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Ontogeny of Xenopus NK cells in the absence of MHC class I antigens
Developmental and Comparative Immunology 27 (2003) 715–726 www.elsevier.com/locate/devcompimm
Ontogeny of Xenopus NK cells in the absence of MHC class I antigens Trudy L. Hortona, Rebecca Stewarta, Nicholas Cohenb, Laura Raub, Pamela Ritchiea, Martin D. Watsona, Jacques Robertb, John D. Hortona,* b
a School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, UK Department of Microbiology and Immunology, University of Rochester Medical Center, New York 14642, USA
Received 14 November 2002; accepted 28 January 2003
Abstract This paper explores the ontogeny of NK cells in control and early-thymectomized (Tx) Xenopus laevis through phenotypic analysis of cells expressing the NK cell antigen 1F8 and by performing in vitro cytotoxic assays. Dual color flow cytometry reveals that a few 1F8positive splenocytes first emerge in late larval life, at , 7-weeks post-fertilization. This is about 2-weeks after the time when surface MHC class Ia expression can first be detected. The proportion of splenocytes expressing 1F8 remains very low in 3 – 4 month-old froglets, but by 1 year there is a sizeable 1F8positive population, which is proportionally elevated in Tx frogs. The ontogeny of NK cell function is monitored by a 5 h DNA fragmentation (JAM) assay. Control and Tx larval splenocytes (from either 5- or 7-week-old tadpoles) fail to kill MHC-deficient thymus-derived tumor cell targets. Such in vitro killing is still relatively poor in 3 – 4 month froglets, compared with high levels of tumor cell cytotoxicity mediated by splenocytes from older frogs. Immunoprecipitation studies identify that the major ligand for the 1F8 mAb is a 55 kDa polypeptide. Finally, further evidence is provided that 1F8positive lymphocytes are indeed bona fide NK cells, distinct from T cells, since purified 1F8positive splenocytes from Tx Xenopus fail to express fully rearranged TCRb V region transcripts. We conclude that NK cells fail to develop prior to MHC class I protein expression and, therefore, do not contribute to the larval immune system, whereas they do provide an important backup for T cells in the adult frog by contributing to anti-tumor immunity. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Amphibians; NK cell ontogeny; NK cell-associated 1F8 antigen; Xenopus; Thymectomy; Tumor immunity
1. Introduction Abbreviations: GFM, growth factor-rich medium (supernatant from concanavalin A-stimulated cells); MHC, major histocompatibility complex; NK cell, natural killer cell; PMA, phorbol myristate acetate; Tx, early thymectomized; 1F8 mAb, anti-Xenopus NK cell monoclonal antibody. * Corresponding author. Tel.: þ44-191-374-3359; fax: þ 44-191374-2417. E-mail address: [email protected] (J.D. Horton).
Cells that are deficient in major histocompatibility complex (MHC) class I protein expression (e.g. certain tumors and virally-infected cells [1,2] cannot be killed by cytotoxic T lymphocytes (CTL) whose T cell receptors (TCRs) are MHC-restricted. Such targets, however, can be effectively lysed by natural
0145-305X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0145-305X(03)00040-5
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killer (NK) cells that are important effectors of innate immunity. Although NK cells lack rearranging TCRs, they do possess a diverse array of NK surface receptors [3 – 6]. Some of these NK receptors transduce inhibitory signals upon binding classical (class Ia) [7] or non-classical (class Ib) [8] MHC class I molecules; others transmit activating signals on recognizing residual MHC class I ligands, class I-like ligands [9] or ligands on MHC I-negative cells. There is now an increasing awareness that NK cells are also important regulators of adaptive immunity [5] and that some NK receptors are expressed by either CD4positive or CD8positive T cells (called NK/T cells) [10]. Despite major progress in the field of NK cell biology, our understanding of the origin and development of these cells remains in its infancy [6]. Because evolutionary conservation of immunological features likely indicates their central importance, we have utilized a comparative model system to explore NK cell ontogeny. Although Xenopus is evolutionarily distinct from mammals (a common ancestor between mammals and amphibians dates back more than 250 million years), its immune system is relatively similar [11]. Adult Xenopus thymectomized (Tx) early in larval life have been used recently to identify NK cells and study NK cell function at the amphibian level [12 –14]. Tx Xenopus have a permanent deficit in bona fide T cells, as was recently confirmed by molecular approaches that probed for TCRb expression [15]. NK cells are found in both control and Tx Xenopus, but are increased in proportion in spleen, liver and intestine following early larval thymectomy [14]. Candidate NK cells in adult Xenopus were initially identified as non-T/non-B lymphocytes that, following 48 h culture in growth factor-rich medium (GFM), display natural cytotoxicity towards MHC class I-deficient lymphoid tumor target cells, but not against MHC class Ipositive lymphoblasts [14]. Availability of Tx Xenopus was instrumental in the generation of monoclonal antibodies (e.g. mAb 1F8) specific for candidate NK cells. These mAbs have been used to isolate NK cells of adult frogs, study their cell surface antigen expression and lymphoid tissue distribution, and explore the mechanism of NK cytotoxicity towards MHC class Ideficient lymphoid tumor cell targets [14,16]. Xenopus is intriguing in that neither classical nor non-classical MHC class I proteins are expressed on
most larval cells until close to metamorphosis; this contrasts with the ubiquitous surface expression of class I proteins in adult frogs [17,18]. This animal, therefore, provides a unique model system to examine whether NK cells can emerge in an environment lacking MHC class I proteins, the presumed inhibitory ligands for these innate immune cells. In this paper, we explore NK cell ontogeny in control and Tx Xenopus by phenotypically analyzing 1F8 antigen expression, and by performing in vitro cytotoxic anti-tumor assays using larval and adult effector splenocytes. We also provide additional biochemical characterization of the Xenopus 1F8 antigen by immunoprecipitation, and present definitive evidence of a 1F8positive lymphoid NK cell population distinct from T cells.
