Adaptive cell-mediated cytotoxicity against allogeneic targets by CD8-positive lymphocytes of rainbow trout (Oncorhynchus mykiss)

Developmental and Comparative Immunology 27 (2003) 323–337 www.elsevier.com/locate/devcompimm

Adaptive cell-mediated cytotoxicity against allogeneic targets by CD8-positive lymphocytes of rainbow trout (Oncorhynchus mykiss ) Uwe Fischera,*,1, Katrin Utkea, Mitsuru Ototakeb, Johannes Martinus Dijkstrab, Bernd Ko¨llnerc,1 a

Institute of Infectiology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany b Inland Station, National Research Institute of Aquaculture, Tamaki, Mie 519-0423, Japan c Institute of Virus Diagnostics, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany Received 15 December 2001; revised 9 September 2002; accepted 17 September 2002

Abstract Rainbow trout surface-(s)IgM2 leukocytes exhibited cell-mediated cytotoxicity (CMC) against allogeneic cells. This is described in concordance with a characterization of gene expression in the effector cells. Peripheral blood leukocytes (PBL) isolated from trout grafted with allogeneic tissue lysed allogeneic target cells (erythrocytes or cells of the RTG-2 cell line) in in vitro assays. The PBL were magnetically separated into different subpopulations using monoclonal antibodies (mabs) specific to thrombocytes, IgM, granulocytes and monocytes. Of the isolated subpopulations only the sIgM2 lymphocytes were capable of lysing allogeneic targets. The separated PBL fractions were characterized by RT-PCR analysis using specific primers for the amplification of trout IgM heavy chain constant region (CH1), T cell receptor alpha chain (TCRa), CD8a and major histocompatibility complex (MHC) class I gene fragments. Most importantly, CD8a was expressed only by the sIgM2 population. Combined with the requirement for sensitization to detect CMC, this strongly suggests T cell involvement in fish as in higher vertebrates. The involvement of CD8a-positive cytotoxic T cells in allograft rejection was supported by additional in vivo and in vitro observations. CD8a expression was barely detectable in the blood of unsensitized trout or trout that received xenografts, but was easily detected in the blood of allogeneically stimulated trout. Furthermore, CD8a expression in sIgM2 lymphocytes from immunized trout was secondarily enhanced by addition of allogeneic targets in vitro. Collectively, these functional and genetic data suggest that fish possess specific cytotoxic cells with phenotype and gene expression pattern similar to those of cytotoxic T cells in higher vertebrates. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cell-mediated cytotoxicity; Leukocytes; CD8; T cells; Allogeneity; Flow cytometry; Magnetic cell sorting; RT-PCR; Marker expression; Rainbow trout; Oncorhynchus mykiss

* Corresponding author. Tel.: þ 49-38351-7105; fax: þ 4938351-7226. E-mail address: [email protected] (U. Fischer). 1 The two authors contributed equally to this work.

1. Introduction In mammals, cell-mediated killing of allografts is performed primarily by cytotoxic T cells but is

0145-305X/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 3 0 5 X ( 0 2 ) 0 0 1 0 0 - 3

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dependent on the type of grafted tissue, with natural killer cells sometimes playing a limited role [1,2]. It is generally accepted that mammalian T cells recognize a combination of major histocompatibility complex (MHC) with endogenous peptide ligand via the T cell receptor (TCR) [3]. The interaction of TCRs with a complex of foreign MHC molecules and foreign peptides results in acute graft rejection. Chronic rejection may occur when MHC of an allogeneic graft donor and the graft recipient are identical. In this case, allogeneic minor histocompatibility antigens are processed and the resulting peptides are presented by the MHCs of the graft recipient to T cells [4]. Thus, mammalian T cells are the main effector cells in both acute and chronic allorejection. Natural killer (NK) cells may contribute to allograft rejection in the absence [5] or in the presence of allospecific antibodies via antibody dependent cell-mediated cytotoxicity (ADCC) [6]. NK cells play a more important role in xenograft rejection [1,2]. Allograft rejection was first described in fish decades ago but characterization of the cell types involved has been limited. A fundamental characteristic of T cells in comparison to NK cells is their specificity. There is a large amount of indirect evidence indicating the involvement of T cells in fish CMC. For example, peripheral blood leukocytes (PBL) from sensitized triploid ginbuna carp induce graft-versus-host reaction (GVHR) in tetraploid recipients sharing three common alleles [7,8]. In vitro studies have shown that piscine leukocytes are able to lyse modified autologous cells [9], allogeneic cell lines [10 –16], allogeneic erythrocytes [17] and virus-infected syngeneic cells [18 –20]. Sensitization was necessary to generate TCR-positive cytotoxic cell clones from channel catfish immunized with allogeneic cells [16,19]. Specificity was shown since in vitro cell-mediated cytotoxicity (CMC) was observed only against cells used for immunization [13,15 – 17]. Few functional studies have investigated T cell activity in context with MHC class I restriction and virus-infected fish. Using a limited set of rainbow trout donors and target cell lines, immunization of donors and MHC class I matching of target cells seemed to be necessary for the killing of virusinfected cells by PBL [19]. Furthermore, a study using ginbuna crucian carp showed that PBL from virusinfected fish efficiently killed syngeneic cells infected

