Distinct subpopulations of gy T cells are present in normal and tumor-bearing human liv

Dis inc subpopula ions o gyT cells a e p esen in no mal and umo -bea ing human li e Tony Kenna, a Lucy Golden-Mason, a Suzanne No is, b John E. Hega y, c,d,1 Cliona O’Fa elly, a,d,1 and De ek G. Dohe y e, * ,1 a Educa ion and Resea ch Cen e, S . Vincen ’s Uni e si y Hospi al, Dublin, I eland b Hepa ology Cen e, S . James’s Hospi al, Dublin, I eland c Li e Uni , S . Vincen ’s Uni e si y Hospi al, Dublin, I eland d Conway Ins i u e o Biomolecula and Biomedical Resea ch, Uni e si y College, Dublin, I eland e Ins i u e o Immunology and Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h, Co., Kilda e, I eland Recei ed 2 Feb ua y 2004; accep ed wi h e ision 17 May 2004 Abs ac gyT cells a e hough o media e immune esponses a epi helial su aces. We ha e quan i ied and cha ac e ized hepa ic and pe iphe al blood gyT cells om 11 no mal and 13 un esol ed umo -bea ing human li e specimens. gyT cells a e en iched in no mal li e (6.6% o T cells) ela i e o ma ched blood (0.9%; P= 0.008). The majo i y exp ess CD4  CD8  pheno ypes and many exp ess CD56 and/o CD161. In i o, hepa ic gyT cells can be induced o kill umo cell lines and elease in e e on-g, umo nec osis ac o -a, in e leukin-2 and in e leukin- 4. Analysis o Vgand Vychain usage indica ed ha Vy3 + cells a e expanded in no mal li e s (21.2% o gyT cells) compa ed o blood (0.5%; P= 0.001). Tumo -bea ing li e s had signi ican expansions and deple ions o gyT cell subse s bu no mal cy oly ic ac i i y. This s udy iden i ies no el popula ions o li e T cells ha may play a ole in immuni y agains umo s. D2004 Else ie Inc. All igh s ese ed. Keywo ds: Tumo immuni y; Li e ; Human; gyT cells; Cy okines; Cy o oxici y In oduc ion gyT cells accoun o small numbe s o pe iphe al blood T cells bu accumula e a epi helial su aces and a si es o in ec ion [1]. Mos gyT cells a e nega i e o CD4 and CD8 and a e capable o ecognizing an igen wi hou he need o majo his ocompa ibili y complex (MHC) es ic ion ( e iewed in Re s. [2,3]. Thei T cell ecep o s (TCRs) ecognize ela i ely conse ed s uc u es on pa hogens and hos cells including non-pep ide me aboli es and hea shock p o eins. Pe iphe al blood gyT cells can apidly lyse an igen- bea ing a ge cells and can kill a ange o umo cell lines in i o. They also can apidly elease Th1 o Th2 cy okines which can egula e di e en ia ion and ac i a ion o compo- nen s o he adap i e immune sys em. gyT cells a e he e o e hough o b idge inna e and adap i e immune esponses [2,3]. Dis inc subse s o gyTcells, based on hei TCR g- and y- chain a iable gene segmen usage, a e di e en ially dis i- bu ed in di e en issues and show d ama ic changes wi h age. In adul humans, he Vg9Vy2 TCR is p edominan ly ound among pe iphe al blood gyT cells, whe eas he majo i y o in es inal gyTcells exp ess Vy1 chains associa ed wi h one o se e al g-chains [4–6]. Mu ine and human s udies ha e p o ided e idence ha he selec i e accumula- ions o gyT cell subse s a di e en body loca ions a e he esul o pe iphe al selec ion and expansion by locally exp essed an igens [4,7,8]. O he s udies, howe e , ha e sugges ed ha selec ion is no equi ed o p oduce in a ian gyTCRs and ha hese ea angemen s a e p og ammed [9,10].Vg9Vy2 T cells ecognize o ganic phosphoes e s, alkylamines, and nucleo ide conjuga es ha a e cons i u i ely exp essed by hos cells and mic obial pa hogens [11–13]. They also ecognize hea shock p o eins and pu a i e umo an igens [14–16].Vy1 + gyT cells can ecognize he s ess- 1521-6616/$ - see on ma e D2004 Else ie Inc. All igh s ese ed. doi:10.1016/j.clim.2004.05.003 * Co esponding au ho . Ins i u e o Immunology and Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h, Maynoo h, Co., Kilda e, I eland. Fax: +353-1-7083845. E-mail add ess: de ek.g.dohe [email protected] (D.G. Dohe y). 