2. Materials and methods 2.1. Rearing and thymectomy of Xenopus Outbred and MHC homozygous F strain X. laevis were reared in our laboratory at 23 8C as described elsewhere [19]. Thymectomy of larvae by microcautery was carried out at 6 –7 days of age, when the thymus is at a rudimentary stage of differentiation [19, 20]. Controls were non-operated siblings. Ontogenetic studies were carried out on 5-week (Nieuwkoop and Faber [21] stage 54/55) and 7-week (stage 56/58) larvae and on 3 –4 month-old froglets. Adults were 12– 24 months of age when assayed. 2.2. Splenocyte culture Splenocyte suspensions were prepared in cold Ca2þ and Mg2þ-free HBSS supplemented with 1% FCS (HBSS/FCS) and adjusted to amphibian tonicity. Adult splenocytes were enriched for lymphoid cells by centrifugation over Ficoll (density 1.077); froglet and larval splenocytes were not Ficoll-enriched. Prior to assay, splenocytes were cultured for 48 h in B3B7 tumor medium [12] supplemented with 25% GFM derived from supernatants of Con A-stimulated X. laevis splenocytes from which Con A had been removed [13]. Cells were maintained at 27 8C in 5% CO2, adult and froglet cells in 1 ml aliquots in 24-well plates (, 2 £ 106 leukocytes/ml), larval cells in 200 ml aliquots in flat-bottom 96-well plates
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(, 1 £ 106/ml). Cells from individual animals were always cultured separately. However, at the time of assay, pooling of larval splenocytes was necessary to provide sufficient lymphocyte numbers for the assay. Each experiment involved , 10 –20 spleens from 5week-old larvae, , 7 –10 spleens from 7-week-old tadpoles. Assays were carried out on unpooled splenocytes from individual froglets and adults. 2.3. Magnetic cell sorting 24 h-cultured splenocytes were incubated with either anti-CD5 (2B1 mAb [22]), or 1F8 mAb [14], followed by MACS microbeads coated with antimouse IgG (Multenyi Biotec) and immunomagnetically sorted as described elsewhere [14]. The purity of sorted splenocytes was determined by flow cytometry. The T cell- and NK cell-enriched populations, and depleted populations, were then cultured separately for an additional 24 h prior to assay. 2.4. Cytotoxicity assays The B3B7 thymic lymphoid tumor cell line (derived from the thymus of F strain Xenopus), that express neither classical class I (class Ia) nor class II MHC proteins [16], served as targets. The main cytotoxicity protocol used was the DNA fragmentation (JAM) assay [23,24], where targets were prepared by incubation of 1 £ 106 B3B7 cells for 3 h with 5 mCi/ml 3HTdR (S.A. ¼ 5 Ci/mmol) and washed twice before use. Effector lymphoid cells (100 ml) were incubated for 5 h with 100 ml 3HTdR-labelled targets (1 £ 104 cells) in 96-well plates prior to harvesting and scintillation counting. Specific killing was determined as follows: % DNA loss ¼ ðT 2 EÞ 2 ðT 2 SÞ 4 T 2 ðT 2 SÞ £ 100; where T ¼ total incorporated label (dpm) in targets at 0 h, S ¼ retained DNA in targets after 5 h culture without effectors and E ¼ experimentally retained DNA in presence of effectors. The 51Cr release assay used to monitor killing by adult Xenopus splenocytes has been described elsewhere [14]. 2.5. Flow cytometry The primary mAb used was 1F8 hybridoma supernatant (mouse IgG2b anti-Xenopus NK [14]),
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which was detected by use of FITC-conjugated rabbit anti-mouse Ig (DAKO). The control mouse mAb was CT3, an anti-chicken CD3 of IgG1 isotype [25]. Secondary, PE-labeled anti-Xenopus mAbs used were D8 (IgG anti-IgM [26]), 2B1 (IgG1 anti-CD5 [22]), F17 (IgM anti-CD8 [26]) and D12 (IgG; identifies a minor Ignegative subset [26]). The PE control was mouse IgG conjugated to PE (DAKO). Flow cytometry was performed as detailed elsewhere [14], gates being set by forward and side scatter to delineate lymphoid cells and exclude dead cells, the latter being confirmed by propidium iodide staining. Per sample 5000– 10,000 gated cells were analyzed on a Coulter XL flow cytometer. 2.6. RT-PCR Cytoplasmic RNA from 1 £ 106 of each splenocyte population studied was reverse transcribed following methodology described elsewhere [23]. One microliter of each reverse transcriptase (RT) reaction was amplified (40 cycles) using primer pairs specific for T cell receptor (TCR) families Vb1 or Vb8. Elongation factor-a (Ef-a) primers acted as the RT control. Gels were blotted and the membranes hybridized with a (32P)TCRb constant cDNA probe. TCRb primers specific for different V families and the C region were synthesized based on published Xenopus sequences [27]. Ef-a primers were as previously published [28]. 2.7. Immunoprecipitation Studies on biotinylated cells. The following cell populations were studied: 48 h medium-cultured or PMA/ionomycin-stimulated (10 ng/ml PMA, 200 ng/ml ionomycin for 16 h [23]) splenocytes from control Xenopus, and both 1F8-enriched and 1F8-depleted medium-cultured splenocytes from Tx animals. 5 £ 106 cells were resuspended in APBS containing 0.5 mg/ml biotin (Vector) and rotated for 30 min at 4 8C. Cell pellets were thoroughly washed in 5 mg/ml lysine, then lysed in 1% NP-40 lysis buffer containing protease inhibitors. Lysates were first precleared with a cocktail of anti-Xenopus Ig mAbs [14]. Equal amounts of lysate were then subjected to overnight immunoprecipitation using protein A sepharose CL-4B beads (Sigma) coated with 1F8 mAb or beads coated with negative control IgG2b
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mouse mAb (anti-Aspergillus niger glucose oxidase (DAKO)). After boiling for 5 min, antibody-captured lysates were subjected to SDS – PAGE and surface proteins then probed with streptavidin-horseradish peroxidase (Vector) and detected by chemiluminescence. Studies on 35S-labelled cells. Medium-cultured or PMA/ionomycin-stimulated splenocytes (see above) were incubated (27 8C, 4 £ 106/ml) in cysteine/methionine-free RPMI medium for 30 min, then overnight in RPMI containing 10 mCi/ml 35Smethionine. Cells were then washed twice in APBS to remove unincorporated 35S-methionine, and incubated 1 h on ice with either 1F8 mAb (anti-NK) or X71 mAb (IgG2a anti-thymocyte) [29] negative control. After washing, cells were resuspended in ice-cold NP-40 lysis buffer and the lysate was incubated 1 h with protein A or G beads to precipitate mAb/surface protein complexes. All the pellets containing immunoprecipitated antigens were washed twice in both Net-NON and Net-N, before being resuspended in SDS loading buffer (^ b-mercaptoethanol), then boiled for 5 min before being subjected to SDS – PAGE. Radiolabeled/immunoprecipitated proteins were detected by exposure of X-ray film.
Fig. 1. TCRb expression by splenocyte populations from control (C) and thymectomized (Tx) adult Xenopus following 48 h culture in GFM. At 24 h, cultured control splenocytes were either left unsorted (U) or MACS-sorted into CD5positive (þ ) and CD5negative (– ) populations; Tx splenocytes were left unsorted (U) or MACS-sorted into 1F8positive (þ) and 1F8negative ( –) populations. Cytoplasmic RNA from these populations was subjected to RT-PCR using primers specific for Vb8 and Ef-a as described in Section 2. The last lane in the upper panel is a negative control without cDNA, that was not included in the lower panel.
controls, unseparated splenocytes from Tx frogs, or the 1F8positive and 1F8negative Tx populations. 3. Results 3.1. 1F8positive NK cells are a lymphoid subset distinct from T cells Fig. 1 illustrates the outcome of a representative experiment searching for TCR transcripts in various splenocyte populations cultured for 48 h in GFM. CD5positive and CD5negative cells, together with unseparated splenocytes, were first obtained from control Xenopus. Immunomagnetic sorting also provided 1F8positive, 1F8negative and unseparated splenocytes from Tx frogs. Subsequent TCR experiments were performed as described above. Fig. 1 reveals that Vb8 transcripts (and also Vb1 transcripts—data not shown) were found only in unseparated and CD5positive populations from control Xenopus. Vb8 (and Vb1) transcripts were not detectable in either CD5negative splenocytes from
3.2. Characterization of 1F8 antigen Immunoprecipitation with biotinylated cells. Surface protein recognized by 1F8 mAb was first characterized by studies on surface-biotinylated splenocytes from control and Tx Xenopus. Lysates from 1F8-enriched (MACS-sorted) splenocytes from Tx frogs yielded two distinct bands, at , 45 and 55 kDa, following immunoprecipitation with 1F8 mAb, but not with either commercial isotype control (IgG2b) mAb (Fig. 2A) or XT1 mAb (anti-Xenopus T cell subset of IgG2b isotype) [30], data not shown. Similar results were obtained several times with cells from different outbred individuals. The 1F8-specific (low intensity) bands immunoprecipitated from the lysate recovered from 1F8-depleted Tx cells likely reflect the incomplete removal of all 1F8positive cells in this preparation (FACS data not shown). The 1F8specific bands were barely detectable in unseparated
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Fig. 2. Characterization of 1F8 antigen by immunoprecipitation of surface-biotinylated or biosynthetically-labeled spleen cells. (A) Cell populations assayed were splenocytes from control adult Xenopus (following 48 h culture in medium alone (‘med’) or following transient exposure to PMA/ionomycin (PMA)) and medium-cultured Tx splenocytes that had been enriched for 1F8positive cells by MACS (1F8positive) or partially depleted of 1F8positive cells (1F8negative). Lysates were pre-cleared with anti-Xenopus Ig mAbs and then immunoprecipitated using protein A beads coated with either 1F8 capture mAb or control IgG2b mAb. After boiling, antibody-captured lysates were subjected to SDS–PAGE and surface proteins probed with streptavidin-horseradish peroxidase and detected by chemiluminescence. 1F8-specific bands are seen at ,45 and 55 kDa. (B) Medium-cultured or PMA/ionomycin-treated (see above) splenocytes were cultured overnight in medium containing 35S methionine and cysteine (108 cpm/ml, 4 £ 106 cells/ml). Labeled cells were incubated with 1F8 mAb or control anti-thymocyte mAb (X71), then lysed and complexes of mAbs and cell surface antigens precipitated with protein A or G beads. After several washes, precipitated surface proteins were separated on SDS–PAGE under non-reducing or reducing conditions. 1F8-specific bands are seen at 55 kDa.