with the same virus but not with other viruses. In the same study the killing of allogeneic cells was much less efficient and was not influenced by virus infection [20]. The cellular equivalents of CD8þ cytotoxic T cells and NK cells of higher vertebrates have not yet been properly described in fish. However, there is evidence for their existence since leukocytes of cartilaginous and bony fish express TCR [21 –26] and CD8 [27] gene homologues and a monoclonal antibody has been described as a possible NK cell marker in channel catfish [28]. Sequences encoding MHC molecules (the ligands of TCRs) have been reported for more than 25 fish species [29 – 32] including rainbow trout [33 – 36]. There is additional evidence that the presentation of foreign peptides via the MHC class I pathway may be similar to that of higher vertebrates since genes for low molecular mass proteins, transporter associated with antigen processing and b2 microglobulin have been reported in rainbow trout [34,37]. Furthermore, TCR analysis in fish indicates that as in mammals, T cells are clonally selected after immunization [38]. Although these lines of evidence suggest the existence of piscine CD8-positive cytotoxic T cells with functions similar to those in mammals, the reports have not been conclusive. For example, CD8 expression has not been linked to cells exhibiting CMC as the separation of leukocyte fractions from immunized fish for analysis of specific CMC has been difficult. The strongest evidence for the existence of cytotoxic T cells in fish may come from the TCRpositive channel catfish cell clones discussed above [16]. However, these clones were generated by multiple passages in vitro, and additional evidence closer to the in vivo response is desirable. One problem in studying T lymphocyte-mediated cytotoxicity is the potential interference of NK cell activity. A number of studies indicate the presence in fish of NK-like cells that can kill independent of the presence of antibodies. In several fish species, spontaneous killing of allogeneic [10,14], xenogeneic [39,40], or virus-infected syngeneic targets [41] has been shown to vary among individuals or among organs from which leukocytes were isolated. In addition, highly concentrated piscine neutrophilic granulocytes were reported to spontaneously kill xenogeneic and also autologous cells in vitro

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[42 – 44]. It appears that in multiple fish species, more than one cell type can be involved in spontaneous killing. In channel catfish, non-specific cytotoxic cells (NCC) isolated from the spleen were distinguished from NK-like cells isolated from the blood [28]. While NK-like cells may spontaneously kill allogeneic cells in catfish [10,14], this phenomenon was not observed in carp as the induction of cytotoxic cells always required previous sensitization [17,18]. The present study was carried out to characterize alloreactive cytotoxic activity of distinct rainbow trout leukocyte subpopulations and to screen for the expression of immunologically relevant molecules in those subpopulations. Induction of alloreactive cytotoxic cells required previous allogeneic sensitization in vivo and cytotoxic cells were shown to be sIgM2 while expressing TCR and CD8 transcripts. The alloreactive cytotoxic cells showed enhanced CD8 expression after secondary in vitro stimulation with the allotarget. Thus, the present study provides in vitro and in vivo evidence for the existence of CD8-positive T cells in fish.

2. Materials and methods 2.1. Fish Two inbred strains of rainbow trout (Oncorhynchus mykiss ), Autumn spawners and Spring spawners, were kindly provided by Dr Anders of the States Institute for Fisheries of Mecklenburg-Western Pomerania. The strains were kept under closed-colony inbred conditions for at least seven generations, and for each generation eggs from up to seven females were inseminated with sperm from up to 25 males. Homozygous isogeneic rainbow trout (clone C25) were derived from Nagano Prefectural Experimental Station of Fisheries, Akashina, Nagano, Japan. The clone was produced by gynogenesis over two generations with suppression of mitosis and meiosis in the first and second generations, respectively. Clonality was confirmed by DNA fingerprinting (unpublished data). Autumn spawners were used as donors for grafts and target cells. Spring spawners and C25 fish were used as graft recipients and for the generation of effector cells. Autumn spawners weighing between

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700 and 1000 g and effector cell donors weighing between 100 and 200 g (Spring spawners) or 300 and 400 g (C25 trout) were held at 15 and 18 8C, respectively, in 400 l tanks in a partially recirculating water system and were fed with commercial dry pellets. 2.2. Preparation of skin grafts, erythrocytes, RTG-2 cells and xenogeneic cells Skin grafts were taken from Autumn spawners by cutting a triangular piece of tissue (4 mm per side) from the posterior end of the operculum or by removing the distal portion of a fin between rays. For the isolation of erythrocytes, Autumn spawners were first anaesthetized in benzocaine solution (Sigma, 50 ng/ml). Then, approximately 0.3 ml of blood was drawn from the caudal vein using a syringe treated with heparin (Sigma; 1000 IU/ml in phosphate buffered saline without magnesium salts). The blood was added to 10 ml of mixed medium (MM) with the following formulation: Iscov’s DMEM/Ham’s F12 (Gibco) at a ratio of 1:1, supplemented with insulin, transferrin, sodium selenite media supplement (Sigma, 1 vial/5 l medium) and 10% fetal bovine serum (FBS). After centrifugation (200 £ g, 4 8C, 10 min), the buffy coat was removed and the remaining erythrocytes were adjusted to 107 cells/ml. RTG-2 cells were grown in minimal essential medium (MEM) supplemented with 15% FBS for 2 days until a complete monolayer formed. Cells were detached from the flask with trypsin, washed and adjusted to a final concentration of 1.5 £ 107 cells/ml. Bovine blood was collected by puncture of the jugular vein. PBL were prepared by density gradient centrifugation, adjusted to a final concentration of 107 cells/ml as described below for trout PBL and used as xenogeneic grafts. 2.3. Sensitization of effector cell donor fish Spring spawners (Group 1, n ¼ 66) were sensitized using skin grafts and erythrocytes originating from Autumn spawners. For skin grafts, a 4 mm incision was made on the dorso-lateral side of the body, space was made by detaching the skin from the underlying muscle tissue using a lancet, and the graft was carefully inserted. For erythrocyte grafts, 5 £ 106

