cells embedded in epidermis .
animal models provide further opportunities to dissect the molecular requirements for induction of cd49a and determine the role of cd49a in ensuring effective trm cell - mediated immunity .
the patchy appearance and fixed recurrence of several inflammatory skin diseases implies the activity of trm cells . in vitiligo ,
there , they are well - positioned to eradicate repopulating melanocytes . in contrast , in active psoriasis , with up to 100-fold more trm cells within the hypertrophic epidermis compared to healthy skin ( cheuk et  al . , 2014 ) , il-17-producing trm cells were profoundly expanded .
moreover , il-17 or ifn- production by distinct trm cells subsets was generally maintained even in the context of the vigorous tissue inflammation .
co - expression of il-17 and ifn- was detected in some cd8cd103cd49a and cd8cd103cd49a trm cells .
however , we could not induce il-17 production by cd8cd103cd49a trm cells sorted from healthy skin following il-23 , il-6 , and il-1 stimulation .
thus , we speculate that during differentiation trm cells require tcr engagement in addition to il-17 polarizing inflammatory cues to display the level of functional plasticity we observe in psoriasis . in resolved psoriasis where numbers of trm  cells are similar to healthy skin
, we previously described enrichment of cd8cd103ccr6il-23r trm cells poised to il-17 production ( cheuk et  al . , 2014 ) .
in contrast , ifn--producing cd8cd103cd49a trm cells were enriched in vitiligo , further supporting the notion of a dichotomy of trm cell subsets according to cd49a expression even in the setting of skin inflammation and immunopathology ( figure  s6o ) . given
the role of il-2 and il-15 in potentiating both il-17 and cytotoxic effector functions in trm cells , blockade of the downstream jak / stat pathway represents a promising therapeutic target for skin diseases putatively caused by aberrant trm cell activation .
interestingly , a case report suggests clinical efficacy of toficitinib , a small molecule inhibitor of jak1 and jak3 , on vitiligo ( craiglow and king , 2015 ) .
systemic administration of il-15r blocking antibodies prevented hair loss in a mouse model of alopecia areata ( xing et  al . , 2014 ) , a disease characterized by aberrant killing of hair follicles .
most patients present with  limited burden of disease that cause considerable suffering , yet do not require systemic treatment .
future development of topical treatments aiming at controlling pathogenic trm cells in  situ would offer an attractive therapeutic strategy , with our data highlighting  chain signaling an attractive target . in summary , our data reveal that human skin contains different subset of epidermal trm cells poised toward cytotoxicity and ifn- or il-17 production , respectively .
inflammatory cytokines unleashed tcr engagement - dependent cellular cytotoxicity by cd49a trm cells from healthy skin , whereas activated , perforin- and granzyme - expressing cd49a trm cells accumulated in  vitiligo lesions .
further insights into the dynamics of the composition , retention , and activation of expanded , functionally specialized trm cell populations might contribute to improved management of both infections and chronic inflammatory skin diseases .
human peripheral blood and healthy skin samples from trunk were obtained from reconstructive skin surgery at advita clinic , stockholm , sweden , and karolinska university hospital , solna .
cervical tissue samples were obtained from non - malignant and non - inflammatory surgical specimens , st .
gut biopsies were obtained from the ileum of healthy patients with hereditary predisposition for colorectal cancer ( lynch syndrome ) at karolinska university hospital , huddinge .
patients with non - segmental vitiligo , or plaque psoriasis were collected at the swedish psoriasis association clinic or the dermatology clinic at karolinska university hospital , solna ( table s3 ) . resolved psoriasis was collected as previously described ( cheuk et  al . , 2014 ) .
lesional vitiligo biopsies were sampled from within 1  cm to and non - lesional at least 10  cm away from lesional borders .
ethical permits 2012/50 - 31/2 , 2015/0041 - 31 , 2015/933 - 32 , 2012/1900 - 31/1 , 2013/1800 - 32 , 2015/1078 - 32 , regional ethical committee of stockholm .