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splenocytes from control frogs. However, biotinlabeling was also performed with control splenocytes stimulated for 2 days with low doses of PMA and ionomycin, since this treatment increases surface 1F8 expression (see FACS data in [23]). The , 45 and 55 kDa 1F8-specific bands in control splenocytes became clear after such signal enhancement (Fig. 2A), whereas control mAbs failed to immunoprecipitate such bands. The 45 and 55 kDa 1F8-specific bands were evident in gels run under both reducing and nonreducing conditions (data not shown). Studies on 35S-labelled cells. Immunoprecipitations to monitor endogenously-produced proteins were carried out with unseparated splenocytes from outbred Xenopus cultured overnight in the presence of 35 S-labelled methionine and cysteine. To increase the 1F8 signal, biosynthetic labeling was also carried out on splenocytes cultured for 2 days with PMA and ionomycin. Pulsed cells were incubated with capture mAbs before lysis, to detect possible surface heterodimers. Fig. 2B reveals that the 1F8 mAb, but not antithymocyte mAb (X71) used as negative control, precipitates one unique band of , 55 kDa from the cell surface, both under non-reducing as well as reducing conditions. A stronger signal of a single 55 kDa polypeptide at the cell surface was detected after PMA/ionomycin stimulation (Fig. 2B, panel 3). 3.3. Ontogeny of NK cells in control and Tx Xenopus Phenotypic studies. Flow cytometry on splenocytes from 1 to 2 year-old adult (control and Tx) Xenopus that were first cultured for 48 h in GFM (a procedure that increases 1F8 expression and promotes NK-like killing [14]) confirmed that 1F8positive splenocytes lack the surface IgM of B cells and the surface markers characteristic of T cells (CD5 and CD8), but frequently express the 56 kDa antigen recognized by mAb D12 (Fig. 3A). A subset of 1F8positive/CD5lo splenocytes was evident in some adult spleens (data not shown). FACS analysis of pooled spleens from 5week-old (stage 54/55) control and Tx Xenopus larvae revealed no 1F8 staining of viable splenocytes following 48 h culture in GFM (data not shown). Dual color flow cytometric data on splenocytes pooled from 7-week larvae (stage 56 – 58) or from 3 to 4 month froglets, following 48 h culture in GFM, is also shown in Fig. 3. A few splenocytes expressing
the 1F8 antigen (of lower fluorescence intensity (1F8lo) than seen in adults) can be visualized for the first time in 7-week-old larvae (Fig. 3C), especially in Tx tadpoles; such 1F8lo cells are IgMnegative, CD5negative and CD8negative, but a few (3%) coexpress the D12 antigen. The proportion of 1F8lo cells remains unchanged in 3– 4 month old froglets (Fig. 3B), where a few double-positive 1F8lo/CD5positive cells were noted in addition to the 1F8lo/D12positive population. 1F8positive cells were not found in the thymus at any stage of development (data not shown). Functional studies. Previous studies had employed 51 Cr-release to identify the anti-tumor cytotoxic potential of purified 1F8positive NK cells in adult Xenopus [12 –15]. The JAM assay, developed for mice by Matzinger [24], monitors DNA loss by fragmentation. This assay is more sensitive than a chromium-release assay for determining cellmediated killing, since DNA fragmentation (DNA ladder formation) is a more reliable criterion for judging apoptotic death. Further, the JAM assay, as adapted and successfully used with Xenopus [23], requires only 1 £ 104 3HTdR-labelled tumor targets, compared with 5 £ 104 targets necessary in the 51Crrelease assay. The JAM assay indeed proved to be more sensitive than 51Cr release in monitoring killing of B3B7 tumor cell targets following their 5 h coculture with 1– 2 year-old adult splenocytes from control and Tx frogs (Fig. 4, top left). Consistent with findings from 51Cr release studies, JAM assay analyses of MACS-separated splenocytes identified the 1F8positive population as the one able to effect target cell DNA fragmentation; T-cells, B-cells and 1F8negative cells were unable to induce DNA fragmentation in B3B7 tumor targets (data not shown). We took advantage of the high sensitivity of the JAM assay for our ontogenetic studies. JAM assays were initially carried out on 5-week (stage 54/55) and 7-week (stage 56/58) control and Tx Xenopus larvae (Fig. 4, bottom). Assays involved culturing of splenocytes from individual larvae for 48 h in GFM (to promote NK cell killing) prior to pooling of between 7 and 20 spleens for assay (Section 2). No DNA fragmentation (, 10% specific killing) of B3B7 targets was observed in any group of experimental larvae, even at 20:1 or 40:1 E:T ratio. This was true even for pooled spleens taken from control 7-week-old tadpoles that had been injected with B3B7 cells 10 days
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Fig. 3. Dual color flow cytometric analysis comparing cell surface antigen expression on splenocytes from control and Tx Xenopus at various stages of development. Following 48 h culture in GFM, cells were stained first with 1F8 (anti-NK) hybridoma supernatant (detected by FITCconjugated anti-mouse Ig), then with either PE-conjugated D8 (anti-IgM), 2B1 (anti-CD5), F17 (anti-CD8) or D12 (identifies a 56 kDa epitope) mAb. Quadrants set to exclude 98% cells stained with control reagents from positive analysis. Data shown are representative of three separate experiments on individual adult and froglet spleens and several pools of larval spleens.
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Fig. 4. Cytotoxicity towards B3B7 lymphoid tumor targets effected by splenocytes from control and Tx Xenopus at various stages of development. Splenocytes from individual animals were cultured for 48 h in GFM prior to 5 h cytotoxicity assay. Cytotoxicity of adult splenocytes (top left) was compared using JAM (1 £ 104 3HTdR-labelled tumor targets) and 51Cr-release (5 £ 104 51Cr-labelled tumor targets) assays. Cytotoxicity of splenocytes from froglets and larvae was monitored by JAM assay only. Data on adults and froglets show mean (^SEM)% killing at various E:T ratios from three separate experiments on individual animals. Larval data shows percentage killing of tumor targets mediated by splenocytes pooled from ,10 to 20 stage 54/55 larvae or ,7–10 stage 56/58 larvae. One pool of 7-week-old larval splenocytes (see bottom left ‘Control inj’) came from tadpoles injected intraperitoneally with 5 £ 103 B3B7 cells 10 days prior to assay.