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erythrocytes re-suspended in 0.5 ml of MM were intravenously (i.v.) injected. Spring spawners in Group 1 were sensitized to allogeneic grafts at least three times, beginning with skin grafts then followed by erythrocyte injections repeated at one-week intervals. The erythrocytes and skin tissues grafted to each recipient were always taken from the same donor. Sensitization of Spring spawners against RTG2 cells (Group 2, n ¼ 8) was performed three times by i.v. injection of 5 £ 106 cells in 0.3 ml MM per fish at 1-week intervals. Group 3 consisted of three C25 clonal trout injected three times at three-week intervals with erythrocytes from a single Autumn spawner, and three additional C25 trout injected with MM following the same schedule. The fish designated Group 4 consisted of five C25 trout injected twice at three-week intervals with 5 £ 106 bovine PBL, and four additional C25 trout injected with medium only following the same schedule. 2.4. Preparation of effector cells from peripheral blood Effector cells were isolated between one and two weeks after the last sensitization. Blood (4 – 6 ml) was collected from anaesthetized fish by puncture of the caudal vein and immediately diluted into a five-fold volume of cold MM. Ten milliliters of prediluted blood was layered onto 3 ml of Percoll (Pharmacia)/Hank’s solution (Sigma) with a density of 1.075 g/cm3 , and centrifuged (650 £ g, 4 8C, 30 min). PBL banding at the interface were collected, washed twice with MM (200 £ g, 4 8C, 10 min), stained with trypan blue (Sigma) and counted using a Thoma hemocytometer and a phase contrast microscope to determine the number of live and dead cells. Effector cells were defined as viable cells with lymphocyte morphology.

cytotoxicity assays were re-suspended in MM containing 0.2% bovine serum albumin (BSA; Sigma). 2.6. Hemoglobin release assay To quantify the killing activity of alloreactive PBL a hemoglobin release assay was carried out as previously described [17]. In this assay effector cells are co-cultured with erythrocyte targets and hemoglobin released from killed erythrocytes into the supernatant is measured colorimetrically using 3,30 ,5,50 -tetramethylbenzidine (TMB). Briefly, 96well plates were set up with control wells for target cell spontaneous release (TSR: erythrocytes þ MM), target cell maximum release (TMR: erythrocytes þ MM þ Triton X100, Sigma), effector cell spontaneous release (ESR: PBL þ MM), medium background (Me: MM) and a volume correction control (VCC: Medium þ Triton X100). Experimental (EXP) wells were filled with varying numbers of effector PBL and a constant number of target erythrocytes (2 £ 104 cells per well) to achieve different effector to target cell (E/T) ratios. In each experiment triplicate wells with final volumes of 200 ml per well were analyzed. After setup, plates were centrifuged (5 min, 200 £ g, 4 8C) and incubated in a CO2 incubator with a humidified atmosphere at 20 8C for 5 h. Triton X100 (10 ml, Sigma) was added to the TMR and VCC wells 45 min before harvesting, for which 100 ml of supernatant from each well was transferred to another 96-well flat-bottom microtiter plate (Greiner). A substrate solution of TMB with H2O2 was freshly prepared and 100 ml of the substrate was added to each well. Plates were incubated for 30 min and optical density was determined using a microplate reader (BIO-RAD, model 450) at 405 nm. The percentage of specific cytotoxicity was calculated using the following formula: % SPECIFIC CYTOTOXICITY ¼

2.5. Preparation of target cells Erythrocytes and RTG-2 cells were prepared as described above and adjusted to final concentrations of 2 £ 105 and 4 £ 105 cells per ml MM, respectively. Erythrocytes and RTG-2 cells designated for in vitro

ðEXP 2 ESRÞ 2 TSR £ 100% ðTMR 2 VCCÞ 2 TSR

2.7. LDH release assay In this assay the amount of LDH released into the supernatant from killed RTG-2 cells was quantified.

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Plates were set up according to the design described for the hemoglobin release assay except that 4 £ 104 RTG-2 cells per well were used as target cells. LDH release was analyzed using a commercially available test kit (Roche). The test was carried out according to the manufacturer’s protocol and supernatants from test wells were analyzed with an microplate reader (see above) at 490 nm. Results were calculated according to the formula described for the hemoglobin release assay. 2.8. Magnetic cell sorting In order to characterize the cytotoxic capacity of different effector cell subpopulations, PBL from sensitized donors of trout Groups 1 and 3 were immunomagnetically separated. Briefly, 1 £ 107 PBL/ml were incubated with primary monoclonal antibodies (mabs) (below) for 30 min on ice. After washing twice in MM the cell pellet was re-suspended in 80 ml MM. Cells were then incubated with 20 ml of goat-anti-mouse-Ig microbeads (Miltenyi Biotec, Germany) for 30 minutes. The magnetically labeled PBL were resuspended in 2 ml of MM and applied to a separator column attached to a MiniMACSw separator (Miltenyi Biotec, Germany). Unlabeled cells flowing through the column were collected. After one washing the column was detached from the magnetic separator and the labeled cells were eluted from the column using 1 ml MM. Cell fractions were counted and analyzed for purity using a FACSCalibur (Becton Dickinson) flow cytometer. An anti-mouse-Ig(Fab2)FITC (Medac, Germany) conjugate was added to the magnetically-sorted fractions of cells and histogram plots for forward scatter and green fluorescence were recorded. PBL from Group 1 trout (n ¼ 18) were separated into sIgM2 PBL and sIgMþ lymphocytes (B-cells) for cytotoxicity assays using three anti-rainbow trout IgM mabs (clone 4c10, H-chain specific [45], clone 1.14 [46] and clone N2, L-chain specific [47], 1:1:1, v/v). PBL obtained from C25 fish (Group 3) were first labeled with an anti-thrombocyte mab to isolate thrombocytes and thrombocyte-free PBL. The latter fraction was then magnetically sorted by a cocktail of anti-monocyte (mab 45) [48], anti-granulocyte/ monocyte (mab Mm) [49] and anti-rainbow trout

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IgM mabs (4C10, 1.14, N2) ([45 – 47], respectively) resulting in a fraction containing B cells, monocytes and neutrophilic granulocytes and another fraction of sIgM2 lymphocytes. These three fractions (thrombocytes; B cells/monocytes/granulocytes; sIgM2 lymphocytes) were analyzed both in CMC assays against allogeneic erythrocytes and by RT-PCR. The number of cells available in magnetically separated PBL subpopulations was closely related to their percentages in the whole PBL. Therefore, different maximum E/T ratios were possible for each fraction in CMC assays. 2.9. RT-PCR amplification of IgM, TCRa, CD8a and MHC class I gene fragments from magnetically sorted PBL For Group 1 fish, mRNA was isolated from 107 PBL or magnetically separated PBL fractions using an mRNA extraction kit (Purescript, Gentra Systems) and transcribed into cDNA using Oligo(d)T primers from the Superscript II reverse transcriptase kit (Gibco) according to the manufacturer’s protocol. 125 ng of cDNA was used for each PCR reaction with Taqe polymerase (Promega) according to the manufacturer’s recommendations. PCR conditions for all reactions were 94 8C for 3 min, 30 cycles at 94 8C for 1 min, 60 8C for 30 s, 72 8C for 1 min and finally 72 8C for 10 min. For experiments with Group 3 PBL, total RNA was isolated from 107 cells from ESR wells after CMC assays (PBL subpopulations only) and from EXP wells (PBL subpopulations cultured in the presence of allogeneic targets) with TRIzol reagent (Invitrogen). The resulting RNA pellet was dissolved in 50 ml DEPC water. RT-PCR was performed using a Onestep-RT-PCR kit (Qiagen) with 1 ml RNA in a 12 ml reaction mixture. PCR conditions were set up according to the manufacturer’s manual (reverse transcription: 30 min/50 8C; initial PCR activation step: 15 min/95 8C; denaturation: 1 min/94 8C; annealing 1 min/55 8C; extension 1 min/72 8C (40 cycles of the last three steps); final extension 10 min/72 8C). To obtain semi-quantitative results RNA was isolated from 107 cells and lower cycle numbers were applied. Table 1 shows the primers used for RT-PCR analysis.

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Table 1 Primers used for RT-PCR. References used for primer generation are listed in square brackets. S, forward primer; AS, reverse primer Gene fragment

Primer

Sequence

Product length

IgM [50] TCRa [22] CD8a [27] MHC class I a 3 [34,35,51] b-actin AF157514a

CH1S CH1AS TCRaS TCRaAS CD8aS CD8aAS MHCIS MHCIAS b-AcS b-AcAS

50 -AGGAAGTTTCCACAGCGTCCAT 50 -TACTGGGCCATGCATCTCTG 50 -GGCAGGTCTCTCACTGTCCTT 50 -CAACCTGGCTGTAGTAGCTG 50 -ATGAAAATGGTCCAAAAGTGGATGC 50 -GGTTAGAAAAAGTCTGTTGTTGGCTATTAGG 50 -CCTCTCCAGTGACMTGCCACGCGAC 50 -YRASTTGAACCASACACTGATACT 50 -GCTGTCTTCCCCTCCATCGTC 50 -GGCAGGGGTGTTGAAGGTCTC

412 bp

a

618 bp 682 bp 241 bp 309 bp

GeneBank accession number.

2.10. Statistics Results of cytotoxicity assays were statistically compared by an f-test followed by a two-tailed Student’s t-test using MS Excel 5.0 (Microsoft Corp.) and were considered to be significantly different at p # 0.05.