1 These au ho s con ibu ed equally o he di ec ion o his s udy. www.else ie .com/loca e/yclim Clinical Immunology 113 (2004) 56– 63 inducible MICA and MICB molecules ha a e up egula ed as a esul o umo ans o ma ion and i us in ec ion [17,18] al hough i appea s ha his ecogni ion is no media ed by he TCR [19].Vy1 + Tcells ha e also been shown o ecognize he lipid-p esen ing molecule, CD1c [20]. The li e appea s o ha e an impo an immuno egula- o y ole, being able o selec i ely induce immuni y o ole ance o an igens [21]. The adul human li e con ains se e al popula ions o lymphocy es ha exhibi apid an i- gen-non-speci ic cy o oxici y and Th1/Th2 and egula o y cy okine sec e ion [22–24]. These include many gyT cells. He e, we ha e quan i ied and cha ac e ized hepa ic gyT cells om no mal and umo -bea ing human li e speci- mens, wi h espec o Vgand Vy-chain usage, T and NK cell ma ke exp ession, cy okine sec e ion, and cy o oxic po en- ial. Ou da a indica e ha he li e has unique epe oi es o gyT cells ha include cells capable o an i umo cy o o- xici y in i o and whose numbe s a e changed in pa ien s wi h hepa ic malignancy. Ma e ials and me hods Tissue specimens Wedge li e biopsies (50–100 mg) we e ob ained om 11 heal hy dono o gans (8 male and 3 emale; mean age 39.5 yea s; age ange 18–63 yea s) a he ime o li e ansplan a ion. Li e biochemis y and his ology we e no mal in all cases. Li e issue was ob ained om 13 pa ien s unde going esec ion o hepa ic me as ases o colonic o igin (7 male and 6 emale; mean age 60.2 yea s; age ange 35–75 yea s). Wedge biopsies om umo -bea - ing issue we e aken app oxima ely 10 cm om he umo ma gin and appea ed his ologically no mal. All o gan dono s and pa ien s we e nega i e o hepa i is A, B, C, y, and E i uses. E hical app o al o his s udy was ob ained om he Resea ch and E hics Commi ee a S . Vincen ’s Uni e si y Hospi al, Dublin, I eland. Single cell suspensions o hepa ic mononuclea cells (HMC) we e p epa ed as p e iously desc ibed [25].gyT cells we e isola ed om esh HMC p epa a ions using mAb-coa ed magne ic beads (Mil enyi Bio ec, Be gisch Gladbach, Ge many) acco ding o manu ac u e ’s p o ocols. The pu i y o isola ed ac ions was assessed by low cy ome y and only p epa a ions wi h pu i ies o 95% o g ea e we e used o unc ional s udies. An ibodies and low cy ome y Monoclonal an ibodies (mAb) speci ic o human CD3 (clone SK7), CD4 (SK3), CD8ah(SK1), CD56 (NCAM16.2), gyTCR (11F2), ahTCR (WT31), CD161 (DX12), IFN-g(25723.11), TNF-a(6401.1111), IL-2 (5344.1111), o IL-4 (8D4-8) we e ob ained om Bec on Dickinson (Ox o d, UK). Unconjuga ed mAbs speci ic o TCR V- egions Vy1 (clone 9R.12), Vy2 (Immu389), Vy3 (P11.5B), Vg2-3-4 (23D12), Vg8(R4.5),andVg9 (Immu360) we e ob ained om Immuno ech (Ma seille, F ance). The exp ession o hese cell su ace an igens by eshly isola ed HMC and pe iphe al blood mononuclea cells (PBMC) was de ec ed by mAb s aining and h ee-colo low cy ome