prior to assay in an attempt to elevate NK-like killing [12]. Replicate experiments on additional outbred and MHC homozygous F strain Xenopus 7-week-old larvae confirmed their inability to kill B3B7 tumor targets in vitro. Moreover, such cytotoxicity was still relatively poor (only , 20% specific killing at highest E:T ratios used) when splenocytes from individual 3– 4 month-old froglets were tested (Fig. 4 top right). 4. Discussion 4.1. Molecular characterization of 1F8þ cells from adult, Tx Xenopus Previous two color flow cytometric data [14] on lymphocytes purified from spleen, liver and kidney of
adult Xenopus suggested that the mAb 1F8 constitutively labels a lymphocyte subset of NK-like cells (surface CD5negative or CD5lo and IgMnegative) that is distinct from T and B cells. Furthermore, 1F8positive NK-like cells were identified as the lymphoid population, which after culture in GFM, were able to kill MHC-deficient tumor cell targets, but not MHC class I-expressing lymphoblasts [14], whereas the recently-discovered minor population of CD8positive NK/T cells do not kill class I-negative targets [23]. We now report that Xenopus 1F8positive splenocytes purified from Tx frogs do not manufacture fully rearranged TCRb V region transcripts. 1F8positive cells, therefore, represent a lymphoid population we consider to be bona fide NK cells, distinct from T and NK/T cells, both of which express rearranged TCRb
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V region transcripts [23]. It is possible that NK cells can still manufacture sterile TCRb C region transcripts, which might help explain previous work where TCRb transcripts were detected in unseparated lymphocyte populations from Tx frogs, albeit at very low levels compared with controls [15]. Presence of abortive TCR transcripts in NK cells would not be surprising in view of compelling evidence that NK cells and T cells can develop from a common progenitor [31]. Although the 1F8 mAb clearly identifies cells that are functionally and structurally NK cells, the characterization of the 1F8 antigen itself is still incomplete, in part because the lack of established NK cell lines makes it difficult to obtain sufficient purified antigen to biochemically analyze. Previously published Western blotting experiments [14] suggested that the 1F8 molecule was a 70 –85 kDa polypeptide. In the current study, immunoprecipitation experiments of both biosynthetically-labeled and surfacebiotinylated lysates reveals that 1F8 binds to a monomeric 55 kDa polypeptide. 1F8 also precipitates a smaller molecule of 45 kDa from surface-biotinylated lysates. That these differences reflect allelic polymorphism is problematic since they have been reproduced several times with cells from different outbred animals. The fact that the 55 kDa molecule was detected by immunoprecipitation under various conditions (e.g. biotinylation, biosynthetic labeling, before lysis, under non-reducing and reducing SDS – PAGE conditions) indicates that it is a real antigenic determinant recognized by 1F8. No other polypeptide was detected from biosynthetically-labeled cells, even after PMA/ionomycin stimulation that up-regulates 1F8 surface signal (as determined by flow cytometry [23]). Therefore, it is unlikely that the 45 kDa molecule precipitated from surface-biotinylated lysates represents a different 1F8 antigen. It is possible that biotinylation allows the detection of a degraded or processed fragment that is not well labeled by 35S (i.e. poor in methionine and cysteine residues), or that this 45 kDa polypeptide is coprecipitated with the 1F8 antigen as a result of its biotinylation (e.g. change of conformation, increased stickiness). The 70 – 85 kDa molecule previously detected by Western blotting [14] was most likely an artifact resulting from an interaction of the 1F8 mAb with boiled material. Further molecular studies
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and the cloning and sequencing of the 1F8 NKassociated antigen are clearly needed, especially in view of our finding [23] that the surface protein recognized by 1F8 mAb plays a biological role in antitumor immunity. In order to clone the 1F8 gene, purified 1F8 protein will be obtained by immunoprecipitation and used to generate a polyclonal antibody against the 1F8 antigen, which could then be used to screen a cDNA expression library. 4.2. Ontogeny of NK cells and tumor immunity in Xenopus In mammals, interactions between inhibitory receptors expressed by developing NK cells and MHC class I proteins expressed by autologous cells are believed to play a crucial role in the education of NK cells and their development of self-tolerance [32]. However, it has recently been shown in humans [33] that immature NK cells express activatory receptors before MHC-specific inhibitory receptors, implying that in early ontogeny, ligands other than MHC may be crucial in NK inhibition. Whether NK cells can develop in the absence of MHC class I expression remains unclear. Although functional NK cells are found in class I-deficient mouse models, such b2m2/2 and TAP2/2 animals express low levels of class I molecules on their cell surfaces [32]. In this paper, we have explored whether NK cells develop in the naturally occurring MHC class I-negative environment of larval Xenopus, but were only able to detect 1F8positive cells in the spleen just prior to metamorphosis, at , 7-weeks (stage 56/58). This is long after T and B cells can be detected in this organ during the second week of larval life [20]. The surprisingly late appearance of NK cells in Xenopus comes , 2-weeks after the ontogeny of MHC class Ia expression, which is first detectable at the splenocyte surface at 5-weeks [34] when low levels of class Ia transcripts are also initially expressed in various non-lymphoid tissues [18]. The initial emergence of 1F8lo cells in 7-weekold larvae precedes MHC class Ib expression that occurs only after metamorphosis [18]. (Class II MHC expression is seen on a range of cell types from early in larval life [35].) The above phenotypic studies suggesting initial appearance of NK cells in late larval life is not precisely mirrored by the results from the JAM assays.
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Thus, GFM-cultured splenocytes from control or Tx (5- and 7-week-old) tadpoles, or even from B3B7injected larvae, failed to effect DNA fragmentation of B3B7 tumor targets. These findings using the sensitive JAM assay confirm and extend previous preliminary data [13], derived from a 51Cr-release cytotoxicity assay, that showed cultured splenocytes from 6 to 7week control larvae failed to kill B3B7 targets. The very low percentage of 1F8lo splenocytes detectable by FACS in late larvae may be competent to kill, but their cytotoxicity is masked when unseparated splenocytes are assayed. Clearly, 1F8-enrichment studies are needed, but they would necessitate using prohibitively large numbers of (Tx) larvae to collect sufficient numbers of effectors. Significant, albeit low, levels of B3B7 tumor killing by splenocytes (i.e. . 10% of the level of killing that can be detected in the JAM assay following 5 h co-culture of tumor cells with 20– 40-fold excess red blood cells (unpublished observations)) are first seen only in 4 month-old froglets. Previous studies have shown that splenocytes from 6 month-old Xenopus (some 3.5 months postmetamorphosis) are almost as effective killers of B3B7 targets as splenocytes from year-old adults [13]. It is conceivable that the poor cytotoxic potential displayed by larvae and 4 month-old froglets relates to the low intensity of 1F8 antigen expression. In humans, only those NK cells expressing high levels of natural cytotoxicity receptors efficiently kill tumor cell lines [6]. Another possibility to consider is that NK cells may become fully differentiated and functional only after metamorphosis, in parallel with the postmetamorphic maturation of the immune system characterized by: new lymphoid histogenesis in the thymus and spleen [20,36]; the expression of MHC class II by peripheral T cells [37]; the reappearance of detectable T cell alloreactivity in mixed lymphocyte culture [38]; and the development of an effective alloimmunity to lymphoid tumor [39,40]. The developmental emergence of fully-effective NK killing activity at , 6 months of age has to be integrated with earlier findings on the ontogeny of in vivo alloimmune reactivity of Xenopus towards injected tumor cells [39]. Thus ff2 thymic lymphoid tumor cells injected into F strain tadpoles and young post-metamorphic F froglets up until nearly 4 months of age initiates tumor growth in the vast majority of cases, whereas when
injected into 6 month and older frogs, tumors failed to develop. The development of this alloimmunity to ff2 tumor was originally considered to be most likely linked to the emergence of T cell functions in the early post-metamorphic period, rather than to NK cell development [39]. Whether NK cells functionally contribute to an effective anti-tumor defense by the adult immune system remains to be determined. Although we know that 1F8positive cells are the lymphoid subset that kills tumor targets in vitro, and that this killing is dependent on NK cell pre-culture in GFM (TCGF-rich culture medium), a role of NK cells in in vivo killing of tumors remains to be demonstrated conclusively. Thus, early-thymectomized F strain adult frogs, with substantial numbers of NK cells in their lymphoid tissues, are susceptible to growth of both injected ff2 thymus-derived lymphoid tumor cells (MHCIapositive cells from F strain Xenopus) [40] and MHC Ianegative 15/0 tumor cells (our unpublished observations), in contrast to thymus-intact F strain frogs, in which such tumors fail to grow. This is consistent with the notion that T cell-mediated tumor immunity plays a fundamental role in the vertebrate immune system [41]. Interesting questions remain, however, as to how T cells can kill the MHC class Ianegative thymus tumor cell targets. Perhaps T cells can visualize MHC class Ib molecules that thymic lymphoid tumor cell lines synthesize [28], or perhaps CTLs recognize thymic lymphocyte tumor-derived gp96 heat shock protein that can elicit potent immunity towards tumor cells [42]. Since injection of adult clonal Xenopus with anti-CD8 mAb impairs the immune response against transplanted syngeneic MHC class Inegative tumors, CD8positive T cells may be crucial effectors in MHC-unrestricted anti-tumor responses [43]. However, NK cells may additionally play a crucial role in such tumor immunity, since we have recently shown that the 1F8 mAb injected into LG15 control Xenopus enhances the rapid growth of 15/0 tumor [23]. NK cells may only function in situations, where T cell-derived growth factors are plentiful (i.e. in controls), whereas the effectiveness of these cells in Tx frogs is compromised because such factors are absent. The question of whether NK cells arose in phylogeny as a backup system for T cells or rather as a surveillance system before the emergence of
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adaptive immunity remains to be resolved. The data presented here suggest that, at least in Xenopus, NK cells are serving a backup role, or at least do not subserve a killing role in the absence of class I molecules.
Acknowledgements We would like to thank Jennifer Gantress, Ana Goyos, Gregory Maniero and Heidi Morales for their collective criticisms and helpful discussions. Research was supported by a project grant from the Leverhulme Trust (to JDH), by grants AI-44011 and CA-76312 from the NIH (to NC) and by NSF grant MCB#0136536 (to JR). R.S. was funded by a Durham University Studentship.