3. Results 3.1. Sensitized PBL efficiently lyse allogeneic cells in vitro Whole PBL were isolated from Spring spawners previously sensitized with either allogeneic erythrocytes and allogeneic skin grafts (Group 1; n ¼ 48) or allogeneic RTG-2 cells (Group 2; n ¼ 8). All effector cells were isolated one week after the last sensitization. In both groups, the level of cytotoxicity against the corresponding target cells (erythrocytes in Group 1 and RTG-2 cells in Group 2) was quite variable, ranging from almost zero (Fig. 1; weak responder against erythrocytes) to more than 50% (Fig. 1(A) and (B); strong responders against erythrocytes and RTG-2 cells, respectively). PBL isolated from 25 out of 48 trout of Group 1 that received transplants were able to kill allogeneic erythrocytes at the highest E/Ts employed (specific release . 5%). PBL from two out of eight trout in Group 2 sensitized by RTG-2 injection showed killing activity toward RTG-2 cells in vitro. Allogeneically sensitized PBL from Group 1 were

unable to kill autologous erythrocytes (three fish) and PBL from unsensitized trout did not exhibit cytotoxicity against allogeneic erythrocytes or RTG-2 cells (three fish and 10 fish, respectively; data not shown). 3.2. Only sIgM2 lymphocytes kill allogeneic erythrocytes PBL isolated from allogeneically sensitized rainbow trout of Group 1 (n ¼ 18) were magnetically separated into sIgMþ lymphocytes (B cells) and sIgM2 PBL using anti-IgM mabs. sIgM2 PBL were morphologically determined by phase contrast microscopy to consist of lymphocytes, thrombocytes and less than 1% granulocytes. Both subpopulations were then tested for their ability to lyse erythrocytes of the allogeneic graft donor. sIgM2 PBL from seven out of 18 individual fish showed marked cytotoxicity, whereas none of the 18 sIgMþ lymphocyte fractions killed allogeneic erythrocytes. Fig. 2 provides an example of a typical experiment consisting of magnetic cell sorting (Fig. 2(A)) followed by CMC assay against allogeneic erythrocytes (Fig. 2(B)). To obtain enriched fractions of sIgM2 lymphocytes, PBL from three C25 trout injected with Autumn spawner erythrocytes and from another three C25 trout injected with the same volume of medium (Group 3) were magnetically sorted into thrombocytes, a mixed fraction of B cells/monocytes/granulocytes, and sIgM 2 lymphocytes using cell-specific mabs (Fig. 3(A)). According to previous FACS analysis with the cell-specific mabs, in unsorted PBL

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Fig. 1. In vitro CMC of whole rainbow trout PBL against allogeneic cells. (A) It shows a hemoglobin release assay with allogeneic erythrocytes (trout Group 1). Spring spawners were sensitized once with allogeneic skin grafts and four times by i.v. injection of allogeneic erythrocytes. Grafts and erythrocytes originated from the same Autumn spawner. Effector PBL were assayed 7 days after the final sensitization. Two representative CMC responses are shown in this figure, one revealing a strong cytotoxic response and a second with no significant cytotoxic response toward allogeneic erythrocytes. (B) It shows a representative LDH release assay with RTG-2 target cells and PBL effectors isolated from a sensitized fish (Group 2) 14 days after three weekly sensitizations with RTG-2 cells. Error bars indicate standard deviation for triplicate tests.

the proportion of thrombocytes ranged 30 –50%, B cells 15 – 30%, monocytes 1 – 5% and granulocytes 1– 8% (data not shown). Consequently, the number of cells in each magnetically separated fraction was different, resulting in different maximum E/T ratios for thrombocytes of 80:1, for the B-cell/monocyte/granulocyte fraction of 20:1 and for the sIgM2 lymphocytes of 5:1. sIgM2 lymphocyte fractions separated from sensitized C25 fish efficiently lysed allogeneic

erythrocytes (Fig. 3(B)). The percentages of specific lysis ranged 46 –53%. sIgM2 lymphocyte fractions from unsensitized C25 fish of Group 3 spontaneously killed allogeneic erythrocytes but to a lesser extent (4 – 18%) at same E/T ratio of 5:1. Neither thrombocytes nor B-cell/monocyte/granulocyte fractions from either sensitized or unsensitized C25 trout killed allogeneic erythrocytes even at high E/T ratios of 80:1 and 20:1, respectively.

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Fig. 2. In vitro CMC of magnetically separated PBL against allogeneic erythrocytes (trout Group 1). (A) It shows a representative result obtained after magnetic separation of rainbow trout PBL collected 8 days after six sensitizations by allogeneic skin grafting combined with erythrocyte injection (Group 1). PBL were separated using anti-IgM mabs; the depleted cell fraction contained 10% positive cells while the enriched fraction consisted of more than 85% B cells. The plots were obtained by FACS analysis and represent FITC fluorescence (FL1-H) against cell counts. Markers (M1) were placed at 2% false positive events for unseparated PBL after incubation with anti-mouse-Ig(Fab2)-FITC conjugate. (B) It shows a representative experiment for an in vitro CMC with magnetically separated PBL from one fish. The fraction of sIgMþ lymphocytes exhibited no cytotoxicity against allogeneic erythrocytes, whereas the sIgM2 PBL fraction was able to lyse erythrocytes of the graft donor in a dose dependent manner. Error bars indicate standard deviation of triplicate tests. Asterisks indicate significantly different values.

3.3. TCR and CD8 are expressed only by sIgM2 lymphocytes Magnetically separated PBL subpopulations used in CMC assays were analyzed for expression of MHC class I, IgM CH1, TCRa and CD8a transcripts. The samples included sIgMþ lymphocytes (B cells) and sIgM2 PBL isolated from Group 1 trout in addition to thrombocytes, a mixed population of B cells/monocytes/granulocytes and

sIgM2 lymphocytes separated from Group 3 trout. For PCR analysis, only magnetically sorted cell fractions with sufficient purity were used (greater than 90% positive cells in the enriched cell fraction and less than 10% positive cells in the depleted fraction). Purity of magnetically sorted cells obtained in a representative experiment with Group 3 fish is shown in Fig. 3(A). RT-PCR analysis was performed with cells from at least three individual fish from Groups 1, 3 and 4.

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Fig. 3. In vitro CMC of magnetically separated PBL against allogeneic erythrocytes (trout Group 3). (A) It shows the cell fractions after magnetic separation of rainbow trout PBL from a C25 trout 7 days after the last sensitization with allogeneic erythrocytes. PBL were magnetically sorted by a two-step separation protocol. After the first separation step using anti-thrombocyte mabs, the depleted cell fraction contained less than 5% thrombocytes (upper left plot) and the enriched fraction consisted of more than 95% thrombocytes (upper right plot). After the second separation step using a cocktail of anti-IgM, anti-monocyte and anti-granulocyte mabs, the depleted population contained less than 10% fluorescent cells and the enriched B cell/monocyte/granulocyte population contained more than 90% fluorescent cells. The figure shows representative separation results for PBL from a sensitized fish. Plots were recorded as shown in Fig. 2(A). (B) It shows the summarized results of CMC experiments with three allogeneically sensitized and three unsensitized fish. CMC experiments were performed with allogeneic erythrocytes from the Autumn spawner previously used for sensitization. 1 ¼ sIgM2 lymphocytes from unsensitized trout; 2 ¼ sIgM2 lymphocytes from sensitized trout; 3 ¼ thrombocytes from unsensitized trout; 4 ¼ thrombocytes from sensitized trout; 5 ¼ B cells/monocytes/granulocytes from unsensitized trout; 6 ¼ B cells/monocytes/granulocytes from sensitized trout. At maximum possible E/T ratios, only the sIgM2 lymphocytes were able to kill allogeneic erythrocytes (columns 1 and 2; E/T ¼ 5:1). Previous sensitization of the effector cell donor against allogeneic erythrocytes significantly enhanced the killing activity of sIgM2 lymphocytes. Thrombocytes (columns 3 and 4; E/T ¼ 80:1) and B cells/monocytes/granulocytes (columns 5 and 6; E/T ¼ 20) did not kill. Error bars represent standard deviation of CMC releases from three fish.

Gene fragments of IgM CH1, TCRa and CD8a were amplified from unsorted PBL (Fig. 4). MHC class I gene fragments were amplified from all PBL fractions and served as an internal positive cDNA control. From the sIgMþ lymphocyte (B cell) population, gene fragments of IgM CH1 but not for TCRa and CD8a were amplified, whereas PCR with cDNA from sIgM2 PBL yielded positive signals with primers specific for TCRa and CD8a

but not for IgM CH1. Neither IgM CH1 nor TCRa or CD8a gene products were detected in thrombocytes. RT-PCR-based detection of specific mRNA transcripts was positive for the mixed population of B cells/granulocytes/monocytes with IgM CH1 primers but not with TCRa and CD8a primers, and sIgM2 lymphocytes yielded positive results with TCRa and CD8a primers but not with IgM CH1 primers (Fig. 4).

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Fig. 4. RT-PCR with magnetically separated rainbow trout PBL from sensitized fish. The figure shows representative PCR results obtained from a single fish from Group 1 for PBL, sIgMþ lymphocytes and sIgM2 PBL, and another fish from Group 3 for thrombocytes, the mixed population of B cells/monocytes/granulocytes and sIgM2 lymphocytes using primers specific for IgM CH1, TCRa CD8a and MHC class I (M, marker).

3.4. CD8 expression is enhanced following allogeneic but not xenogeneic stimulation Levels of CD8a expression were examined for Group 3 rainbow trout effector cell PBL fractions separated from sensitized and unsensitized trout following the in vitro CMC assays. Higher levels of CD8a expression were observed in sIgM2 lymphocytes isolated from sensitized trout compared to those from unsensitized trout (Fig. 5, lanes 1 and 4,

respectively). B cells/monocytes/granulocytes and thrombocytes were CD8a negative. In order to show that allogeneic but not xenogeneic sensitization of rainbow trout stimulates the expression of CD8 in blood, PBL were isolated from C25 trout 21 days after the second sensitization with allogeneic (Group 3) and xenogeneic (Group 4) cells. One-step RT-PCR using RNA isolated from allogeneically sensitized PBL yielded strong CD8a signals whereas little or no signal was received from

Fig. 5. RT-PCR with magnetically separated PBL subpopulations from Group 3 trout sensitized by allogeneic grafts. The figure shows representative PCR results obtained from one in vivo sensitized fish and another unsensitized fish using primers specific for CD8a (upper box) and b-actin as a positive control (lower box). Some cell fractions were secondarily sensitized by allogeneic erythrocytes in vitro (lanes 2; 5; 7; 10; 12 and 15). Note that CD8 expression is higher in sensitized sIgM2 lymphocytes than in unsensitized sIgM2 lymphocytes (lanes 1 and 4, respectively) and that secondary incubation with erythrocytes resulted in upregulation of CD8 transcripts in sensitized sIgM2 lymphocytes (lanes 1 and 2, respectively). This in vitro upregulation was not observed in unsensitized sIgM2 lymphocytes. M: marker; lane 1: sIgM2 lymphocytes from sensitized trout; lane 2: sIgM2 lymphocytes from sensitized trout þ allogeneic erythrocytes in vitro; lane 3: allogeneic erythrocytes; lane 4: sIgM2 lymphocytes from unsensitized trout; lane 5: sIgM2 lymphocytes from unsensitized trout þ allogeneic erythrocytes in vitro; lane 6: B cells/monocytes/granulocytes from sensitized trout; lane 7: B cells/monocytes/granulocytes from sensitized trout þ allogeneic erythrocytes in vitro; lane 8: allogeneic erythrocytes; lane 9: B cells/monocytes/granulocytes from unsensitized trout; lane 10: B cells/monocytes/granulocytes from unsensitized trout þ allogeneic erythrocytes in vitro; lane 11: thrombocytes from sensitized trout; lane 12: thrombocytes from sensitized trout þ allogeneic erythrocytes in vitro; lane 13: allogeneic erythrocytes; lane 14: thrombocytes from unsensitized trout; lane 15: thrombocytes from unsensitized trout þ allogeneic erythrocytes in vitro; lane 16: water control.

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amounts of the various effector cell preparations for RT-PCR. This provided the opportunity to examine re-stimulation of effector cells with allogeneic cells in experimental wells. sIgM2 lymphocytes from allogeneically sensitized trout cultured in the presence of allogeneic erythrocytes in experimental wells expressed more CD8a mRNA than the same cells cultured in the absence of erythrocytes (ESR control wells). sIgM2 lymphocytes from unsensitized fish cultured with allogeneic erythrocytes did not show enhanced expression of CD8a when compared to the same cells cultured in the absence of allogeneic erythrocytes (Fig. 5). b-actin products were successfully amplified from all samples tested. 4. Discussion

Fig. 6. RT-PCR with whole PBL from rainbow trout sensitized with allogeneic erythrocytes (trout Group 3; (A)) or with xenogeneic (bovine) PBL (trout Group 4; (B)) using primers specific for CD8 (upper box; M, marker) and b-actin (lower box). PBL were isolated 21 days after the second sensitization. Lane 1: sensitized PBL (trout 1); lane 2: sensitized PBL (trout 2); lane 3: sensitized PBL (trout 3); lane 4: unsensitized PBL (trout 1); lane 5: unsensitized PBL (trout 2); lane 6: unsensitized PBL (trout 3); lane 7: water.

xenogeneically sensitized PBL (Fig. 6(A), lanes 1– 3 and Fig. 6(B), lanes 1 – 3, respectively). Low levels of CD8 expression were again detected with unsensitized PBL (lanes 4 – 6). b-actin products were amplified from all samples tested indicating adequate RNA quality and PCR conditions. 3.5. Allogeneic re-stimulation of sIgM2 lymphocytes results in enhanced expression of CD8 in vitro For fish of Group 3, magnetically sorted cells were first used in CMC assays, then collected from the assay plate and RNA was isolated from equal

It was shown in this study that rainbow trout sIgM2 lymphocytes from sensitized fish are able to kill allogeneic cells. Immunomagnetic separation was used to enrich sIgMþ (B) lymphocytes, thrombocytes or a mixture of B-lymphocytes, monocytes and neutrophilic granulocytes. These fractions were not able to kill allogeneic cells in vitro. After a two-step separation protocol in which thrombocytes, B-lymphocytes, monocytes and neutrophilic granulocytes were removed from whole PBL, the remaining sIgM2 lymphocytes should have contained only NK-like and T cells. Using data from the CMC experiments alone, it is impossible to conclude whether the allospecific cytotoxicity was executed by NK-cells, T cells or both cell types. However, ADCC or lysis of erythrocytes by antibodies against blood group antigens was most likely absent in the assays described in this paper since serum antibodies were removed from separated PBL by multiple washing and separation steps. sIgM2 lymphocytes from unsensitized rainbow trout exhibited clearly detectable cytotoxicity although it was significantly lower than that of sIgM2 lymphocytes from sensitized trout. Thus, NK-like cells, which have been shown to exhibit spontaneous cytotoxicity against allogeneic targets without the need of sensitization in channel catfish [10,14] and rainbow trout [41], may have partially contributed to the cytotoxicity in our experiments. In mammals, CMC executed by CD8þ T cells requires previous sensitization of the effector cell donor [52,53]. The same requirement has been

