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Stem Cells 2004;22:1101-1110 www.StemCells.com
© 2004 AlphaMed Press

Absence of Major Histocompatibility Complex Class I on Neural Stem Cells Does Not Permit Natural Killer Cell Killing and Prevents Recognition by Alloreactive Cytotoxic T Lymphocytes In Vitro

Michele Mammolentia, Shyam Gajavellib, Pantelis Tsoulfasb, Robert Levya

a Department of Microbiology and Immunology and
b Department of Neurosurgery, University of Miami School of Medicine, Miami, Florida, USA

Key Words. Neural stem cells • T-cell recognition • Natural killer recognition Myosin heavy chain expression • Cytotoxicity

Correspondence: Robert B. Levy, Ph.D., University of Miami School of Medicine Department of Microbiology and Immunology, P.O. Box 016960 (R-138), Miami, FL 33101, USA. Telephone: 305-243-4542; Fax: 305-243-6903; e-mail: rlevy{at}med.miami.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Potential applications of neural stem cells (NSCs) for transplantation requires understanding myosin heavy chain (MHC) expression and the ability of T cells and natural killer (NK) cells to recognize this progenitor population. Cells from the cortices of day-13 embryonic (E13) B6 (H-2b) mice were explanted and cultured to expand NSCs. Analysis of P2-P17–cultured cells using anti-MHC class I/II monoclonal antibodies (mAbs) showed marginal expression of both products. Although recombinant murine interferon-gamma (rmIFN{gamma}) exposure did not alter the multipotential capacity of these stem cells, titration of mrIFN{gamma}NSC cultures demonstrated that MHC molecules could be strongly upregulated after addition of 3 ng/ml rmIFN{gamma} for 60 hours. To assess the susceptibility of NSCs with low or absent versus high levels of MHC expression to lysis by cytotoxic T lymphocyte (CTL) and NK populations, untreated and rmIFN{gamma}-treated NSC target cells were examined. Untreated NSCs were not recognized by BALB/c (H-2d) allospecific anti-H-2b CTL, consistent with the mAb findings; however, upregulation of MHC products on both early and later passaged NSCs resulted in their efficient lysis by CTL. NK cells were prepared from syngeneic B6 or allogeneic BALB/c mice. Although NK cells effectively killed control YAC-1 target cells, these effectors did not kill MHC-deficient (or expressing) NSC targets. Thus, similar to hematopoietic, embryonic, and mesenchymal stem cell populations, unmanipulated NSCs are not readily killed by T and NK cells. These findings suggest that following transplant into syngeneic or allogeneic recipients, NSCs may exhibit diminished susceptibility to clearance by host T- and NK-cell populations.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Stem cell populations can be identified in the central nervous system (CNS) of both embryonic and adult mammals, including mice [1,2]. These neural stem cell (NSC) populations have the capacity to produce both neuronal and glial progeny in vitro, including neurons, astrocytes, and oligo-dendroglial cells [1,3]. In addition, several studies have reported that murine and human NSCs have the potential to transdifferentiate and produce hematopoietic and other progeny [4–7]. However, several groups have challenged these results [8–10]. Furthermore, hematopoietic stem cell (HSC) transdifferentiation into nonhematopoietic cells is also an extremely rare event, and the results have not been reproduced [11–13]. Notably, in vivo transdifferentiation in murine experiments was reported following transplant into allogeneic myosin heavy chain (MHC) disparate recipients [4]. One question that arises following transplant of any progenitor population is how such cells will be surveyed by the recipient immune system. Because both T cells and natural killer (NK) cells have the potential to reject foreign progenitor cell populations [14–16], the expression of cell-surface MHC gene products is presumed to be a crucial factor in transplant outcome.