y (FACScan, Bec on Dickinson) and analyzed using CellQues so wa e (Bec on Dickinson). To a oid compe i ion o binding si es on he gyTCR, he mAb ha ecognizes all gyTCRs was no used in conjunc ion wi h mAbs speci ic o indi idual Vgo VyTCR chains. Ins ead, cells we e cos ained wi h he pan-gyand an i-CD3 mAbs, and an i-CD3 wi h indi idual Vgo VymAbs in sepa a e ubes. De ec ion o in acellula cy okines Cy okine p oduc ion by gyT cells a e s imula ion in i o was examined by low cy ome y. To elimina e non- speci ic au o luo escence om o he li e cell popula ions, gyT cells we e i s pu i ied om o al HMC by immuno- magne ic bead sepa a ion. Hepa ic gyT cells (1 10 6 cells/ ml) we e s imula ed wi h 10 ng/ml pho bol my is a e ace a e (PMA) (Sigma Co., Poole, UK) plus 1 Ag/ml ionomycin (Sigma) o 4 h in 24-well pla es a 37jCin5%CO 2 .As con ols, uns imula ed cells we e ea ed simila ly. B e eldin A (10 Ag/ml, Sigma), an inhibi o o p o ein ansloca ion om he endoplasmic e iculum o he Golgi appa a us, was added o he cells o he 4-h incuba ion. Cells we e hen s ained wi h mAbs speci ic o su ace CD3 and in acellula IFN-g, TNF-a, IL-2, o IL-4 and de ec ed by h ee-colo low cy ome y as desc ibed p e iously [24]. Cy o oxici y assays Magne ic bead-pu i ied gyT cells we e used as e ec o cells in cy o oxici y assays agains K562 and Daudi a ge cell lines. Na u al cy o oxici y and lymphokine-ac i a ed killing (LAK) we e examined using gyT cells ha we e p ecul u ed o 3 days in he absence o p esence o 50 uni s/ ml IL-2, espec i ely. Cy o oxici y was assessed in s anda d 4-h 51 C elease assays using 2000 51 C -labeled a ge cells incuba ed wi h pu i ied gyT cells a E/T a ios o 1:1, 5:1, 25:1, and 50:1 [24]. Speci ic lysis was calcula ed om he amoun s o 51 C eleased in o supe na an s using he o - mula: % speci ic lysis = (cpm o sample cpm o spon aneous elease) 100 / (cpm o maximum elease  cpm o spon aneous elease). Spon aneous elease was de e mined by incuba ing he a ge cells in he absence o e ec o cells and maximum elease was ob ained by incu- ba ion o a ge s wi h 0.1% T i on X-100. S a is ical analysis Resul s we e exp essed as median and anges. The Spea man Rank and Mann–Whi ney U es s o non-pa a- me ic da a we e used o analyze esul s. P alues o <0.05 we e conside ed o be signi ican . T. Kenna e al. / Clinical Immunology 113 (2004) 56–63 57 Resul s cd T cells accumula e in human li e Flow cy ome y was used o de e mine he equencies o human T cells ha exp ess he TCR gyhe e odime in 11 his ologically no mal dono li e s and ma ched blood sam- ples (Fig. 1A). Fo his pa o he s udy, cells we e s ained ex i o wi h no p io manipula ion. Fig. 1B shows ha he e is a signi ican en ichmen o gyT cells in no mal human li e ela i e o blood, since his TCR was ound o be exp essed by a median o 0.93% ( ange 0.24–2.7%) o pe iphe al blood CD3 + cells and 6.6% ( ange 4.0–13.8%) o hepa ic CD3 + cells (P= 0.008). Pheno ypic cha ac e iza ion o cd T cells in no mal human li e The pheno ypes o gyT cells in eshly isola ed ma ched li e and pe iphe al blood samples we e u he analyzed by h ee-colo low cy ome y. Simila o ci cula ing gyT cells, he majo i y o hepa ic gyT cells we e nega i e o CD4 and CD8 (median 85.5%; ange 79.1–92.0%; Table 1). Less han 5% o hepa ic gyT cells exp essed CD4 (4.2%; ange 0.8– 12.6%), while a signi ican ly highe p opo ion o hepa ic gy T cells we e CD8 + (8.9%; ange 