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Ontogeny of Xenopus NK cells in the absence of MHC class I antigens Trudy L. Hortona, Rebecca Stewarta, Nicholas Cohenb, Laura Raub, Pamela Ritchiea, Martin D. Watsona, Jacques Robertb, John D. Hortona,* b
a School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, UK Department of Microbiology and Immunology, University of Rochester Medical Center, New York 14642, USA
Received 14 November 2002; accepted 28 January 2003
Abstract This paper explores the ontogeny of NK cells in control and early-thymectomized (Tx) Xenopus laevis through phenotypic analysis of cells expressing the NK cell antigen 1F8 and by performing in vitro cytotoxic assays. Dual color flow cytometry reveals that a few 1F8positive splenocytes first emerge in late larval life, at , 7-weeks post-fertilization. This is about 2-weeks after the time when surface MHC class Ia expression can first be detected. The proportion of splenocytes expressing 1F8 remains very low in 3 – 4 month-old froglets, but by 1 year there is a sizeable 1F8positive population, which is proportionally elevated in Tx frogs. The ontogeny of NK cell function is monitored by a 5 h DNA fragmentation (JAM) assay. Control and Tx larval splenocytes (from either 5- or 7-week-old tadpoles) fail to kill MHC-deficient thymus-derived tumor cell targets. Such in vitro killing is still relatively poor in 3 – 4 month froglets, compared with high levels of tumor cell cytotoxicity mediated by splenocytes from older frogs. Immunoprecipitation studies identify that the major ligand for the 1F8 mAb is a 55 kDa polypeptide. Finally, further evidence is provided that 1F8positive lymphocytes are indeed bona fide NK cells, distinct from T cells, since purified 1F8positive splenocytes from Tx Xenopus fail to express fully rearranged TCRb V region transcripts. We conclude that NK cells fail to develop prior to MHC class I protein expression and, therefore, do not contribute to the larval immune system, whereas they do provide an important backup for T cells in the adult frog by contributing to anti-tumor immunity. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Amphibians; NK cell ontogeny; NK cell-associated 1F8 antigen; Xenopus; Thymectomy; Tumor immunity
1. Introduction Abbreviations: GFM, growth factor-rich medium (supernatant from concanavalin A-stimulated cells); MHC, major histocompatibility complex; NK cell, natural killer cell; PMA, phorbol myristate acetate; Tx, early thymectomized; 1F8 mAb, anti-Xenopus NK cell monoclonal antibody. * Corresponding author. Tel.: þ44-191-374-3359; fax: þ 44-191374-2417. E-mail address: [email protected] (J.D. Horton).
Cells that are deficient in major histocompatibility complex (MHC) class I protein expression (e.g. certain tumors and virally-infected cells [1,2] cannot be killed by cytotoxic T lymphocytes (CTL) whose T cell receptors (TCRs) are MHC-restricted. Such targets, however, can be effectively lysed by natural
0145-305X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0145-305X(03)00040-5
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killer (NK) cells that are important effectors of innate immunity. Although NK cells lack rearranging TCRs, they do possess a diverse array of NK surface receptors [3 – 6]. Some of these NK receptors transduce inhibitory signals upon binding classical (class Ia) [7] or non-classical (class Ib) [8] MHC class I molecules; others transmit activating signals on recognizing residual MHC class I ligands, class I-like ligands [9] or ligands on MHC I-negative cells. There is now an increasing awareness that NK cells are also important regulators of adaptive immunity [5] and that some NK receptors are expressed by either CD4positive or CD8positive T cells (called NK/T cells) [10]. Despite major progress in the field of NK cell biology, our understanding of the origin and development of these cells remains in its infancy [6]. Because evolutionary conservation of immunological features likely indicates their central importance, we have utilized a comparative model system to explore NK cell ontogeny. Although Xenopus is evolutionarily distinct from mammals (a common ancestor between mammals and amphibians dates back more than 250 million years), its immune system is relatively similar [11]. Adult Xenopus thymectomized (Tx) early in larval life have been used recently to identify NK cells and study NK cell function at the amphibian level [12 –14]. Tx Xenopus have a permanent deficit in bona fide T cells, as was recently confirmed by molecular approaches that probed for TCRb expression [15]. NK cells are found in both control and Tx Xenopus, but are increased in proportion in spleen, liver and intestine following early larval thymectomy [14]. Candidate NK cells in adult Xenopus were initially identified as non-T/non-B lymphocytes that, following 48 h culture in growth factor-rich medium (GFM), display natural cytotoxicity towards MHC class I-deficient lymphoid tumor target cells, but not against MHC class Ipositive lymphoblasts [14]. Availability of Tx Xenopus was instrumental in the generation of monoclonal antibodies (e.g. mAb 1F8) specific for candidate NK cells. These mAbs have been used to isolate NK cells of adult frogs, study their cell surface antigen expression and lymphoid tissue distribution, and explore the mechanism of NK cytotoxicity towards MHC class Ideficient lymphoid tumor cell targets [14,16]. Xenopus is intriguing in that neither classical nor non-classical MHC class I proteins are expressed on
most larval cells until close to metamorphosis; this contrasts with the ubiquitous surface expression of class I proteins in adult frogs [17,18]. This animal, therefore, provides a unique model system to examine whether NK cells can emerge in an environment lacking MHC class I proteins, the presumed inhibitory ligands for these innate immune cells. In this paper, we explore NK cell ontogeny in control and Tx Xenopus by phenotypically analyzing 1F8 antigen expression, and by performing in vitro cytotoxic anti-tumor assays using larval and adult effector splenocytes. We also provide additional biochemical characterization of the Xenopus 1F8 antigen by immunoprecipitation, and present definitive evidence of a 1F8positive lymphoid NK cell population distinct from T cells.
2. Materials and methods 2.1. Rearing and thymectomy of Xenopus Outbred and MHC homozygous F strain X. laevis were reared in our laboratory at 23 8C as described elsewhere [19]. Thymectomy of larvae by microcautery was carried out at 6 –7 days of age, when the thymus is at a rudimentary stage of differentiation [19, 20]. Controls were non-operated siblings. Ontogenetic studies were carried out on 5-week (Nieuwkoop and Faber [21] stage 54/55) and 7-week (stage 56/58) larvae and on 3 –4 month-old froglets. Adults were 12– 24 months of age when assayed. 2.2. Splenocyte culture Splenocyte suspensions were prepared in cold Ca2þ and Mg2þ-free HBSS supplemented with 1% FCS (HBSS/FCS) and adjusted to amphibian tonicity. Adult splenocytes were enriched for lymphoid cells by centrifugation over Ficoll (density 1.077); froglet and larval splenocytes were not Ficoll-enriched. Prior to assay, splenocytes were cultured for 48 h in B3B7 tumor medium [12] supplemented with 25% GFM derived from supernatants of Con A-stimulated X. laevis splenocytes from which Con A had been removed [13]. Cells were maintained at 27 8C in 5% CO2, adult and froglet cells in 1 ml aliquots in 24-well plates (, 2 £ 106 leukocytes/ml), larval cells in 200 ml aliquots in flat-bottom 96-well plates
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(, 1 £ 106/ml). Cells from individual animals were always cultured separately. However, at the time of assay, pooling of larval splenocytes was necessary to provide sufficient lymphocyte numbers for the assay. Each experiment involved , 10 –20 spleens from 5week-old larvae, , 7 –10 spleens from 7-week-old tadpoles. Assays were carried out on unpooled splenocytes from individual froglets and adults. 2.3. Magnetic cell sorting 24 h-cultured splenocytes were incubated with either anti-CD5 (2B1 mAb [22]), or 1F8 mAb [14], followed by MACS microbeads coated with antimouse IgG (Multenyi Biotec) and immunomagnetically sorted as described elsewhere [14]. The purity of sorted splenocytes was determined by flow cytometry. The T cell- and NK cell-enriched populations, and depleted populations, were then cultured separately for an additional 24 h prior to assay. 2.4. Cytotoxicity assays The B3B7 thymic lymphoid tumor cell line (derived from the thymus of F strain Xenopus), that express neither classical class I (class Ia) nor class II MHC proteins [16], served as targets. The main cytotoxicity protocol used was the DNA fragmentation (JAM) assay [23,24], where targets were prepared by incubation of 1 £ 106 B3B7 cells for 3 h with 5 mCi/ml 3HTdR (S.A. ¼ 5 Ci/mmol) and washed twice before use. Effector lymphoid cells (100 ml) were incubated for 5 h with 100 ml 3HTdR-labelled targets (1 £ 104 cells) in 96-well plates prior to harvesting and scintillation counting. Specific killing was determined as follows: % DNA loss ¼ ðT 2 EÞ 2 ðT 2 SÞ 4 T 2 ðT 2 SÞ £ 100; where T ¼ total incorporated label (dpm) in targets at 0 h, S ¼ retained DNA in targets after 5 h culture without effectors and E ¼ experimentally retained DNA in presence of effectors. The 51Cr release assay used to monitor killing by adult Xenopus splenocytes has been described elsewhere [14]. 2.5. Flow cytometry The primary mAb used was 1F8 hybridoma supernatant (mouse IgG2b anti-Xenopus NK [14]),
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which was detected by use of FITC-conjugated rabbit anti-mouse Ig (DAKO). The control mouse mAb was CT3, an anti-chicken CD3 of IgG1 isotype [25]. Secondary, PE-labeled anti-Xenopus mAbs used were D8 (IgG anti-IgM [26]), 2B1 (IgG1 anti-CD5 [22]), F17 (IgM anti-CD8 [26]) and D12 (IgG; identifies a minor Ignegative subset [26]). The PE control was mouse IgG conjugated to PE (DAKO). Flow cytometry was performed as detailed elsewhere [14], gates being set by forward and side scatter to delineate lymphoid cells and exclude dead cells, the latter being confirmed by propidium iodide staining. Per sample 5000– 10,000 gated cells were analyzed on a Coulter XL flow cytometer. 2.6. RT-PCR Cytoplasmic RNA from 1 £ 106 of each splenocyte population studied was reverse transcribed following methodology described elsewhere [23]. One microliter of each reverse transcriptase (RT) reaction was amplified (40 cycles) using primer pairs specific for T cell receptor (TCR) families Vb1 or Vb8. Elongation factor-a (Ef-a) primers acted as the RT control. Gels were blotted and the membranes hybridized with a (32P)TCRb constant cDNA probe. TCRb primers specific for different V families and the C region were synthesized based on published Xenopus sequences [27]. Ef-a primers were as previously published [28]. 2.7. Immunoprecipitation Studies on biotinylated cells. The following cell populations were studied: 48 h medium-cultured or PMA/ionomycin-stimulated (10 ng/ml PMA, 200 ng/ml ionomycin for 16 h [23]) splenocytes from control Xenopus, and both 1F8-enriched and 1F8-depleted medium-cultured splenocytes from Tx animals. 5 £ 106 cells were resuspended in APBS containing 0.5 mg/ml biotin (Vector) and rotated for 30 min at 4 8C. Cell pellets were thoroughly washed in 5 mg/ml lysine, then lysed in 1% NP-40 lysis buffer containing protease inhibitors. Lysates were first precleared with a cocktail of anti-Xenopus Ig mAbs [14]. Equal amounts of lysate were then subjected to overnight immunoprecipitation using protein A sepharose CL-4B beads (Sigma) coated with 1F8 mAb or beads coated with negative control IgG2b
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mouse mAb (anti-Aspergillus niger glucose oxidase (DAKO)). After boiling for 5 min, antibody-captured lysates were subjected to SDS – PAGE and surface proteins then probed with streptavidin-horseradish peroxidase (Vector) and detected by chemiluminescence. Studies on 35S-labelled cells. Medium-cultured or PMA/ionomycin-stimulated splenocytes (see above) were incubated (27 8C, 4 £ 106/ml) in cysteine/methionine-free RPMI medium for 30 min, then overnight in RPMI containing 10 mCi/ml 35Smethionine. Cells were then washed twice in APBS to remove unincorporated 35S-methionine, and incubated 1 h on ice with either 1F8 mAb (anti-NK) or X71 mAb (IgG2a anti-thymocyte) [29] negative control. After washing, cells were resuspended in ice-cold NP-40 lysis buffer and the lysate was incubated 1 h with protein A or G beads to precipitate mAb/surface protein complexes. All the pellets containing immunoprecipitated antigens were washed twice in both Net-NON and Net-N, before being resuspended in SDS loading buffer (^ b-mercaptoethanol), then boiled for 5 min before being subjected to SDS – PAGE. Radiolabeled/immunoprecipitated proteins were detected by exposure of X-ray film.
Fig. 1. TCRb expression by splenocyte populations from control (C) and thymectomized (Tx) adult Xenopus following 48 h culture in GFM. At 24 h, cultured control splenocytes were either left unsorted (U) or MACS-sorted into CD5positive (þ ) and CD5negative (– ) populations; Tx splenocytes were left unsorted (U) or MACS-sorted into 1F8positive (þ) and 1F8negative ( –) populations. Cytoplasmic RNA from these populations was subjected to RT-PCR using primers specific for Vb8 and Ef-a as described in Section 2. The last lane in the upper panel is a negative control without cDNA, that was not included in the lower panel.
controls, unseparated splenocytes from Tx frogs, or the 1F8positive and 1F8negative Tx populations. 3. Results 3.1. 1F8positive NK cells are a lymphoid subset distinct from T cells Fig. 1 illustrates the outcome of a representative experiment searching for TCR transcripts in various splenocyte populations cultured for 48 h in GFM. CD5positive and CD5negative cells, together with unseparated splenocytes, were first obtained from control Xenopus. Immunomagnetic sorting also provided 1F8positive, 1F8negative and unseparated splenocytes from Tx frogs. Subsequent TCR experiments were performed as described above. Fig. 1 reveals that Vb8 transcripts (and also Vb1 transcripts—data not shown) were found only in unseparated and CD5positive populations from control Xenopus. Vb8 (and Vb1) transcripts were not detectable in either CD5negative splenocytes from
3.2. Characterization of 1F8 antigen Immunoprecipitation with biotinylated cells. Surface protein recognized by 1F8 mAb was first characterized by studies on surface-biotinylated splenocytes from control and Tx Xenopus. Lysates from 1F8-enriched (MACS-sorted) splenocytes from Tx frogs yielded two distinct bands, at , 45 and 55 kDa, following immunoprecipitation with 1F8 mAb, but not with either commercial isotype control (IgG2b) mAb (Fig. 2A) or XT1 mAb (anti-Xenopus T cell subset of IgG2b isotype) [30], data not shown. Similar results were obtained several times with cells from different outbred individuals. The 1F8-specific (low intensity) bands immunoprecipitated from the lysate recovered from 1F8-depleted Tx cells likely reflect the incomplete removal of all 1F8positive cells in this preparation (FACS data not shown). The 1F8specific bands were barely detectable in unseparated
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Fig. 2. Characterization of 1F8 antigen by immunoprecipitation of surface-biotinylated or biosynthetically-labeled spleen cells. (A) Cell populations assayed were splenocytes from control adult Xenopus (following 48 h culture in medium alone (‘med’) or following transient exposure to PMA/ionomycin (PMA)) and medium-cultured Tx splenocytes that had been enriched for 1F8positive cells by MACS (1F8positive) or partially depleted of 1F8positive cells (1F8negative). Lysates were pre-cleared with anti-Xenopus Ig mAbs and then immunoprecipitated using protein A beads coated with either 1F8 capture mAb or control IgG2b mAb. After boiling, antibody-captured lysates were subjected to SDS–PAGE and surface proteins probed with streptavidin-horseradish peroxidase and detected by chemiluminescence. 1F8-specific bands are seen at ,45 and 55 kDa. (B) Medium-cultured or PMA/ionomycin-treated (see above) splenocytes were cultured overnight in medium containing 35S methionine and cysteine (108 cpm/ml, 4 £ 106 cells/ml). Labeled cells were incubated with 1F8 mAb or control anti-thymocyte mAb (X71), then lysed and complexes of mAbs and cell surface antigens precipitated with protein A or G beads. After several washes, precipitated surface proteins were separated on SDS–PAGE under non-reducing or reducing conditions. 1F8-specific bands are seen at 55 kDa.
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splenocytes from control frogs. However, biotinlabeling was also performed with control splenocytes stimulated for 2 days with low doses of PMA and ionomycin, since this treatment increases surface 1F8 expression (see FACS data in [23]). The , 45 and 55 kDa 1F8-specific bands in control splenocytes became clear after such signal enhancement (Fig. 2A), whereas control mAbs failed to immunoprecipitate such bands. The 45 and 55 kDa 1F8-specific bands were evident in gels run under both reducing and nonreducing conditions (data not shown). Studies on 35S-labelled cells. Immunoprecipitations to monitor endogenously-produced proteins were carried out with unseparated splenocytes from outbred Xenopus cultured overnight in the presence of 35 S-labelled methionine and cysteine. To increase the 1F8 signal, biosynthetic labeling was also carried out on splenocytes cultured for 2 days with PMA and ionomycin. Pulsed cells were incubated with capture mAbs before lysis, to detect possible surface heterodimers. Fig. 2B reveals that the 1F8 mAb, but not antithymocyte mAb (X71) used as negative control, precipitates one unique band of , 55 kDa from the cell surface, both under non-reducing as well as reducing conditions. A stronger signal of a single 55 kDa polypeptide at the cell surface was detected after PMA/ionomycin stimulation (Fig. 2B, panel 3). 3.3. Ontogeny of NK cells in control and Tx Xenopus Phenotypic studies. Flow cytometry on splenocytes from 1 to 2 year-old adult (control and Tx) Xenopus that were first cultured for 48 h in GFM (a procedure that increases 1F8 expression and promotes NK-like killing [14]) confirmed that 1F8positive splenocytes lack the surface IgM of B cells and the surface markers characteristic of T cells (CD5 and CD8), but frequently express the 56 kDa antigen recognized by mAb D12 (Fig. 3A). A subset of 1F8positive/CD5lo splenocytes was evident in some adult spleens (data not shown). FACS analysis of pooled spleens from 5week-old (stage 54/55) control and Tx Xenopus larvae revealed no 1F8 staining of viable splenocytes following 48 h culture in GFM (data not shown). Dual color flow cytometric data on splenocytes pooled from 7-week larvae (stage 56 – 58) or from 3 to 4 month froglets, following 48 h culture in GFM, is also shown in Fig. 3. A few splenocytes expressing
the 1F8 antigen (of lower fluorescence intensity (1F8lo) than seen in adults) can be visualized for the first time in 7-week-old larvae (Fig. 3C), especially in Tx tadpoles; such 1F8lo cells are IgMnegative, CD5negative and CD8negative, but a few (3%) coexpress the D12 antigen. The proportion of 1F8lo cells remains unchanged in 3– 4 month old froglets (Fig. 3B), where a few double-positive 1F8lo/CD5positive cells were noted in addition to the 1F8lo/D12positive population. 1F8positive cells were not found in the thymus at any stage of development (data not shown). Functional studies. Previous studies had employed 51 Cr-release to identify the anti-tumor cytotoxic potential of purified 1F8positive NK cells in adult Xenopus [12 –15]. The JAM assay, developed for mice by Matzinger [24], monitors DNA loss by fragmentation. This assay is more sensitive than a chromium-release assay for determining cellmediated killing, since DNA fragmentation (DNA ladder formation) is a more reliable criterion for judging apoptotic death. Further, the JAM assay, as adapted and successfully used with Xenopus [23], requires only 1 £ 104 3HTdR-labelled tumor targets, compared with 5 £ 104 targets necessary in the 51Crrelease assay. The JAM assay indeed proved to be more sensitive than 51Cr release in monitoring killing of B3B7 tumor cell targets following their 5 h coculture with 1– 2 year-old adult splenocytes from control and Tx frogs (Fig. 4, top left). Consistent with findings from 51Cr release studies, JAM assay analyses of MACS-separated splenocytes identified the 1F8positive population as the one able to effect target cell DNA fragmentation; T-cells, B-cells and 1F8negative cells were unable to induce DNA fragmentation in B3B7 tumor targets (data not shown). We took advantage of the high sensitivity of the JAM assay for our ontogenetic studies. JAM assays were initially carried out on 5-week (stage 54/55) and 7-week (stage 56/58) control and Tx Xenopus larvae (Fig. 4, bottom). Assays involved culturing of splenocytes from individual larvae for 48 h in GFM (to promote NK cell killing) prior to pooling of between 7 and 20 spleens for assay (Section 2). No DNA fragmentation (, 10% specific killing) of B3B7 targets was observed in any group of experimental larvae, even at 20:1 or 40:1 E:T ratio. This was true even for pooled spleens taken from control 7-week-old tadpoles that had been injected with B3B7 cells 10 days
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Fig. 3. Dual color flow cytometric analysis comparing cell surface antigen expression on splenocytes from control and Tx Xenopus at various stages of development. Following 48 h culture in GFM, cells were stained first with 1F8 (anti-NK) hybridoma supernatant (detected by FITCconjugated anti-mouse Ig), then with either PE-conjugated D8 (anti-IgM), 2B1 (anti-CD5), F17 (anti-CD8) or D12 (identifies a 56 kDa epitope) mAb. Quadrants set to exclude 98% cells stained with control reagents from positive analysis. Data shown are representative of three separate experiments on individual adult and froglet spleens and several pools of larval spleens.
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Fig. 4. Cytotoxicity towards B3B7 lymphoid tumor targets effected by splenocytes from control and Tx Xenopus at various stages of development. Splenocytes from individual animals were cultured for 48 h in GFM prior to 5 h cytotoxicity assay. Cytotoxicity of adult splenocytes (top left) was compared using JAM (1 £ 104 3HTdR-labelled tumor targets) and 51Cr-release (5 £ 104 51Cr-labelled tumor targets) assays. Cytotoxicity of splenocytes from froglets and larvae was monitored by JAM assay only. Data on adults and froglets show mean (^SEM)% killing at various E:T ratios from three separate experiments on individual animals. Larval data shows percentage killing of tumor targets mediated by splenocytes pooled from ,10 to 20 stage 54/55 larvae or ,7–10 stage 56/58 larvae. One pool of 7-week-old larval splenocytes (see bottom left ‘Control inj’) came from tadpoles injected intraperitoneally with 5 £ 103 B3B7 cells 10 days prior to assay.