810  m thick sections were stained as previously described ( cheuk et  al . , 2014 ) with primary antibodies to collagen iv ( clone col-94 , abcam ) , melana ( clone ep1422y , abcam ) , keratin 5/6 ( cloned516 , dako ) , cd8 ( ab4055 , abcam ) , cd49a ( clone 550594 , bd bioscience ) , and cd3 ( clone cd3 - 12 , abcam ) .
images were acquired by zeiss lsm700 and lsm800 ( zeiss ) and analyzed with fiji - imagej .
whole - skin punch biopsies and cervix samples were incubated in 5u dispase ( life technologies ) overnight at 4c followed by manual separation of  epidermis and cervical epithelium from dermis or cervical submuocsa respectively followed by 90  min incubation in collagenase iii ( 3  mg / ml ; worthington ) with dnase ( 5  g / ml ; roche ) in rpmi 1640 .
dermis and submucosa were further processed by medicon tissue disruptor ( bd biosciences ) as previously described ( cheuk et  al . , 2014 ) .
ileum biopsies were digested in collagenase ii ( 0.25  mg / ml ; sigma - aldrich ) with dnase ( 0.2  mg / ml ; roche ) in imdm ( life technologies ) for 3045  min .
complete rpmi medium was added and the cell suspension were subsequently passed through a 40  m ( gut ) / 70  m ( skin or cervix ) cell strainer ( bd bioscience ) . peripheral blood mononuclear cells ( pbmcs )
p815 cells were purchased from atcc and maintained in complete medium ( rpmi 1640 supplemented with 10% fetal bovine serum [ fbs ] , l - glutamine ; all hyclone ) .
recombinant il-15 , il-1 , il-2 , il-6 , il-7 , il-23 , il-12 and ifn- ( all r&d systems ) were stored at 80c .
human collagen iv , phorbol 12-myristate 13-acetate ( pma ) , and ionomycin were purchased from sigma .
sorting was performed within 2 - 4  hr using moflo xdp ( beckman coulter ) cell sorter or bd faczjazz ( bd ) .
details on flow cytometry and sorting experiments , as well as  functional evaluations are provided in the supplemental experimental procedures .
dna was extracted ( puregene , qiagen ) and the tcr- cdr3 regions were sequenced and mapped ( immunoseq , adaptive biotech ) .
rna was purified from cell - qiazol lysate ( mirneasy , qiagen ) and quality was checked ( bioanalyzer rna 6000 , agilent ) before library construction ( smart - seq , clontech ) and sequencing ( illumina hiseqtm 2000 ) .
the cytotoxicity assay was performed and analyzed as described previously ( chiang et  al . , 2013 ) .
in brief , sorted and rested cell populations were used as effector cells against cr - labeled p815 target cells supplemented with  0.5  g / ml of anti - cd3 antibody ( clone s4.1 , invitrogen ) with effector - to - target ratios between 10 and 0.3 .
cr release in supernatant was measured on a -counter ( wizard , perkinelmer ) and specific lysis was calculated as previously described ( chiang et  al . , 2013 ) . statistical analysis and graphical illustration of numerical data
was performed by using either prism ( v6 , graphpad ) , jmp 13 or rstudio .
, a.i . , and l.e . provided reagents and clinical material : m.e . ,
l.e . supervised ex  vivo preparation and analysis of human skin cells , y.t.b .
 summarytissue - resident memory t ( trm ) cells form a heterogeneous population that provides localized protection against pathogens . here 
 , we identify cd49a as  a marker that differentiates cd8 + trm cells on a compartmental and functional basis . in human skin epithelia , cd8+cd49a+ trm cells produced interferon- , whereas cd8+cd49a trm cells produced interleukin-17 
 ( il-17 ) . 
 in addition , cd8+cd49a+ trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme b when stimulated with il-15 , thereby promoting a strong cytotoxic response . in skin from patients with vitiligo , where melanocytes are eradicated locally , cd8+cd49a+ trm cells that constitutively expressed perforin and granzyme b accumulated both in the epidermis and dermis . 
 conversely , cd8+cd49a trm cells from psoriasis lesions predominantly generated il-17 responses that promote local inflammation in this skin disease . 
 overall , cd49a expression delineates cd8 + trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases . 