Identification of Novel Human NK Cell Progenitor Subsets

International Journal of Molecular Sciences

Article Identiﬁcation of Novel Human NK Cell Progenitor Subsets

Priyanka Sathe 1,2, Swee Heng Milon Pang 1,2, Rebecca Delconte 1,2 ID , Ngaire Elwood 3,4 and Nicholas D. Huntington 1,2,* 1 The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; [email protected] (P.S.); [email protected] (S.H.M.P.); [email protected] (R.D.) 2 Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia 3 Cord Blood Research, Murdoch Children’s Research Institute, Melbourne, VIC 3052, Australia; [email protected] 4 Department of Paediatrics, University of Melbourne, Melbourne, VIC 3010, Australia * Correspondence: [email protected]; Tel.: +61-3-9345-2409

Received: 31 October 2017; Accepted: 8 December 2017; Published: 15 December 2017

Abstract: Understanding the pathways and regulation of human haematopoiesis, in particular, lymphopoiesis, is vital to manipulation of these processes for therapeutic purposes. However, although haematopoiesis has been extensively characterised in mice, translation of these ﬁndings to human biology remains rudimentary. Here, we describe the isolation of three progenitor subsets from human foetal bone marrow that represent differential stages of commitment to the natural killer (NK) cell lineage based on IL-15 responsiveness. We identify CD7 as a marker of IL-15 responsive progenitors in human bone marrow and ﬁnd that this expression is maintained throughout commitment and maturation. Within the CD7+ fraction, we focussed on the lineage potential of three subsets based on CD127 and CD117 expression and observed restricted lymphoid and biased NK cell potential amongst subsets. We further demonstrate the presence of subsets similar in both phenotype and function in umbilical cord blood and the bone marrow of humanised mice, validating these as appropriate sources of progenitors for the investigation of human haematopoiesis. Overall, we describe several stages in the process of lymphopoiesis that will form the basis of investigating the regulators of this process in humans.

Keywords: human haematopoiesis; lymphopoiesis; NK cell progenitors

1. Introduction Despite extensive progress in our understanding of the pathways and regulation of murine haematopoiesis, our knowledge of these processes in humans remains undeveloped. In particular, while the myeloid differentiation pathway has been described [1,2] the intermediate stages in the process of lymphopoiesis are less clear. Understanding these intermediate stages is a critical precursor to elucidating the regulators of lymphopoiesis in humans, information that would help better understand immunodeﬁciencies and becomes increasingly important as more therapies seek to manipulate the numbers or function of lymphoid cells, in particularly natural killer (NK) cells in cancer. Human common lymphoid progenitors (CLP) have been variously described as CD7+, CD10+ or CXCR4+, with varying levels of CD117 and CD127 expression having been reported [3–6]. Further downstream, progenitors with T and NK cell potential have been described [7]. Neither B nor myeloid cell potential was detected, although it is unclear whether the assay used, foetal thymic organ culture, was appropriate for detection of these lineages. Whether such a T/NK cell committed progenitor exists remains to be determined.

Int. J. Mol. Sci. 2017, 18, 2716; doi:10.3390/ijms18122716 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 2716 2 of 11

Recently, Renoux et al. identified a downstream NK cell-restricted progenitor within the Lin−CD34+CD7+CD10+ fraction as a human equivalent of the murine NK cell progenitor (NKP) [8]. Both lymphoid primed multipotent progenitors (LMPP) and “CLP-like” cells gave rise to these progenitors in vitro, suggesting the NKP described is a stage in the physiological process of NK cell development. To track these intermediate stages in which loss of myeloid, T and B cell potential and thus commitment to the NK cell lineage occurs, we have focused on the only factor known to be essential for human and murine NK cell development, IL-15 [9–12], sensitivity to which marks murine NK cell lineage commitment [13–15]. Here, we track in vitro IL-15 responsive progenitors to identify progenitors with NK potential. We characterise the progeny of IL-15 responsive CD34+ progenitors and validate the role of these intermediate progenitors in the pathway to NK cell differentiation, including characterisation of their potential for alternate lineages. Further, we have characterised progenitors from a variety of sources of human tissue, finding their lineage potential broadly conserved and validating a new model of humanised immune system mice for studying lymphopoiesis. Overall, we describe multiple populations of IL-15 responsive progenitors, traceable by uniform expression of the T and NK cell marker CD7, and differential expression of CD117, a marker whose downregulation represents progression into terminal differentiation. Further, we describe conservation of these stages of commitment across different sources of human progenitors. We expect these findings will provide a framework for future studies investigating the regulation of human lymphopoiesis.

2. Results

2.1. CD7 Identifies the IL-15 Responsive Population Amongst CD34+ Progenitors We previously reported an expansion of mature CD7+CD56+ NK cells in the spleen and also noted a robust expansion of lineage− cells in the bone marrow (BM) of human immune system mice following treatment with IL-15 receptor agonists [11]. The CD7+ population in human cord blood and foetal liver has previously been postulated to represent a heterogeneous mix of progenitors, including the common lymphoid progenitor. NK cells develop from CD34+CD38+ progenitors from various sources after several weeks in stem cell factor (SCF) and IL-15 in vitro [16]. CD7 is expressed on a subset of lin−CD34+ progenitors in humans thus we purified total CD34+ from foetal bone marrow and monitored CD7, CD34 expression amongst the IL-15 responsive cells in vitro. In response to IL-15 alone, only the IL-15 responsive cells (as determined by cell proliferation/dilution of carboxyfluorescein succinimidyl ester (CFSE) expressed CD7 after 8 days of culture (Figure1A) indicating that either only the CD34+CD7+ population responds to IL-15 or IL-15 induces CD7 on responsive progenitors. In contrast, in vitro culture of identical progenitors in IL-7 + IL-15 and Notch-ligand resulted in the proliferation of both CD7+ and CD7− progenitors, arguing against IL-15 purely inducing CD7 on responsive progenitors (Figure1A). Consistent with a recent report [ 17], Notch signalling and IL-7 appear to impair commitment towards the NK cell lineage as evidenced by reduction in proliferating CD7+ progenitors. We next assessed if CD7+ progenitors derived from cord blood conserved this preferential sensitivity to IL-15. Since lin−CD34+ progenitors are rare, we assessed the ability of total lin− cord blood cells to respond to IL-15 or thrombopoetin, fms-like tyrosine kinase-3 ligand and SCF (TPO/FLT3L/SCF). While CD7+ cells did not proliferate in response to TPO/FLT3L/SCF, CD7− cells proliferated extensively. Consistent with data from foetal bone marrow, we observed an equally biased response to IL-15 by CD7+ cells as primarily CD7+ cells survived and proliferated after 7 days of culture (Figure1B). A small subset of CD7 − progenitors was also found to proliferate in these cultures. Whether this reflects a separate IL-15 responsive pathway or a population of early progenitors which have yet to acquire CD7 will be an interesting subject for further investigation. This period of in vitro cultivation was not sufficient to induce NK cell or T cell lineage markers (CD56, NKp46, CD161, CD3; data not shown) suggesting they represent IL-15 responsive progenitors. Taken together, this data Int. J. Mol. Sci. 2017, 18, 2716 3 of 11 Int. J. Mol. Sci. 2017, 18, 2716 3 of 10

+ responsiveconﬁrms that progenitors. human CD34 Takencells together respond, this to data IL-15 confirms and responsive that human cells CD34 preferentially+ cells respond express to IL-15 CD7, anda marker responsive of mature cells NK preferentially and T cells. express CD7, a marker of mature NK and T cells.

Figure 1. (A) CD7+ progenitors respond to IL-15. CD34+ progenitors purified from human foetal liver + + Figurewere labelled 1. (A) CD7 with carboxyfluoresceinprogenitors respond succinimidyl to IL-15. CD34 ester progenitors (CFSE) and purified cultured from for 8 dayshuman in IL-15foetal aloneliver wereor IL-15 labelled + IL-7 +with Notch-ligand. carboxyfluorescein FACS plots succinimidyl are representative ester (CFSE) of 2 experiments. and cultured (B) Umbilicalfor 8 days cord in bloodIL-15 alonecells were or IL-15 isolated + IL-7 and + labelledNotch-ligand. with cell FACS trace pl violetots are (CTV) representative and cultured of 2 in experiments. either FLT3L ( +B) SCF Umbilical + TPO cordor IL-15 blood alone cells for were 7 days. isolated Cultures and labelled were analysed with cellfor trace expression violet (CTV) of CD7 and andcultured CTV. in FACS either plots FLT3L are +representative SCF + TPO or ofIL-15 three alone experiments. for 7 days. Cultures were analysed for expression of CD7 and CTV. FACS plots are representative of three experiments. 2.2. The Lin−CD7+ Fraction Consists of Multiple Subsets Defined by CD117 and CD127 2.2. The Lin−CD7+ Fraction Consists of Multiple Subsets Defined by CD117 and CD127 We previously reported an expansion of mature CD7+CD56+ NK cells in the spleen and also noted + + a robustWe previously expansion ofreported CD7+CD56 an expansion− cells in theof mature BM of humanCD7 CD56 immune NK systemcells in (HIS)the spleen mice followingand also + − notedtreatment a robust with IL-15expansion receptor of agonistsCD7 CD56 [11 ].cells The in CD7 the+ BMpopulation of human in humanimmune cord system blood (HIS) and foetalmice + followingliver has previously treatment with been IL-15 postulated receptor to agonists represent [11]. a heterogeneous The CD7 population mix of progenitors, in human cord including blood and the foetalcommon liver lymphoid has previously progenitor. been po Tostulated determine to represent whether a the heterogene CD7+ IL-15ous responsivemix of progenitors, population including in HIS + theBM common was a similar lymphoid mix ofprogenitor. lymphoid To progenitors, determine wewhether further the characterised CD7 IL-15 responsive this population population based onin HISmarkers BM was who a havesimilar previously mix of lymphoid reported progenitors, to be differentially we further expressed characterised on progenitors this population at differential based ondegrees markers of lineage who have restriction; previously CD117 reported (c-kit) andto be CD127 differentially (IL-7Rα). expressed Lineage negative on progenitors progenitors at differential in human degreesfoetal bone of lineage marrow restriction; contain a CD7CD117+ fraction (c-kit) whichand CD127 co-express (IL-7R MHC-IIα). Lineage (HLA-DR) negative and progenitors CD244.2 (2B4). in + humanWithin thefoetal Lin bone−CD7 marrow+CD244.2 contain+ population a CD7 wefraction found which three co-express distinct subsets MHC-II with (HLA-DR) the Lin−CD7 and+ CD244.2fraction, − + + − + (2B4).based Within on CD117 the andLin CD127CD7 CD244.2 expression; population CD117high weCD127 found− ,three CD117 distinctintCD127 subsets+ and with CD117 thelow LinCD127CD7− high − int + fraction,cells (Figure based2A). Similarly,on CD117 these and three CD127 subsets expression; were observed CD117 at an almostCD127 identical, CD117 frequencyCD127 gating and low − CD117on Lin−CD7CD127+CD34 cells+ indicating (Figure 2A). that Similarly, Lin−CD7 these+HLA-DR three+ suCD244.2bsets were+ CD34 observed+ human at foetalan almost bone identical marrow − + + − + + + + frequencyprogenitors gating contain on three Lin CD7 distinctCD34 subsets indicating based on that CD127 Lin CD7 and CD117HLA-DR expressionCD244.2 (Figure CD342B). human In contrast, foetal bonemature marrow NK cells progenitors from the foetal contain bone three marrow distinct (CD7+ CD56subsets+CD244.2 based +onCD3 CD127−) failed and to CD117 express expression CD117 nor + + + − (FigureCD127 (Figure2B). In2 C).contrast, To investigate mature NK whether cells thesefrom IL-15the foetal responsive bone HISmarrow BM progenitors(CD7 CD56 reflectedCD244.2 genuine CD3 ) faileddevelopmental to express intermediates, CD117 nor CD127 we searched (Figure for 2C). similar Toprogenitors investigate in whether foetal BM, these the IL-15 primary responsive site of human HIS BM progenitors reflected genuine developmental intermediates, we searched for similar progenitors in foetal BM, the primary site of human lymphopoiesis, and umbilical cord blood (UCB), a commonly

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Int. J. Mol. Sci. 2017, 18, 2716 4 of 10 lymphopoiesis, and umbilical cord blood (UCB), a commonly used source of human haematopoietic high − int + low − tissue.used We source found of phenotypicallyhuman haematopoietic identical tissu CD117e. We CD127found phenotypically, CD117 CD127 identicaland CD117CD117highCD127CD127−, as low + − + + wellCD117 as CD117intCD127CD127+ and CD117progenitorslowCD127 within− as well the Linas CD117CD7 lowCD244.2CD127+ progenitorsfraction of bothwithin humanised the Lin−CD7 mouse+ boneCD244.2 marrow+ fraction (Figure of2 D)both and humanised cord blood mouse (Figure bone2E). marrow (Figure 2D) and cord blood (Figure 2E).

+ FigureFigure 2. 2.Identification Identification of of CD7 CD7+progenitor progenitor subsetssubsets in humans. (A (A) )Mononuclear Mononuclear cells cells were were isolated isolated fromfrom 13-week 13-week foetal foetal bone bone marrow marrow (BM)(BM) andand analysedanalysed by by flow flow cytometry cytometry for for expression expression of oflineage lineage − + + markersmarkers (CD3, (CD3, CD56, CD56, CD19, CD19, CD14) CD14) and and CD7, CD7, HLA-DR, HLA-DR, CD244.2 CD244.2 (2B4). (2B4). LinLin−CD7CD7+CD244.2CD244.2+ progenitorsprogenitors high − int + low − werewere further further characterised characterised as as CD117 CD117highCD127CD127−, ,CD117 CD117intCD127CD127+, CD117, CD117lowCD127CD127−. Percentages. Percentages are arerepresentative representative of offour four different different donors; donors; (B) (MononuclearB) Mononuclear cells cellswere were isolated isolated from 13-week from 13-week foetal BM foetal BMand and analysed analysed by by flow flow cytometry cytometry for for expression expression of oflineage lineage markers markers (CD3, (CD3, CD56, CD56, CD19, CD19, CD14) CD14) and and CD7CD7 and and CD34. CD34. Lin Lin−CD7−CD7+CD244.2+CD244.2++ progenitors were further characterised as CD117highhighCD127CD127−, −, CD117CD117intintCD127CD127++,, CD117CD117lowlowCD127CD127−.− Percentages. Percentages are are representative representative of offour four different different donors; donors; (C) ( C) CD56CD56+CD3+CD3−− NK cells from from 13-week 13-week foetal foetal BM BM were were analysed analysed by flow by cytometry flow cytometry for expression for expression of CD7, of CD7,CD244.2, CD244.2, CD117 CD117 and and CD127. CD127. FACS FACS plots plots are are representative representative of of four four donors. Lin−CD7CD7+CD244.2+CD244.2+ + progenitorsprogenitors from from ( D(D)) humanised humanised mouse bone bone marrow marrow or or(E) ( Eumbilical) umbilical cord cord blood blood was analysed was analysed for forthe the CD127/CD117 CD127/CD117 progenitor progenitor subsets subsets identified identified in in(A). (A FACS). FACS plots plots are are representative representative of ofthree three biologicalbiological replicates. replicates.

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2.3.2.3. Differential Differential Lineage Lineage Potential Potential of of Progenitors Progenitors WeWe had identified identified IL-15 responsive responsive cells cells that that coul couldd be be found found in in multiple multiple sources sources of of human human tissue. tissue. ToTo determine whether these were indeed NK cell pr progenitors,ogenitors, and and more more broadly, the developmental potentialpotential of of these these cells, cells, we we sorted sorted ea eachch fraction fraction from from human human foetal foetal BM, BM, as this as this is the is thephysiological physiological site ofsite human of human lymphopoiesis, lymphopoiesis, and tested and testedtheir lineage their lineage potential potential both in vivo both andin vivo in vitro.and Allin vitro three. Allfractions three efficientlyfractions efficientlygave rise to gave CD56 rise+NKp46 to CD56+ NK+NKp46 cells +inNK vitro cells as inexpected vitro as (Figure expected 3A). (Figure We were3A). unable We were to observeunable toT cell observe differentiation T cell differentiation in these in vitro in these assaysin vitro whileassays all subsets while gave all subsets rise to gaveB cells rise (Figure to B cells3B). Interestingly,(Figure3B) . Interestingly, despite their despite expression their of expression CD7, long of postulated CD7, long to postulated be a marker to beof alymphoid marker of restriction, lymphoid − therestriction, CD117high theCD127 CD117− fractionhighCD127 efficientlyfraction generated efficiently CD14 generated+ monocytes CD14 (Figure+ monocytes 3C). Neither (Figure 3theC). CD117 Neitherint orthe CD117 CD117lowint progenitorsor CD117low retainedprogenitors this myeloid retained potential, this myeloid consistent potential, with a consistent model in withwhich, a modelas in the in murinewhich, assystem, in the decreasing murine system, levels decreasingof CD117 denoted levels of increasing CD117 denoted lineage increasing restriction. lineage restriction.

− + FigureFigure 3. Lineage potential of foetal BMBM progenitors.progenitors. LinLin−CD7CD7+ progenitorsprogenitors were were sorted sorted from from foetal foetal high − int + low − BMBM as as CD117 CD117highCD127CD127, −CD117, CD117CD127intCD127or +CD117or CD117CD127lowCD127. (A)− Progenitors.(A) Progenitors were cultured were cultured on MS-5 on + + stromalMS-5 stromal cells with cells IL-15 with IL-15and analysed and analysed after after7 days 7 daysof culture of culture for the for presence the presence of CD56 of CD56NKp46+NKp46 NK+ cells;NK cells; (B,C ()B Progenitors,C) Progenitors were were cultured cultured on onMS-5 MS-5 stromal stromal cells cells with with IL-7 IL-7 and and analysed analysed after after 7 7 days days of of cultureculture for the presence of ((B)) CD10CD10++CD19+ BB cells cells and and ( (CC)) CD10 CD10−CD14CD14+ +monocytesmonocytes and and macrophages. macrophages. FACSFACS plots are representative of 2 experiments.

+ 2.4.2.4. CD7 CD7+ ProgenitorProgenitor Subset Subset Potential Potential in in HIS HIS BM BM and and Cord Cord Blood Blood − + WeWe had found subsetssubsets withinwithin thethe LinLin−CD7CD7+ populationpopulation that that displayed displayed progressive progressive lineage lineage high low restriction,restriction, suggesting a temporal progressionprogression fromfrom the the CD117 CD117high population toto thethe CD117CD117lowfractionfraction inin the the process process of of human human lymphopoiesis. lymphopoiesis. Given Given the inherent the inherent difficulty difﬁculty in obtaining in obtaining human human foetal tissue, foetal andtissue, the andpresence the presence of phenotypically of phenotypically similar progenitors similar progenitors in models in of modelshuman oflymphopoiesis, human lymphopoiesis, including umbilicalincluding cord umbilical blood cord(UCB) blood and our (UCB) newly and described our newly HIS described BM, we HISwanted BM, to we determine wanted towhether determine this high − processwhether thisof lineage process ofrestriction lineage restriction was conserved was conserved in the in UCB the UCB and and HIS HIS BM BM CD117CD117highCD127−, , CD117intCD127+ and CD117lowCD127− progenitors. We therefore sorted each fraction from cord blood or HIS BM (Figure 4), and analysed their responses to various cytokine stimuli. We found that no population from either cord blood or HIS BM gave rise to any progeny in response to granulocyte

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CD117intCD127+ and CD117lowCD127− progenitors. We therefore sorted each fraction from cord bloodInt. J. orMol. HIS Sci. 2017 BM, 18 (Figure, 2716 4), and analysed their responses to various cytokine stimuli. We found6 of 10 that no population from either cord blood or HIS BM gave rise to any progeny in response to granulocytemacrophage macrophage colony stimulating colony stimulatingfactor (GM-CSF) factor [18], (GM-CSF) suggesting [18], a suggestinglack of myeloid a lack potential of myeloid in any potentialof these inprogenitors. any of these This progenitors. discrepancy This not discrepancywithstanding, not cord withstanding, blood or HIS cord BM bloodderived or progenitors HIS BM derivedfollowed progenitors the same followed broad process the same of broad lymphoid process lineage of lymphoid commitment lineage as commitment their foetal as bone their marrow foetal − bonecounterparts. marrow counterparts. Both the CD117 BothhighCD127 the CD117− andhigh CD117CD127intCD127and+ CD117fractionsintCD127 efficiently+ fractions generated efﬁciently B cells, T generatedcell and BNK cells, cells, T cell with and B NKcell cells,potential with peaking B cell potential (by proportion peaking (byof progeny) proportion in ofthe progeny) CD117int inCD127 the − − CD117population.intCD127 population.

− + FigureFigure 4. Lineage4. Lineage potential potential of HISof HIS BM BM and and umbilical umbilical cord cord blood blood (UCB) (UCB) progenitors. progenitors. Lineage Lineage−CD7CD7+ progenitorsprogenitors were were sorted sorted from from HIS HIS BM BM or UCBor UCB as CD117as CD117highCD127highCD127−, CD117−, CD117intCD127intCD127+ +or or CD117CD117lowlowCD127CD127− −as as indicated. indicated. Progenitors Progenitors were were cultured cultured on on OP9 OP9 stromal stromal cells cells with with IL-7 IL-7 or or IL-15 IL-15 and and analysedanalysed after after 14 14 days days for for the the presence presence of CD45of CD45+CD19+CD19+ B+ cellsB cells or CD45or CD45+CD56+CD56+ NK+ NK cells cells respectively respectively oror on on OP9-DL1 OP9-DL1 stromal stromal cells cells with with IL-7 IL-7 and and analysed analysed after after 21 days21 days for for the the presence presence of CD45of CD45+CD3+CD3+ T+ T cells.cells. FACS FACS plots plots are are representative representative of 2of experiments. 2 experiments.

2.5.2.5. Frequency Frequency of of Lymphoid Lymphoid Progenitors Progenitors within within CD7 CD7 Populations Populations WeWe had had demonstrated demonstrated a broadly a broadly similar similar pattern pattern of lineageof lineage commitment commitment inthe in the Lin Lin−CD7−CD7+ fraction+ fraction thatthat could could be be tracked tracked by by differential differential expression expression of of CD117 CD117 and and CD127. CD127. However,However, fromfrom these assays, it it was unclearunclear whetherwhether thesethese werewere homogeneoushomogeneous populations populations with with the the potentials potentials seen seen in in bulk bulk assays, assays, oror whether whether a subset a subset of eachof each population population was was giving giving riseto rise the to lineages the lineag wees observed we observedin vitro inand vitroin vivoand .in Tovivo. determine To determine the degree the ofdegree heterogeneity of heterogeneity within with thesein populations, these populations, thus better thus better characterising characterising the lymphoidthe lymphoid progenitors, progenitors, we ﬁrst we sought first sought to quantify to quanti the frequencyfy the frequency of progenitors of progenitors with potential with potential for each for lymphoideach lymphoid lineage. lineage. Since ourSince bulk our culturebulk culture assays assays had had shown shown HIS HIS BM BM to closely to closely reﬂect reflect the the lineage lineage potentialpotential of of foetal foetal BM BM fractions, fractions, we we analysed analysed the the CD117 CD117highhigh, CD117 CD117intint andand CD117 CD117lowlow fractionsfractions from from HIS HISBM BM in inlimit limit dilution dilution assays assays under under the the conditions conditions that that supported supported the the growth growth of of T T cells, cells, NK cells or B B cells. WeWe found found that that the the frequency frequency of of progenitors progenitors with with B, B, T andT and NK NK cell cell potential potential varied varied between between the the subsetssubsets (Figure (Figure5). 5). Consistent Consistent with with the the results results seen seen in ourin our bulk bulk assays, assays, the the CD117 CD117intCD127intCD127+ fraction+ fraction containedcontained the the greatest greatest frequency frequency of B cellof progenitors.B cell progenitors. This population This population had a B-cell had precursor a B-cell frequency precursor offrequency approximately of approximately twice the CD117 twicehigh thefraction, CD117high while fraction, the CD117 whilelow thepopulation CD117low population had few to nohad B-cell few to progenitors.no B-cell progenitors. Interestingly, Interestingly, T-cell potential T-cell seemed potential to correlate seemed withto correlate this trend, with while this NKtrend, progenitor while NK progenitor frequency trended inversely to B-cell potential. However, we found a low frequency of progenitors for any lineage amongst all the progenitor fractions, suggesting a high level of heterogeneity in all the progenitor populations. Elucidating the nature of this heterogeneity will require single cell analysis of progenitor potential.

Int. J. Mol. Sci. 2017, 18, 2716 7 of 11 frequency trended inversely to B-cell potential. However, we found a low frequency of progenitors for any lineage amongst all the progenitor fractions, suggesting a high level of heterogeneity in all the progenitor populations. Elucidating the nature of this heterogeneity will require single cell analysis of Int.progenitor J. Mol. Sci. potential.2017, 18, 2716 7 of 10

FigureFigure 5. Frequency ofof lymphoidlymphoid progenitorprogenitor potentialpotentialwithin within lin-CD7 lin-CD7+ +fractions. fractions. LineageLineage−−CD7+ progenitors were sorted fromfrom HISHIS BMBM as as CD117 CD117highhighCD127CD127−−, ,CD117 CD117intintCD127CD127+ +oror CD117 CD117lowlowCD127CD127− as− indicated.as indicated. Varying Varying numbers numbers of prog ofenitors progenitors from from27 cells 27 to cells 1 cell to per 1 cell well per were well cultured were cultured on OP9 stromal on OP9 cellsstromal with cells IL-7 with or IL-15 IL-7 and or IL-15analysed and after analysed 14 days after for 14 the days presence for the of presenceCD45+CD19 of CD45+ B cells+CD19 or CD45+ B+ cellsCD56 or+ NKCD45 cells+CD56 respectively+ NK cells or respectivelycultured on OP9-DL1 or cultured stromal on OP9-DL1 cells with stromal IL-7 and cells analysed with IL-7 after and 21 analysed days for after the presence21 days forof theCD45 presence+CD3+ T of cells. CD45 Wells+CD3 with+ T cells.more Wellsthan 2 with cells more were than scored 2 cells as positive, were scored and asprogenitor positive, frequencyand progenitor estimated frequency using estimated Extreme using ExtremeLimitingLimiting Dilution Dilution Analysis Analysis (ELDA) (ELDA) software (https://www.elda.at/portal27/eldaportal/con(https://www.elda.at/portal27/eldaportal/content?contentid=10007.682446&viewmode=contenttent?contentid=10007.682446&viewmode=content). ).

3.3. Discussion Discussion NKNK cellscells are are short short lived lived innate innate effector effector lymphocytes lymphocytes that spontaneously that spontaneously react against react transformed against transformedand pathogen and infected pathogen tissues. infected The peripheral tissues. The pool periph of matureeral pool NK of cells mature requires NK constantcells requires replenishment constant replenishmentfrom bone marrow from progenitors bone marrow [19 progenitors]. Upon commitment [19]. Upon to commitment the NK cell lineage,to the NK NK cell cell lineage, homeostasis NK cell is homeostasisregulated by theis regulated growth factor by the IL-15 growth [20]. We factor here describeIL-15 [20]. novel We stages here indescribe the pathway novel of stages early NKin the cell pathwaycommitment, of early prior NK to expressioncell commitment, of conventional prior to NK expression cell markers of conventional and driven by NK IL-15 cell responsiveness. markers and drivenFurther, by we IL-15 describe responsiveness. conservation Further, of these we patterns describe of lineage conservation commitment, of these albeit patterns with differentialof lineage commitment,stages of commitment, albeit with across differential multiple stages sources, of validatingcommitment, a humanised across multiple immune sources, system (HIS)validating mouse a humanisedas a physiological immune model system for (HIS) lymphopoiesis. mouse as a physiological model for lymphopoiesis. CD7CD7 is a transmembrane protein protein member member of of the the Ig Ig superfamily with with a a potential role role in NK cell adhesionadhesion [21], [21], and and expressed expressed highly highly on on mature mature NK NK cells. cells. We We describe a linear pathway pathway of of restriction restriction toto the the NK NK cell cell lineage lineage that that can can be be tracked tracked by by CD7 CD7 expression expression and and as as is is the the case for for the murine NK NK lineage,lineage, increasing increasing IL-15 IL-15 responsiveness. responsiveness. Intriguingly, Intriguingly, contrary to that described in mice, the earliest IL-15IL-15 responsive responsive human human progenitors progenitors are are not not NK NK cell cell restricted, but but rather rather retain retain both lymphoid and and myeloidmyeloid potential,potential, raising raising questions questions around around the differentialthe differential role of IL-15role of in NKIL-15 cell in lineage NK speciﬁcation.cell lineage specification.Whether this lineageWhether plasticity this lineage has a physiologicalplasticity has role,a physiological and whether role, these and alternate whether potentials these alternate manifest potentialsunder physiological manifest under conditions, physiological are harnessed conditio underns, are emergency harnessed conditionsunder emergencyin vivo ,conditions or represent in vivo,residual or represent potential onlyresidual realised potential in the only presence realised of supraphysiological in the presence of levelssupraphysiological of cytokines, islevels unclear. of + cytokines,Indeed, in is vitro unclear.cultivation Indeed, of in highly vitro puriﬁedcultivation human of highly CD34 purified+ progenitors human in CD34 IL-15 resultsprogenitors in the in clear IL- 15proliferation results in the of aclear fraction proliferatio of thesen progenitorsof a fraction without of these obviousprogenitor inductions without of obvious an NK cell induction commitment of an NKprogram cell commitment in the proliferating program population. in the proliferating population. A common source of variability in studies of human haematopoiesis is that range of progenitor sources used, as access to human tissue varies widely. Further, there remains a need for a model that resembles the physiological pathway of human haematopoietic cell generation as in vivo manipulation of the immune system, such as for cancer immunotherapy, becomes increasingly common. We here demonstrate an identical pattern of lineage restriction in HIS BM as that seen in foetal BM, the primary site of human lymphopoiesis, suggesting the haematopoietic lineages that

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A common source of variability in studies of human haematopoiesis is that range of progenitor sources used, as access to human tissue varies widely. Further, there remains a need for a model that resembles the physiological pathway of human haematopoietic cell generation as in vivo manipulation of the immune system, such as for cancer immunotherapy, becomes increasingly common. We here demonstrate an identical pattern of lineage restriction in HIS BM as that seen in foetal BM, the primary site of human lymphopoiesis, suggesting the haematopoietic lineages that develop in these mice do so along pathways that mirror their physiological counterparts in humans. Thus, we suggest the HIS mouse model as an ideal tool to study the stages of human haematopoiesis. In limit dilution assays, we found surprisingly low precursor potential amongst all precursor fractions. While this may be in part due to the limitations of the assay in efficiently supporting the development of lymphoid cells from all potential progenitors, it also suggested the progenitor fractions are highly heterogeneous. Traditionally, such heterogeneity has been delineated by the further subsetting of the precursor fractions by surface markers. However, the relative lack of available reagents and known surface markers in humans may preclude such fine subsetting. Given recent advances in genomic technology, we suggest the progenitors we have described here form the basis for isolation of progenitors for single cell RNA sequencing, which would provide further insights into progenitor heterogeneity and regulation. Although such work is beyond the scope of this study, our description of the frequency of progenitors with B, NK and T cell lineage in each fraction and our description of the consistency of progenitor potential across different sources of human haematopoietic tissue lays an exciting foundation for correlating progenitor potential with the gene expression profile of cells within these populations, thus identifying regulators of this process.

4. Materials and Methods

4.1. Mice Balb/C immunodeﬁcient mice have been previously described [11,22]. Experimental mice were speciﬁc-pathogen-free (SPF) and all animal work was conducted with standard operating procedures approved by The Walter and Eliza Hall Institute of Medical Research animal ethics committees AEC2015.017 (1/3/2015). Mice with human immune systems were generated by irradiating newborn (3–5 day-old) mice with 3 Gy total body irradiation from a γ source, and were injected intra-hepatically (i.h.) with 5 × 105–3× 106 CD34+ human cord blood cells obtained from the Bone Marrow Donor Institute/Cord Blood Research Unit (Murdoch Children’s Research Institute, Parkville, VIC, Australia) following approval from The Walter and Eliza Hall Institute of Medical Research Human Research Ethics Committee.

4.2. In Vitro Assays of Progenitor Potential Progenitors were isolated as indicated and were cultured on either MS-5 or OP9 stromal cells with IL-7 (50 ng/mL) to determine B cell potential; on MS-5 stromal cells with granulocyte macrophage colony stimulating factor (GM-CSF (25 ng/mL) to determine myeloid potential; on either MS-5 or OP9 stromal cells with IL-15 (50 ng/mL) to determine NK cell potential; or on OP9-DLI stromal cells with IL-7 (50 ng/mL) to determine T cell potential. Cells were split 1:2 onto fresh stromal cells and fresh cytokine media if required. Myeloid assays were analysed after 7 days for the presence of CD33+CD14+ monocytes and CD33+CD16+ granulocytes; B cell and NK cell assays were analysed after 14 days for the presence of CD45+CD19+ B cells or CD45+CD56+ or NKp46+ NK cells respectively; and T cell assays analysed at 21 days for the presence of CD45+CD3+ T cells. All cultures were analysed by flow cytometry as described below. Foetal bone marrow cultures were performed in 48 well plates in DMEM/10% AB serum + IL-15 (50 ng/mL) or on OP9-DL1 with IL-7 (50 ng/mL) and IL-15 (50 ng/mL). Cells were purified as indicated and labelled with 5 µM carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, USA). Int. J. Mol. Sci. 2017, 18, 2716 9 of 11

4.3. Flow Cytometry Analysis for Cell-Surface and Intracellular Markers The following anti-human mAb were used to stain cell suspension for FACS analysis: CD3 (SK7), CD4 (SK3), CD34 (581), CD8 (SK1), CD19 (HIB19), CD10 (HI10a), CD38 (HB7), NKp46 (9E2), CD16 (3G8), CD161 (DX12), CD56 (B159), HLA-DR (L243), CD244.2 (2B4), HLA-A/B/C (G46-2.6), CD117 (YB5.B8), CD14 (M5E2), CD122 (Mik-3), TCR-β (T10B9.1A-31), CD127 (hIL-7R-M21), CD11c (B-ly6), CD7 (M-T701), CD45 (2D1) from (BD Bioscience, CA, USA). All washings and reagent dilutions were done with phosphate buffered saline (PBS) containing 2% foetal calf serum (FCS). All acquisitions were performed using FACSVerse cytometers and cell sorting was perform using FACS ARIA, all machines were interfaced to the FACS-Diva software (BD Bioscience). Data analysis was performed using FlowJo software from TreeStar Inc. (Ashland, OR, USA).

4.4. Cell Preparation Umbilical cord blood was obtained from the Bone Marrow Donor Institute Cord Blood Bank (Murdoch Children’s Research Institute, Parkville, VIC, Australia). Experiments were approved by The Walter & Eliza Hall Institute of Medical Research Human Research Ethics Committee (HREC12/08). Magnetic enrichment of CD34+ cells (>98% pure) was performed by using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA, USA), after preparation of single-cell suspension and isolation of mononuclear cells by density gradient centrifugation over Ficoll-Hypaque (Nycomed Pharma, Roskilde, Denmark). Cell suspensions were prepared in PBS medium with 2% foetal calf serum. Single cell suspensions of murine organs were prepared as previously described [23]. Human foetal bone marrow was a kind gift from Professor Hergen Spits (AMC, Amsterdam-Zuidoost, The Netherlands) and obtained from elective abortions approved by the Medical and Ethical Committee at AMC-UvA. Human NK cells were puriﬁed from donor blood buffy coat prepared by density gradient centrifugation over Ficoll-Hypaque (Nycomed Pharma, Zürich, Switzerland) using anti-CD56 magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany). Puriﬁed cells (CD34+) were loaded with 5 µM CFSE (Invitrogen, Carlsbad, CA, USA) or 5 µM cell trace violet (CTV) and cultured at 1 × 105 cells/mL in StemSpan (STEMCELLS Technologies, Vancouver, Canada) with 10% human serum and either 50 ng/mL recombinant human IL-15 (rhIL-15; Peprotech, NJ, USA) or human thrombopoietin (TPO), fms-like tyrosine kinase 3 ligand (FLT3L) and stem cell factor (SCF) (100 ng/mL).

4.5. Statistical Analysis Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). All data were subjected to two-tailed unpaired Student t test analysis. Limit dilution assays were analysed using extreme limit dilution assay (ELDA) software (Hu and Smyth, 2009).

Acknowledgments: We acknowledge the Bloemenhove Clinic (Heemstede, The Netherlands) for providing foetal tissues and Hergen Spits (AMC, Amsterdam-Zuidoost, The Netherlands) for the kind gift of foetal bone marrow and reagents. This work is supported project grants from the National Health and Medical Research Council (NHMRC) of Australia (1049407, 1066770, 1057852, 1027472 to N.D.H), an NHMRC CDF2 fellowship (NDH) as well as an NHMRC Independent Research Institute Infrastructure Support scheme grant and a Victorian State Government Operational Infrastructure Scheme grant. NDH is a recipient of research grants from the following organisations: Ian Potter Foundation (Australia), Harry J Lloyd Charitable Trust (USA), Melanoma Research Alliance (USA) and Cancer Research Institute (USA). Author Contributions: Priyanka Sathe and Nicholas D. Huntington designed and performed experiments and wrote the paper; Rebecca Delconte and Swee Heng Milon Pang performed experiments and Ngaire Elwood supplied reagents. Conﬂicts of Interest: The authors declare no conﬂict of interest. Int. J. Mol. Sci. 2017, 18, 2716 10 of 11

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