prior to assay in an attempt to elevate NK-like killing [12]. Replicate experiments on additional outbred and MHC homozygous F strain Xenopus 7-week-old larvae confirmed their inability to kill B3B7 tumor targets in vitro. Moreover, such cytotoxicity was still relatively poor (only , 20% specific killing at highest E:T ratios used) when splenocytes from individual 3– 4 month-old froglets were tested (Fig. 4 top right). 4. Discussion 4.1. Molecular characterization of 1F8þ cells from adult, Tx Xenopus Previous two color flow cytometric data [14] on lymphocytes purified from spleen, liver and kidney of
adult Xenopus suggested that the mAb 1F8 constitutively labels a lymphocyte subset of NK-like cells (surface CD5negative or CD5lo and IgMnegative) that is distinct from T and B cells. Furthermore, 1F8positive NK-like cells were identified as the lymphoid population, which after culture in GFM, were able to kill MHC-deficient tumor cell targets, but not MHC class I-expressing lymphoblasts [14], whereas the recently-discovered minor population of CD8positive NK/T cells do not kill class I-negative targets [23]. We now report that Xenopus 1F8positive splenocytes purified from Tx frogs do not manufacture fully rearranged TCRb V region transcripts. 1F8positive cells, therefore, represent a lymphoid population we consider to be bona fide NK cells, distinct from T and NK/T cells, both of which express rearranged TCRb
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V region transcripts [23]. It is possible that NK cells can still manufacture sterile TCRb C region transcripts, which might help explain previous work where TCRb transcripts were detected in unseparated lymphocyte populations from Tx frogs, albeit at very low levels compared with controls [15]. Presence of abortive TCR transcripts in NK cells would not be surprising in view of compelling evidence that NK cells and T cells can develop from a common progenitor [31]. Although the 1F8 mAb clearly identifies cells that are functionally and structurally NK cells, the characterization of the 1F8 antigen itself is still incomplete, in part because the lack of established NK cell lines makes it difficult to obtain sufficient purified antigen to biochemically analyze. Previously published Western blotting experiments [14] suggested that the 1F8 molecule was a 70 –85 kDa polypeptide. In the current study, immunoprecipitation experiments of both biosynthetically-labeled and surfacebiotinylated lysates reveals that 1F8 binds to a monomeric 55 kDa polypeptide. 1F8 also precipitates a smaller molecule of 45 kDa from surface-biotinylated lysates. That these differences reflect allelic polymorphism is problematic since they have been reproduced several times with cells from different outbred animals. The fact that the 55 kDa molecule was detected by immunoprecipitation under various conditions (e.g. biotinylation, biosynthetic labeling, before lysis, under non-reducing and reducing SDS – PAGE conditions) indicates that it is a real antigenic determinant recognized by 1F8. No other polypeptide was detected from biosynthetically-labeled cells, even after PMA/ionomycin stimulation that up-regulates 1F8 surface signal (as determined by flow cytometry [23]). Therefore, it is unlikely that the 45 kDa molecule precipitated from surface-biotinylated lysates represents a different 1F8 antigen. It is possible that biotinylation allows the detection of a degraded or processed fragment that is not well labeled by 35S (i.e. poor in methionine and cysteine residues), or that this 45 kDa polypeptide is coprecipitated with the 1F8 antigen as a result of its biotinylation (e.g. change of conformation, increased stickiness). The 70 – 85 kDa molecule previously detected by Western blotting [14] was most likely an artifact resulting from an interaction of the 1F8 mAb with boiled material. Further molecular studies
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and the cloning and sequencing of the 1F8 NKassociated antigen are clearly needed, especially in view of our finding [23] that the surface protein recognized by 1F8 mAb plays a biological role in antitumor immunity. In order to clone the 1F8 gene, purified 1F8 protein will be obtained by immunoprecipitation and used to generate a polyclonal antibody against the 1F8 antigen, which could then be used to screen a cDNA expression library. 4.2. Ontogeny of NK cells and tumor immunity in Xenopus In mammals, interactions between inhibitory receptors expressed by developing NK cells and MHC class I proteins expressed by autologous cells are believed to play a crucial role in the education of NK cells and their development of self-tolerance [32]. However, it has recently been shown in humans [33] that immature NK cells express activatory receptors before MHC-specific inhibitory receptors, implying that in early ontogeny, ligands other than MHC may be crucial in NK inhibition. Whether NK cells can develop in the absence of MHC class I expression remains unclear. Although functional NK cells are found in class I-deficient mouse models, such b2m2/2 and TAP2/2 animals express low levels of class I molecules on their cell surfaces [32]. In this paper, we have explored whether NK cells develop in the naturally occurring MHC class I-negative environment of larval Xenopus, but were only able to detect 1F8positive cells in the spleen just prior to metamorphosis, at , 7-weeks (stage 56/58). This is long after T and B cells can be detected in this organ during the second week of larval life [20]. The surprisingly late appearance of NK cells in Xenopus comes , 2-weeks after the ontogeny of MHC class Ia expression, which is first detectable at the splenocyte surface at 5-weeks [34] when low levels of class Ia transcripts are also initially expressed in various non-lymphoid tissues [18]. The initial emergence of 1F8lo cells in 7-weekold larvae precedes MHC class Ib expression that occurs only after metamorphosis [18]. (Class II MHC expression is seen on a range of cell types from early in larval life [35].) The above phenotypic studies suggesting initial appearance of NK cells in late larval life is not precisely mirrored by the results from the JAM assays.
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Thus, GFM-cultured splenocytes from control or Tx (5- and 7-week-old) tadpoles, or even from B3B7injected larvae, failed to effect DNA fragmentation of B3B7 tumor targets. These findings using the sensitive JAM assay confirm and extend previous preliminary data [13], derived from a 51Cr-release cytotoxicity assay, that showed cultured splenocytes from 6 to 7week control larvae failed to kill B3B7 targets. The very low percentage of 1F8lo splenocytes detectable by FACS in late larvae may be competent to kill, but their cytotoxicity is masked when unseparated splenocytes are assayed. Clearly, 1F8-enrichment studies are needed, but they would necessitate using prohibitively large numbers of (Tx) larvae to collect sufficient numbers of effectors. Significant, albeit low, levels of B3B7 tumor killing by splenocytes (i.e. . 10% of the level of killing that can be detected in the JAM assay following 5 h co-culture of tumor cells with 20– 40-fold excess red blood cells (unpublished observations)) are first seen only in 4 month-old froglets. Previous studies have shown that splenocytes from 6 month-old Xenopus (some 3.5 months postmetamorphosis) are almost as effective killers of B3B7 targets as splenocytes from year-old adults [13]. It is conceivable that the poor cytotoxic potential displayed by larvae and 4 month-old froglets relates to the low intensity of 1F8 antigen expression. In humans, only those NK cells expressing high levels of natural cytotoxicity receptors efficiently kill tumor cell lines [6]. Another possibility to consider is that NK cells may become fully differentiated and functional only after metamorphosis, in parallel with the postmetamorphic maturation of the immune system characterized by: new lymphoid histogenesis in the thymus and spleen [20,36]; the expression of MHC class II by peripheral T cells [37]; the reappearance of detectable T cell alloreactivity in mixed lymphocyte culture [38]; and the development of an effective alloimmunity to lymphoid tumor [39,40]. The developmental emergence of fully-effective NK killing activity at , 6 months of age has to be integrated with earlier findings on the ontogeny of in vivo alloimmune reactivity of Xenopus towards injected tumor cells [39]. Thus ff2 thymic lymphoid tumor cells injected into F strain tadpoles and young post-metamorphic F froglets up until nearly 4 months of age initiates tumor growth in the vast majority of cases, whereas when
injected into 6 month and older frogs, tumors failed to develop. The development of this alloimmunity to ff2 tumor was originally considered to be most likely linked to the emergence of T cell functions in the early post-metamorphic period, rather than to NK cell development [39]. Whether NK cells functionally contribute to an effective anti-tumor defense by the adult immune system remains to be determined. Although we know that 1F8positive cells are the lymphoid subset that kills tumor targets in vitro, and that this killing is dependent on NK cell pre-culture in GFM (TCGF-rich culture medium), a role of NK cells in in vivo killing of tumors remains to be demonstrated conclusively. Thus, early-thymectomized F strain adult frogs, with substantial numbers of NK cells in their lymphoid tissues, are susceptible to growth of both injected ff2 thymus-derived lymphoid tumor cells (MHCIapositive cells from F strain Xenopus) [40] and MHC Ianegative 15/0 tumor cells (our unpublished observations), in contrast to thymus-intact F strain frogs, in which such tumors fail to grow. This is consistent with the notion that T cell-mediated tumor immunity plays a fundamental role in the vertebrate immune system [41]. Interesting questions remain, however, as to how T cells can kill the MHC class Ianegative thymus tumor cell targets. Perhaps T cells can visualize MHC class Ib molecules that thymic lymphoid tumor cell lines synthesize [28], or perhaps CTLs recognize thymic lymphocyte tumor-derived gp96 heat shock protein that can elicit potent immunity towards tumor cells [42]. Since injection of adult clonal Xenopus with anti-CD8 mAb impairs the immune response against transplanted syngeneic MHC class Inegative tumors, CD8positive T cells may be crucial effectors in MHC-unrestricted anti-tumor responses [43]. However, NK cells may additionally play a crucial role in such tumor immunity, since we have recently shown that the 1F8 mAb injected into LG15 control Xenopus enhances the rapid growth of 15/0 tumor [23]. NK cells may only function in situations, where T cell-derived growth factors are plentiful (i.e. in controls), whereas the effectiveness of these cells in Tx frogs is compromised because such factors are absent. The question of whether NK cells arose in phylogeny as a backup system for T cells or rather as a surveillance system before the emergence of
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adaptive immunity remains to be resolved. The data presented here suggest that, at least in Xenopus, NK cells are serving a backup role, or at least do not subserve a killing role in the absence of class I molecules.
Acknowledgements We would like to thank Jennifer Gantress, Ana Goyos, Gregory Maniero and Heidi Morales for their collective criticisms and helpful discussions. Research was supported by a project grant from the Leverhulme Trust (to JDH), by grants AI-44011 and CA-76312 from the NIH (to NC) and by NSF grant MCB#0136536 (to JR). R.S. was funded by a Durham University Studentship.
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