6.1%–15.8%) compa ed wi h hose ound in he ci cula ion (1.5%; ange 0.6–2.8%) (P< 0.001; Table 1). A high p opo ion o bo h ci cula ing and hepa ic gyT cells exp essed he NK cell ma ke s CD56 (38.9%; ange 21.3–57.9% in blood and 38.8%; ange 21.1– 58.4% in li e ) and CD161 (36.8%; ange 9.1–59.2% in blood and 36.2%; ange 17.0–54.6% in li e ; Table 1). TCR cand dchain gene segmen usage To de e mine whe he hepa ic gyT cells display any ‘ issue opism’, we examined he exp ession by hepa ic and pe iphe al blood gyT cells o he Vgand Vychains ha ha e p e iously been epo ed o be he mos equen - ly ound in blood, namely Vg2-3-4, Vg8, Vg9, Vy1, Vy2, and Vy3 chains [26,27] (Fig. 2). The Vg2-3-4, Vg8, and Vg9 chains we e ound o be exp essed by simila e- quencies o gyT cells in blood and li e (Fig. 2A). The majo i y o hepa ic gyT cells exp essed Vy2chains (56.4%; ange 29.0–86.4%) as ound in blood, while use o he Vy1 chain by hepa ic gyT cells (8.9%; ange 3.4– 32.7%) was signi ican ly educed compa ed wi h ha seen in he blood (42.7%; ange 5.7–74.2%; P= 0.035; Fig. 2A). S ikingly, a signi ican p opo ion o hepa ic gyT cells we e ound o exp ess he Vy3 chain (21.2%; ange 0–37.2%). In con as , his y-chain is almos ne e exp essed by ci cula ing lymphocy es (0.5%; ange 0– 0.9%; P= 0.001; Figs. 2A and B). Cy okine p oduc ion by hepa ic cd T cells Flow cy ome y was used o examine he cy okine sec e ion p o iles o hepa ic gyT cells in esponse o in i o s imula ion wi h PMA and ionomycin (Fig. 3).To enhance cla i y o mAb s aining, gyT cells we e i s posi i ely selec ed om o al HMC using magne ic beads. Hepa ic gyT cells displayed a s ong Th1 bias. Signi ican p opo ions (median 29.2%; ange 14.6–54.9%) o hepa ic gyT cells p oduced IFN-gin esponse o s imula ion in i o wi h PMA and ionomycin. Smalle p opo ions p oduced IL-2 (12.9%; ange 8.0–17.1%) o TNF-a(5.3%; ange 1.4–15.6%). A median o 4.6% (2.8–6.2%) o hepa ic gyT cells p oduced he Th2 cy okine IL-4. In addi ion, 6.2% (1.7–14.7%) o hepa ic gyT cells om heal hy li e Fig. 1. gyT cell ecep o (TCR) exp ession by eshly isola ed human pe iphe al blood and hepa ic T cells. (A) Rep esen a i e low cy ome y do plo showing ahand gyTCR exp ession by ga ed CD3 + mononuclea cells om he blood and li e o a li e ansplan dono . Numbe s show he pe cen ages o CD3 + cells ha exp ess gyTCRs. (B) Pe cen ages o CD3 + cells in blood and li e s o 11 li e ansplan dono s exp essing gyTCRs. Medians a e shown as ho izon al lines wi h s anda d e o s as e o ba s. *P= 0.008. Table 1 Pheno ypic analysis o pe iphe al blood and hepa ic gyT cells in 11 his ologically no mal li e ansplan dono s Pheno ype F equency in blood F equency in li e P alue % Range % Range CD4 3.6 0.8–5.4 4.2 0.8–12.6 CD8 1.5 0.6–2.8 8.9 6.1–15.8 0.001 DN 92.8 91.9–98.7 85.5 79.1–92.0 CD56 38.8 21.3–57.9 38.9 21.1–58.4 CD161 36.8 9.1–59.2 36.2 17.0–54.6 T. Kenna e al. / Clinical Immunology 113 (2004) 56–6358 spon aneously p oduced IFN-gin he absence o s imula ion while 7.3% (0.5–12.1%) o uns imula ed cells p oduced IL- 2. This spon aneous cy okine p oduc ion may be due o backg ound le els o in i o ac i a ion, o al e na i ely, i may be due o low le el ac i a ion by he mAb-coa ed magne ic beads used o gyT cell pu i ica ion. cd T cells in umo -bea ing li e The numbe s, pheno ypes, and unc ions o hepa ic and pe iphe al blood gyT cells we e also compa ed be ween li e dono s and pa ien s wi h hepa ic malignancy. Fig. 4A shows ha gyT cells a e signi ican ly expanded in he blood o pa ien s wi h hepa ic malignancy (3.9% o o al CD3 + cells s. 0.9%; P= 0.008). While umo -bea ing li e also showed inc eased p opo ions o gyT cells (10.4% s. 6.6%) his inc ease was no signi ican (P= 0.3). The exp ession o he NK cell ma ke s CD56 and CD161 by hepa ic gyT cells was inc eased in malignancy. CD56 was exp essed by a median o 38.9% o gyT cells om his ologically no mal li e s and 59.7% in malignan issue (P= 0.04). CD161 was exp essed by 36.2% o gyT cells om no mal li e s and 47.5% o gyT cell om umo - bea ing li e s ( P= 0.05; Fig. 4B). Analysis o Vgand Vychain usage in umo -bea ing li e e ealed signi ican ly di e en usage compa ed wi h heal hy li e (Fig. 4C). Tumo -bea ing li e displayed p e e en ial usage o he Vy1 gene p oduc s (26.8% o o al gyT cells; P= 0.03), while Vy2 (21.2%) and Vy3 (9.61%) exp ession was signi ican ly educed ( P= 0.03 and 0.02, espec i ely). While almos all gyT cells in he blood and li e o li e ansplan dono s consis en ly s ained posi i e o mAbs speci ic o ei he Vy1, Vy2, o Vy3, app oxima ely 40% o gyT cells in umo -bea ing li e s did no s ain o any o hese mAbs, sugges ing ha agyT cell wi h a p e iously un ecognized TCR y-chain is expanded in he li e s o pa ien s wi h hepa ic malig- nancy. No di e ences we e seen be ween Vgchain usage be ween no mal and umo -bea ing li e s (da a no shown). Cy o oxic ac i i y o hepa ic cd T cells gyT cells om heal hy (n= 4) and umo -bea ing (n=4) li e samples we e pu i ied om eshly isola ed HMC using immunomagne ic beads (Fig. 5A) and used as e ec- Fig. 3. Cy okine p oduc ion by hepa ic gyT cells. Pe cen ages o hepa ic gy T cells ha s ain posi i e o in e e on-g(IFN-g), in e leukin-2 (IL-2), umo nec osis ac o -a(TNF-a) and IL-4 which we e ei he uns imula ed () o s imula ed o 4 h wi h PMA and ionomycin (+). Ho izon al lines indica e medians o ou indi idual samples. Fig. 2. T cell ecep o gand ychain usage by eshly isola ed human pe iphe al blood and hepa ic T cells. (A) Box plo s showing medians (ho izon al lines), in e qua ile anges (shaded a eas) and anges (e o ba s) o pe cen ages o gyT cells in blood and li e s o 11 li e ansplan dono s exp essing Vy1, Vy2, Vy3, Vg2-3-4, Vg8, and Vg9. *P= 0.035; **P= 0.001. (B) Rep esen a i e low cy ome y do plo showing Vy3 exp ession by CD3 + T cells om he blood and li e o a li e ansplan dono . The numbe s show he pe cen ages o CD3 + T cells ha exp ess Vy3. T. Kenna e al. / Clinical Immunology 113 (2004) 56–63 59 o s in cy o oxici y assays agains K562 and Daudi a ge cells. When eshly isola ed gyT cells we e used as e ec o s, hey we e unable o lyse ei he a ge cell line (Fig. 5B). Howe e , a e incuba ion o 3 days wi h 50 uni s/ml IL-2, gyT cells om bo h no mal and umo - bea ing li e s exhibi ed cy o oxici y agains K562 and Daudi a ge s (Fig. 5B), indica ing ha hepa ic gyT cell popula ion con ain p ecu so s o LAK cells. No signi ican di e ence in he cy o oxic ac i i y o gyT cells om no mal o umo -bea ing li e s agains K562 o Daudi a ge cells was obse ed. Discussion gyT cells a e p esen in signi ican numbe s in he epi helia o he small in es ine, skin, lungs and he p egnan u e us bu hey ep esen only a small p opo ion (<5%) o he T cells in pe iphe al blood [2,3]. The esul s o he p esen s udy indica e ha he adul li e also is a si e o accumula ion