Although the absence of MHC expression on the cell surface of a transplanted population may protect against T-cell recognition, the inability to signal inhibitory receptors (i.e., Ly49 and KIR) may result in immune attack by autologous or allogeneic NK populations [17]. The ability to evade both adaptive and innate systems in the recipient could support the initial engraftment of stem cell populations, although maintenance of differentiated MHCs expressing progeny may require different regulatory processes. Recent studies have reported that NK cells do not readily lyse embryonic stem cells [18], and previous investigations failed to detect NK and T cell–mediated killing of hematopoietic stem cell populations [19]. CNS-derived cell populations, including neurons and oligodendroglial cells, express almost undetectable MHC class I and II products [20,21]. The present studies assessed the constitutive and regulated expression of MHC products in embryonic-derived NSCs before analysis of their susceptibility to killing by cytotoxic T lymphocyte (CTL) and NK population. The findings demonstrated that the absence of MHC class I and II expression by early and later passaged NSCs protects the cells from immune recognition by allospecific CD8+ CTL. However, upregulation of MHC class I on these stem cells enables their efficient recognition and lysis by CTL. Nonetheless, the absence or presence of MHC expression did not result in NSC susceptibility to lysis by either syngeneic or allogeneic NK cells. Such findings suggest that stem cells may not be readily killed by T/NK cells via the principal cell-mediated lytic effector pathways. The findings are discussed in relation to how stem cell populations may evade and inhibit immune graft rejection responses after transplant.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
C57BL/6J (H-2b) (B6), BALB/c (H-2d), C5BL/6J–severe combined immunodeficiency (SCID) (H-2b), and BALB/c-SCID (H-2d) mice were obtained from Jackson Laboratory (Bar Harbor, ME). B6.SJL-Cd45aPep-3b/BoyJ (H-2b, Ly5.1) originally obtained from Jackson Laboratory were bred in our facility and used to generate some neurosphere cultures. All mice were maintained in a pathogen-free colony until use. Time-pregnant E10 C57BL/6J mice were obtained and dissected on E13. Embryos were staged using previously defined criteria [22].

Expansion and Differentiation of Neurospheres
NSCs were derived from C57BL/6J (H-2b) mouse embryonic cerebral cortices (gestation day 13; day of conception = day 0). The embryonic cortices were dissected in Ca2+ and Mg2+-free Hank’s balanced salt solution, dissociated, and plated in N2 supplemented with 20 ng/ml of fibroblast growth factor (FGF) 2 or FGF2 and epidermal growth factor (EGF) at 20 ng/ml each on uncoated tissue culture dishes. After 4–5 days in vitro, small neurospheres appear at an average of 50 microns in diameter. This is considered passage zero (P0). Neurospheres were passaged according to Reynolds et al. [23]. After 4–5 days in P0, the spheres were spun down at 800 rpm for 2 minutes and resuspended in 2 ml N2. The spheres were mechanically dissociated into single cells by trituration with a fire-polished Pasteur pipette, counted, and replated at low-density in N2 supplemented with 20 ng/ml of FGF2 or FGF2 and EGF at 20 ng/ml each. This is considered P1. To initiate differentiation, neurospheres were plated on fibronectin or collagen-coated dishes in the presence of 2.5% fetal calf serum without FGF2.

Immunofluorescence and Staining Analysis
The monoclonal antibodies (mAbs), obtained from Pharmingen (San Diego), were as follows: phycoerythrin (PE) anti-mouse I-Ab (AF6-120.1), PE anti-mouse H-2Kb (AF6-88.5), PE anti-mouse Ly-6A/E (Sca-1) (E13-161.7), PE-anti-NK1.1, and PE-DX5. Briefly, fresh E13 cortical cells or carefully triturated, later-passaged (as indicated) NSCs were washed with fluorescence-activated cell sorter (FACS) buffer (phosphate-buffered saline [PBS] containing 1% bovine serum albumin [BSA] and 0.02% sodium azide) and incubated with the appropriate PE-conjugated mAbs for 30 minutes on ice. The cells were again washed and resuspended in 0.5 ml FACS buffer, and 0.5 x 104 cells were collected and analyzed on a FACscan flow cytometer within gates determined by the lack of staining with 7AAD (Pharmingen). Cells fixed with 4% paraformaldehyde were briefly permeabilized with 0.1% Triton-X100 and blocked with 10% normal goat serum, incubated with primary antibody for 45 minutes, washed, and incubated with secondary antibody for 30 minutes. In the last wash, cells were incubated with diamidinophenylindole (DAPI) (0.3 mg/ml) and mounted with Citifluor (2.5% 1,4diazabicyclo[2,2,2]octane, 90% glycerol). The following primary antibodies were diluted and used: 1:150 of anti-nestin mAB (Rat-401), developed by Hockfield and McKay [29] and obtained from Developmental Studies Hybridoma Bank, maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA [18], 1:500 of anti-NCadherin mAB (Transduction Labs, BD Biosciences, CA), 1:1500 of anti-GFAP polyclonal (DAKO, Carpinteria, CA) [24], 1:2000 TuJ1 polyclonal (Covance, Princeton, NJ) [25], undiluted mAb 01 [26], and 1:5 of RIP mAB [27]. Appropriate secondary antibodies, goat anti-mouse Alexa 594 and goat anti-rabbit Alexa 488, were purchased from Molecular Probes (Eugene, OR). For staining using two mAB (e.g., N-cadherin and nestin), Zenon One Alexa 594 kits were used to label the antinestin antibody (Molecular Probes). Images were captured using an Olympus IX70 microscope and an Optronix DEI-75-0 CCD camera connected to a 9600 Power Macintosh equipped with a CG-7 PCI card and Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Confocal images were obtained using a laser-scanning confocal microscope Zeiss LSM510.

Cytotoxic Effector Populations and Cytotoxicity Assay
The generation of anti-H-2b CTL was performed as previously described [28]. Briefly, 4 x 106 responder spleen cells from BALB/c (H-2d) mice in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids (Cellgro, Herndon, VA), glutamine, penicillin-streptomycin (Gibco, Carlsbad, CA), and 2-mercaptoethanol were mixed with 2 x 106 (20 Gy) B6 splenic stimulator cells in a final volume of 2 ml. Cultures were incubated in 24-well plates (Costar, Corning, USA) for 4–6 days at 37°C in 5% CO2 humidified air. NK killing against NSCs was examined using surface immunoglobulin positive (sIg+)–depleted normal and SCID B6 (i.e., syngeneic) and BALB/c (i.e., allogeneic) spleen cells. To augment the level of NK cells in these populations, spleen cells were initially incubated on anti-mouse Ig-coated plastic dishes for 45 minutes at 4°C to remove sIg+ cells. These populations routinely contained 12%–15% NK1.1+ or DX5+ cells. To produce highly enriched B6 NK1.1+ populations, a positive selection procedure was performed using the Miltenyi Macs System (Miltenyi Biotec Inc., Bergisch Gladbach, Germany). The sIg– B6 spleen cells were resuspended in PBS containing 0.5% BSA and labeled with DX5 mAb-conjugated (Miltenyi Biotec Inc.) magnetic beads by incubation for 15 minutes at 4°C. The cells were then washed and resuspended in PBS/0.5% BSA and loaded onto a Macs separation column in a magnetic field. The unlabeled cells were removed by washing, and DX5+ cells were eluted from the column outside of the magnetic field. NK1.1 expression following staining confirmed the enrichment for NK cells. To activate NK cells before assay, an enriched B6 NK population was cultured in medium supplemented with recombinant murine (750 U/ml) IL-2 (Peprotech, Rocky Hill, NJ) for 24 hours. Cells were collected, washed, and tested against target cell populations, as described below.

Cytotoxic activity against NSC and other targets was assessed in a 4-hour chromium (51Cr) release assay, as previously described [28]. Chromium-labeled target cells EL4 (H-2b lymphoma) and P815 (H-2d mastocytoma) were used as positive and negative control targets, respectively, for H-2d anti-H-2b–generated CTL. NSCs (passages P2 through P17) were used as target cells in the CTL assays. Some NSC cultures were supplemented with recombinant murine interferon-gamma (rmIFN{gamma}) (Peprotech, Rocky Hill, NJ) at 30 ng/ml, which was added for 60 hours before use as target cells in the cytotoxicity assay. In addition to untreated and rmIFN{gamma}-treated P2-P17 NSCs,YAC-1 was used as a positive NK-sensitive control target population. All target cells were chromium-labeled, washed, and added at 10,000 cells/well (0.1 ml) in 96-well U-bottom microtiter plates (Costar). T cells or NK effector cells were added to the targets at various E:T ratios in triplicate cultures and incubated for 4 hours at 37°C, 5% CO2 humidified air. Supernatants were harvested, and percentage specific target cell lysis was calculated by using the following formula:


in which experimental CPM is CPM from cultures with effector cells, maximum CPM is CPM of acid lysed target cells, and spontaneous CPM is CPM from supernatants of target cells incubated in the absence of effector cells.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FGF2-expanded neuroepithelial stem cell cultures were established as neurospheres from E13 mouse cerebral cortex. The neurospheres were immunopositive for the neuroepithelial stem cell marker [29] nestin (Fig. 1BGo) and negative for neuronal and macroglia markers (data not shown). Upon plating onto an adherent substrate and serum, the cells differentiate into neurons (stained for type III beta-tubulin, a neuronal marker), oligodendrocytes (stained for O1, an oligo-dendroglial marker [26]), and astrocytes (stained for glial fibrillary acidic protein [GFAP], an astrocytic marker) [24] (Figs. 1C, 1DGo), indicating that the neurospheres had the capacity to differentiate along the main CNS lineages. Continuous passage of neurospheres were obtained following dissociation and reculturing in noncoated dishes. Cells from neurospheres up to 17 passages were used in the experiments reported in this study. Transplant of stem cell populations requires host conditioning to facilitate donor cell engraftment and inhibits immune-mediated rejection by the recipient [30–32]. To investigate the expression of MHC molecules on NSCs, freshly obtained B6 (H-2b) cortical cells from E13 embryos were incubated with directly conjugated anti-MHC class I and II mAbs. Flow cytometric analysis failed to detect staining with either H-2Kb–specific or H-2IAb–specific mAbs, indicating the absence of detectable cell-surface MHC proteins on these CNS cells (Fig. 2AGo). Subsequently, cells from NSC cultures (grown either with FGF2 or FGF2) and EGF (P2 and P15) were also found to lack expression of MHC class I and II products (Fig. 2Go and data not shown). IFN{gamma} is known to modulate MHC class I and II expression in mammalian cells [33–35], including neuroepithelial cells [20,21]. To determine if IFN{gamma} could upregulate MHC antigens in these cells, we incubated NSC (P2) for up to 60 hours in different concentrations of rmIFN{gamma} before staining with the anti-MHC class I–specific and II–specific mAbs. After incubation with rmIFN{gamma} concentrations ranging from 3–30 ng/ml, both MHC class I and II products were readily upreg-ulated in these cultures (Figs. 2C, 2DGo). MHC class I and II were also upregulated on long-term passaged cells, i.e., P9 to P15 (data not shown).



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  		Figure 1. Stem cells from neurospheres give rise to all three central nervous system–derived cell types. (A): Phase contrast of P3 mouse neurospheres. (B): Double fluorescence labeling of P3-undifferentiated neurospheres showing immunoreactivity of N-cadherin in green and nestin in red. (C): P3-differentiated neurospheres were immunostained for type III beta-tubulin (green), a neuronal marker, and GFAP (red), an astrocytic marker. (D): P3-differentiated neurospheres were immunostained with 01 (yellow), an oligodendroglial marker, and GFAP (green). Note the yellow color is due to carbocyanine 3 emission spectrum and not due to overlay. Nuclei were revealed with 4,6-diamidino-2-phenylindole-2-HCI (blue). Abbreviation: GFAP, glial fibrillary acidic protein.

 


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  		Figure 2. Histograms from B6 E13 cortical cells (A) and B6 NSC P-15 (B) show a lack of MHC class I or class II staining. Solid black histograms represent unstained background. Cells stained with MHC class Ia H-2Kb-PE antibody or MHC class IIa H-2IAb-PE antibody are represented by the unfilled histogram. (A): Unstained background MFI = 2.3, class I MFI = 2.9, and class II MFI = 2.6. (B): Unstained background MFI = 3.0, class I MFI = 4.2, and class II MFI = 3.7. P2 B6 NSCs show upregulation of MHC class I (C) and class II (D) by mrIFN{gamma} after culture for 60 hours. Solid histograms represent cells cultured with no IFN{gamma} and stained with the appropriate antibody; class I MFI = 12, class II MFI = 22. Solid line histograms represent cells cultured for 60 hours with 3ng/ml IFN{gamma}; class I MFI = 303, class II MFI = 77. Dashed line histograms represent cells cultured for 60 hours with 10 ng/ml IFN{gamma}; class I MFI = 366, class II MFI = 114. Dotted line histograms represent cells cultured for 60 hours with 30 ng/ml IFN{gamma}; class I MFI = 352, class II MFI = 147. Abbreviations: MFI, mean fluroescence intensity; MHC, major histocompatibility complex; mrIFN{gamma}, recombinant murine interferon-gamma; NSC, neural stem cell.

 
To ensure that treatment of NSC cells with rmIFN{gamma} did not lead to their differentiation into CNS cells and thereby account for the expression of MHC genes, these cultures were stained with markers that define cells derived from the neuroepithelium. Individual cells in P4 neurospheres are clearly detected by costaining for both N-cadherin and nestin (Figs. 3A, 3DGo). After coculture with rmIFN{gamma}, type III beta-tubulin (a neuronal marker), RIP (an oligodendroglial marker), and GFAP (an astrocytic marker) were assessed (Fig. 3Go). These NSC cultures maintained in the presence or absence of IFN{gamma} contained low or undetectable numbers of type III beta-tubulin+ (middle panels) RIP+ (middle panel), and GFAP+ (right panels) populations. Notably, nestin continued to be expressed after rmIFN{gamma} coculture at levels equivalent to NSCs not exposed to the cytokine (right panels). These findings demonstrate that after incubation with rmIFN{gamma}, the NSC cultures did not differentiate toward neuronal, oligodendroglial, or astrocytic lineages.



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  		Figure 3. Confocal images show that the neurosphere characteristics and properties do not change after exposure to INF{gamma}. Neu-rospheres that were exposed for 60 hours to recombinant murine INF{gamma} (D, E, F) or not exposed (A, B, C) were stained with several markers to ensure that the cells did not differentiate. (A, D): Double fluorescence labeling of P4-untreated and -treated neurospheres showing immunoreactivity of N-cadherin in green and nestin in red. (B, E): P4-untreated and -treated neurospheres were immunos-tained with polyclonal type III beta-tubulin (green) or monoclonal anti-RIP (red). (C, F): Double-fluorescence labeling of P4-untreated and -treated neurospheres with a polyclonal anti-GFAP (green) and monoclonal anti-nestin (red). Note the absence of GFAP+ cells in these neurospheres. Abbreviations: GFAP, glial fibrillary acidic protein; INF{gamma}, interferon-gamma.

 
Recognition by CD8+ CTL requires expression of MHC class I products on target cells. Alloantigen reactive CTL from BALB/c spleen cells (H-2d) was generated after in vitro stimulation with B6 (H-2b)-stimulating cells and then assayed against B6 NSC cells obtained from early and later (data not shown) passaged cultures. The CTL effectively lysed control class I expressing EL4 (H-2b) target cells but failed to kill B6 P2 NSC targets (Fig. 4AGo). However, after incubation with rmIFN{gamma}, as described above, the NSC target cells were efficiently lysed by the anti-H-2b CTL (Figs. 4A, 4BGo). Identical results have been obtained with all early and later passaged NSC cultures examined (data not shown). These observations confirmed the phenotypic absence of MHC class I on NSCs and indicated that such stem cells are unlikely to be recognized by host CTL after allogeneic transplant unless these products are upregulated on the stem cell population. Correlation of NSC susceptibility to lysis and MHC expression by the cells was confirmed in an independent experiment in which P2 NSC target cells expressing lower levels of MHC following rmIFN{gamma} culture were not killed as effectively as the control H-2b target cells in the lytic assay (Fig. 4BGo).



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  		Figure 4. Alloreactive cytotoxic T lymphocytes fail to lyse untreated NSC populations but kill mrIFN{gamma}-treated NSC target cells. BALB/c spleen cells cultured with irradiated B6 spleen cells (anti-H-2b) for 5 days lyse EL4 (control H-2b target) ({blacksquare}) and B6 P2 NSCs treated with rmIFN{gamma} (30 ng/ml, see Materials and Methods) ({blacktriangledown}) but not untreated B6 P2 NSCs ({blacktriangleup}). (A, B): Differences in the amount of NSC target cell killing correlated with the amount of MHC class I upregulation on the NSC targets after rmIFN{gamma} treatment (histogram insets, A and B, MHC expression assessed using anti-H-2Kb-PE monoclonal antibody). Abbreviations: MHC, myosin heavy chain; mrIFN{gamma}, recombinant murine interferon-gamma; NSC, neural stem cell.

 
The lack of MHC expression on the NSCs in the present studies would not enable inhibitory signaling by Ly49 receptors. Therefore, NK cell populations from syngeneic B6 and B6-SCID donor mice were tested to examine their ability to kill NSCs. As expected, these effector cells readily killed the NK-sensitive YAC-1 target cells but failed to lyse rmIFN{gamma}-induced MHC class I–positive B6 NSCs (Figs. 5A, 5BGo). However, these NK cells also failed to lyse NSC target cells, which did not express detectable MHC class I (Figs. 5A, 5BGo). To determine if NK effector cells from allogeneic mice had the capacity to recognize B6 NSCs, spleen cells were assessed from normal and BALB/c-SCID mice for cytotoxic activity against NSC. Similar to the results using syngeneic populations, NK killing by this allogeneic population also exhibited strong killing against the YAC-1 target but did not lyse the NSC targets regardless of the absence (or presence) of MHC class I (Fig. 5CGo). Finally, in an independent experiment, an enriched B6 NK1.1+ population was prepared by positive selection (see Materials and Methods), and the cells were activated for 24 hours with rIL-2 before use in a cyto-toxic assay (Fig. 5DGo). This population exhibited extremely potent killing activity against the NK-sensitive YAC-1 target cells but did not lyse the NSC targets.



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  		Figure 5. Syngeneic and allogeneic NK cells do not kill early- or late-passaged NSC target cells. (A): Syngeneic B6 splenic NK cells lysed YAC-1 (control NK-sensitive target) ({blacksquare}) but not untreated P2 NSCs ({blacktriangleup}) or rmIFN{gamma}-treated (as described in Fig. 4Go) P2 NSCs ({blacktriangledown}). (B): Enriched syngeneic B6 NK spleen cells (Miltenyi DX5 beads: 74% NK1.1+, see Materials and Methods) lysed YAC-1 ({blacksquare}) but not untreated P-15 NSCs ({blacktriangleup}) or rmIFN{gamma}-treated P-15 NSC ({blacktriangledown}). (C):Allogeneic BALB/c (H-2d) splenic NK cells (5.6% DX5+, see Materials and Methods) lysed YAC-1 ({blacksquare}) but not untreated P-6 NSCs ({blacktriangleup}) or mrIFN{gamma}-treated P-6 NSCs ({blacktriangledown}). (D): Enriched syngeneic B6 NK cells (Miltenyi DX5 beads: 56.0%, see Materials and Methods) were placed into culture in medium supplemented with recombinant mouse interleukin-2 for 24 hours before assay (see Materials and Methods). Cultures were harvested, and the activated NK cells efficiently lysed YAC-1 ({blacksquare}) but not untreated P15 NSCs ({blacktriangleup}).Abbreviations: mrIFN{gamma}, recombinant murine interferon-gamma; NK, natural killer; NSC, neural stem cell.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The pluripotential capacity of NSCs makes these cells a potentially attractive population for use in CNS transplantation. MHC antigens on cells obtained from all NSC cultures examined were virtually undetectable by flow cytometric analysis or alloreactive CTL recognition. Similarly, other stem cell populations also lack MHC expression, including embryonic and mesenchymal stem cells [18,36]. However, NSCs during early and later passage in vitro could be induced to express these products under appropriate IFN{gamma} signaling conditions, also similar to other stem cell populations [18,36]. These findings examining the multipotential NSCs derived from E13 cortices in our studies contrast reports from a previous study that found that a subpopulation of E10 neuroepithelial cells that gave rise exclusively to neuronal cells failed to express MHC class I in response to mrIFN{gamma} [37].