o gyT cells and ha his o gan has a dis inc dis ibu ion o he mos common TCR g- and y-chains. The majo i y o hepa ic gyT cells exp ess double-nega i e CD4  CD8  pheno ypes in keeping wi h hei abili y o ecognize an igen wi hou he need o MHC- es ic ion, bu CD4 + and CD8 + gyT cells we e also seen. A signi ican p opo ion exp ess he NK cell-associa ed ecep o s (NKR) CD56 and CD161. Hepa ic gyT cells can be induced by cul u e wi h IL-2 o kill he umo cell lines, K562 and Daudi, and upon ac i a ion in i o, hey p oduce a ange o cy okines, including IFN-g, TNF-a, IL-2, and IL-4. Fig. 4. Pe iphe al blood and hepa ic gyT cell pheno ypes in pa ien s wi h hepa ic malignancy. (A) Box plo s showing medians (ho izon al lines), in e qua ile anges (shaded a eas) and anges (e o ba s) o pe cen ages o CD3 + T cells in blood and li e s o 11 his ologically no mal and 13 umo - bea ing li e specimens ha exp ess gyT cell ecep o s. (B and C) Exp ession o CD56, CD161 and CD8 (B) and Vy1, Vy2 and Vy3 T cell ecep o chains (C) by gyT cells om he li e s o 11 his ologically no mal and 13 umo -bea ing li e specimens. *P< 0.05 in all cases. Fig. 5. Hepa ic gyT cells a e capable o lymphokine-ac i a ed killing o K562 and Daudi a ge cells. (A) Rep esen a i e low cy ome y his og am showing gyT cell ecep o (TCR) exp ession by magne ic bead isola ed hepa ic gyT cells (shaded his og am) and unsepa a ed hepa ic mononuclea cells (unshaded). The numbe indica es he pu i y o he gyT cell p epa a ion. (B) Na u al cy o oxici y (no IL-2) and lymphokine ac i a ed killing (+IL-2) o K562 and Daudi a ge cells by magne ic bead-pu i ied gy T cells. The igu e is ep esen a i e o expe imen s using ou his ologically no mal and ou umo -bea ing li e samples. T. Kenna e al. / Clinical Immunology 113 (2004) 56–6360 T cells ha exp ess NKRs and display po en MHC- un es ic ed cy o oxici y and apid cy okine sec e ion ac- coun o abou one hi d o all hepa ic lymphocy es bu <5% o pe iphe al blood lymphocy es [22–24].These include CD1d- es ic ed NKT cells wi h in a ian ahTCRs ha ecognize lipid an igens [28,29]. He e, we show ha hey also include gyT cells. Thus, mul iple ypes o semi- in a ian T cells ha media e immedia e and egula o y immune unc ions accumula e in he li e . In con as o he human ahTCR epe oi e, he ge mline encoded gyTCR epe oi e is small [26,30,31]. As p e i- ously epo ed [4,26,27], we ound ha almos all pe iphe al blood and hepa ic gyT cells a e posi i e o ei he Vg2-3-4, Vg8, o Vg9 and ei he Vy1, Vy2, o Vy3 TCR chains. In bo h issues, he majo i y (usually be ween 50% and 90%) o gyT cells exp ess Vg9andVy2. Howe e , we ound ha Vy3 + T cells accoun o app oxima ely 20% o hepa ic gyT cells bu only abou 0.5% o pe iphe al gyT cells in heal hy indi iduals. The eason o he ex ensi e use o he Vy3 elemen in he li e is unclea . I is known ha he Vy3 gene segmen equen ly ecombines wi h Jaand Cagene seg- men s and ha he Vy3 TCR chain does no appea o selec i ely pai wi h any pa icula Vgchain [26].gyTCR ea angemen s occu ea ly in childhood and pa icula gyT cell subse s accumula e a pa icula body loca ions wi h age [4–6]. Such accumula ions may be p og ammed [9,10] o an igen-d i en [7,8,27] and i he la e is ue, he Vy3 TCR chain is likely o espond o an an igen ound in he li e . Vy3 + T cells a e epo ed o expand in he pe iphe al blood o enal ansplan ecipien s ha de elop cy omegalo i us in ec ion [27]. I is unknown whe he Vy3 + T cells ha e unc ions ha a e di e en om hose o o he gyT cells bu p e ious s udies ha e sugges ed ha di e en gyT cells subse s ha e dis inc cy okine sec e ion pa e ns [3]. While he biased usage o gyTCRs in he li e will e lec a dis inc speci ici y o an igen ecogni ion by gyT cells, u u e s udies using pu i ied Vy3 + T cells a e equi ed o iden i y hei unc ions. The e is a la ge body o e idence o sugges ha gyT cells play a ole in hos de ence agains umo s [32,33].gyT cells display an i umo cy o oxici y in i o [34–36]. Vg9Vy2 + T cells ecognize an igens ound on myeloma and lymphoma cells [15,16] and Vy1 + T cells ecei e cos imula o y signals h ough NKG2D, a ecep o o he nonclassical MHC class I-molecule, MICA, which is up e- gula ed on umo cells o epi helial o igin [17,18].gyT cells a e p esen among umo in il a ing lymphocy es in b eas ca cinoma [37,38], enal ca cinoma [39] panc ea ic cance [40], epi helial umo s [41], melanoma, and sa coma [42]. Addi ionally, di e en pa e ns o Vygene ea angemen ha e been obse ed in pai ed blood and umo -in il a ing lymphocy e samples om pa ien s wi h di e en cance s [43]. Seki e al. [44,45] epo ed ha ac i a ed gyT cells accumula e in he li e s o mice and humans wi h umo s. We ha e ound small o e all expansions o gyT cells exp essing NKRs in he blood and li e s o pa ien s wi h hepa ic malignancy. Howe e , on examina ion o gyT cell subse s, we ound ha he p opo ions o hepa ic gyT cells exp essing he Vy1 + TCR chain, hough o play a ole in he esponse o epi helial umo s [17,18], a e signi ican ly expanded in he li e . Vy1 + T cells a e also expanded in he li e s o pa ien s wi h ch onic hepa i is C i us in ec ion [46], a condi ion ha ca ies an inc eased isk o de eloping hepa ocellula ca cinoma. In con as , Vy2 + cells which a e he mos abundan hepa ic gyT cell subse , and Vy3 + cells which appea o be unique o he li e we e ound in signi ican ly lowe equencies in umo -bea ing li e s. In e es ingly, he p opo ions o gyT cells exp essing Vy1, Vy2, o Vy3 TCR chains in he blood and li e o li e ansplan dono s consis en ly added up o app oxi- ma ely 100%, bu app oxima ely 40% o gyT cells in umo -bea ing li e s did no s ain posi i e o any o hese mAbs, sugges ing ha a gyT cell wi h a p e iously un ecognized TCR y-chain is expanded in he li e s o pa ien s wi h hepa ic malignancy. I is unclea om he p esen s udy whe he he dec eases in Vy2 + and Vy3 + cell numbe s in umo -bea ing li e s a e a esul o in luxes o expansions o Vy1 + and o he gyT cells. I is possible ha changes in he numbe s o gyT cell subse s may ei he p edispose an indi idual o me as a ic li e disease o occu in esponse o he umo . Al hough he p opo ions o gyT cell subse s we e di e en in his ologically no mal and umo -bea ing li e s, o al gyT cells pu i ied om hese issues displayed simila LAK cy o oxic ac i i ies agains K562 and Daudi a ge s in i o. In conclusion, he esul s o he p esen s udy p o ide u he e idence o he p esence o cy o oxic and Th1/Th2 cy okine- eleasing T cells wi h semi-in a ian TCRs in he adul human li e . Hepa ic gyT cells a e p edominan ly Th1-like cells and show biased TCR usage compa ed o blood. Ou da a sugges a ole o hepa ic gyT cells in immuni y agains umo s. Acknowledgmen s This wo k was suppo ed by g an s awa ded by he I ish Heal h Resea ch Boa d and En e p ise I eland. 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