Different exogenous factors can influence the differentiation of NSCs, and therefore IFN{gamma} might have influenced the stem cell phenotype, resulting in the upregulation of MHC gene expression. However, antibody analysis indicated that these cultures maintained the NSC phenotype and culture after IFN{gamma} exposure demonstrated retention of their capacity to generate new neurospheres (data not shown). Although MHC genes can be induced in virtually all cell populations, Sca-1, a member of the Ly6 gene family, is selectively expressed throughout the body, including expression in hema-topoietic stem cells and cells in the CNS [38,39]. Although Sca-1 was not expressed constitutively on fresh cortical cells or on cells in the NSC cultures, after rmIFN{gamma} coculture, Sca-1 was detected by flow cytometric analysis and immunohistochemical staining on a subpopulation (10%–15%) of NSCs (data not shown). In total, the findings demonstrate that IFN{gamma} selectively affected gene expression in NSC cultures; however, the NSC cells retained their phenotype following exposure to this T/NK cell–secreted cytokine.

Mouse hematopoietic stem cells have been examined for susceptibility to lysis by NK cells and were reportedly not killed by this effector population [19]. Despite the inability to induce NK inhibitory (e.g., via Ly49 signaling because of absence of MHC class I expression) signals, NSC cells were also not lysed by syngeneic (including IL-2–activated NK effector cells [Fig. 5DGo] and 7-day LAK-activated effectors [data not shown]) or allogeneic NK cells, presumably because antigens required for NK recognition by activating receptors are not present on these progenitor cells. Thus, these findings are similar to those from previous studies investigating two other stem cell populations, i.e., hema-topoietic and embryonic, which also appear not susceptible to lysis by NK effector cells [18,19]. These findings are interesting because they suggest that several stem cell populations in general may not be readily killed by T- and NK-cell populations. With regard to such findings, it is interesting to consider whether stem cell populations may inherently possess the ability to evade or diminish host immune responses. Several studies have reported the ability of stem cell populations, including mesenchymal stem cells (MSC) and HSCs, to inhibit or downregulate immune responses in vitro and in vivo [40,41]. MSCs have recently been reported to inhibit naive and memory antigen–specific T cells [36]. Other reports have noted a role for FasL in regulating progenitor cell kinetics [42]. Interestingly, notch signaling occurs in CD4+CD25+ regulatory populations [43]. Recent studies have observed that the notch ligand Jagged 1 is expressed in both primitive (CD34+CD38–Lin–) and mature human hematopoietic cells as well as mouse cultured bone marrow stroma [44,45]. Notch signaling in T regulatory cells might therefore provide an additional pathway supporting progenitor cell engraftment by regulating rejection responses.

Notably, several studies, including our own, have reported the ability of cytotoxically defective T cells to resist allogeneic stem/progenitor cell grafts [46–49]. These studies have demonstrated that despite no contribution by perforin, FasL, TNFR, TRAIL, and TWEAK (Zimmerman and Levy, unpublished observations), bone marrow progenitor cell allografts can still be resisted across allogeneic disparities. The NSCs examined in these studies were poorly if at all recognized by cytotoxic T cells or NK cells. Transplant of NSC population into allogeneic (T or NK) or syngeneic (NK) recipients might therefore provide some engraftment advantage for these cells in the absence of immediate recognition and attack by cellular immune responses in the recipient. Recent studies support the notion that CNS progenitor populations are not easily recognized in vivo [50]. NSC populations are presently being examined in transplant models to investigate their capacity to survive and differentiate following in vivo infusion.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported in part by NIH Grants 1R01 RR11576 and 5RO1 HL52461 (R.B.L.), NIH Grant N01-NS-6-2349, The Miami Project to Cure Paralysis, The FaBene Foundation, Wilson Foundation, and Abramson Foundation (P.T.). We acknowledge Karen Del Rio for her careful preparation of this manuscript and the Sylvester Comprehensive Cancer Center for its support of the Flow Cytometry Facility for the phenotypic analysis of cell populations used in these experiments.

Michele Mammolenti and Shyam Gajavelli contributed equally to this study.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received March 10, 2004; accepted for publication May 27, 2004.



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