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reported for in vivo (GVHR) [7] and in vitro CMC [17,18] in ginbuna crucian carp. However, in those experiments whole PBL of unsensitized fish were used as effector cells. In the experiments described in this paper, the cytotoxic cells were markedly enriched by magnetic cell sorting. The need for such an increased concentration may explain why many other authors were not able to show spontaneous CMC of unsensitized PBL against allogeneic cells in vitro. In our experiments CMC levels of sensitized sIgM2 lymphocytes were approximately five times higher than levels observed for unsensitized sIgM2 lymphocytes, suggesting that NK cells were only partially involved in the enhanced CMC. This is based on the assumption that T cells are the main cell type stimulated by sensitization. However, mammalian NK cells can be stimulated by alloantigens [5] in an adaptive response as well as T cells. Therefore, enhancement after sensitization is not sufficient proof for T cell involvement, especially in fish where the cytotoxic cells have not been well characterized. To support the evidence that T cells contributed to the cytotoxicity against allogeneic cells, magnetically enriched thrombocytes, sIgMþ B cells (in some cases together with neutrophilic granulocytes and monocytes) and sIgM2 PBL or lymphocytes were tested for their expression of IgM, TCRa, CD8a and MHC class I gene fragments. Purified thrombocytes were negative for expression of IgM, TCRa and CD8a genes while sIgM þ lymphocytes (B-cells) were positive for IgM expression but not TCRa and CD8a. sIgM2 lymphocytes lacked expression of IgM but were positive for TCRa and CD8a, which is characteristic of mammalian cytotoxic T lymphocytes. In addition, CD8a was not expressed or was expressed only at low levels in whole PBL or sIgM2 lymphocytes from unsensitized fish whereas allogeneic sensitization yielded high levels of CD8a expression. During the in vitro CMC assays the in vivo sensitized sIgM2 lymphocytes were clearly re-stimulated by the erythrocytes that were previously used for sensitization. The level of CD8a expression in those cells was higher than in sensitized sIgM2 lymphocytes cultured in the absence of erythrocytes. In contrast, in vitro contact of unsensitized sIgM2 lymphocytes with allogeneic cells did not result in increased CD8a expression. The mammalian CD8 molecule is a TCR co-receptor of cytotoxic T lymphocytes. TCR molecules of higher vertebrates are

critical for recognition of foreign peptides complexed with MHC class I of target cells. Thus, in our experiments the cells stimulated by allogeneic erythrocytes were presumably specific cytotoxic T cells. We obtained evidence that the CD8 upregulation was a specific response of T cell stimulation and not due to a generally upregulated immune status and stimulation of NK-like cells. Xenogeneic cells are attacked by NK-like cells in mammals, and studies in different fish species using xenogeneic targets indicate similar activities [28,39,40]. We injected clonal rainbow trout with allogeneic or xenogeneic cells. Only PBL from allogeneically sensitized fish expressed high levels of CD8a mRNA, while CD8a expression in PBL from xenogeneically sensitized fish did not exceed the level of expression in unsensitized PBL. This supports the idea that the CD8 upregulation observed in our experiments was caused by direct interaction of allogeneic cells with cytotoxic T cells. This conclusion is in agreement with data obtained for channel catfish. In this species different alloantigendependent cell lines have been cloned [16,54]. One group of allospecific cytotoxic cells expressed TCR genes whereas another group lacked TCR expression. The TCR-positive clones were obtained only from fish sensitized with allogeneic cells, in agreement with the marked CD8 upregulation observed after allogeneic sensitization in rainbow trout. The TCR-negative clones were readily obtained from unsensitized catfish, in agreement with the cytotoxicity observed for IgM2 lymphocytes lacking significant CD8 expression isolated from unsensitized trout. The TCR-negative channel catfish cell clones were obtained by repeated sensitization in vitro with allogeneic targets. Similarly, in trout no CD8 upregulation was obtained by this method and apparently in vitro sensitization alone was not sufficient to significantly stimulate cytotoxic T cells in either trout or catfish. Unfortunately, CD8 expression in the two species cannot be compared because CD8 gene homologues have not yet been detected in channel catfish. A fraction of the mammalian NK population is known to express CD8a homodimers. The function of CD8 in NK cells is not known but indications exist that NK cells bearing CD8a antigens do not use this molecule to trigger cytolysis [55]. In fish, particularly in rainbow trout, additional tools are required to further discriminate NK from T cell function.

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The PCR conditions described in this paper were quite stringent (relatively high annealing temperature, 30 PCR cycles instead of the 36 recommended by the manufacturer) since all three PBL fractions still contained small proportions of contaminating cells from other fractions as shown by FACS analysis. Under less stringent PCR conditions specific products were amplified from PBL fractions for all three genes (data not shown), presumably derived from the contaminating cells. Thrombocytes were always negative with CD8a and TCRa primers even at less stringent PCR conditions but positive with CH1 primers (data not shown); contamination of magnetically enriched cells with B cells is a common problem due to their spontaneous adherence to magnetic separator columns. Variable levels of CMC against allogeneic erythrocytes (Group 1) and RTG-2 cells (Group 2) were observed in our experiments. Although rainbow trout of Groups 1 and 2 were inbred, their genetic background was obviously not homogeneous. Thus, varying genetic differences between the effector cell donor and the target cell donor may have contributed to different CMC levels. Other factors include phenotypic differences between individuals or variation between experiments, since even effector cells from clonal trout of Group 3 (with identical MHC class I) exhibited different cytotoxic activities to the same allogeneic target cells. In ginbuna crucian carp, the cytotoxicity of sensitized leukocytes against third party allogeneic cells was shown to be variable (ranging from negative to moderate), whereas unsensitized leukocytes never killed allogeneic cells [17]. In the experiments described here third party erythrocytes were not used as targets for sensitized effectors, since this would not prove the specificity of the cytotoxic reaction in a system where genetic differences between effector and target cell donors were not characterized. High density neutrophilic granulocytes were excluded from the PBL before immunomagnetic sorting by density gradient separation to prevent a potential source of spontaneous cytotoxicity, as described for this cell population in ginbuna carp [44]. After the first magnetic separation step with PBL of Group 3, small low density neutrophils still remained in the thrombocyte-free PBL (data not shown). These neutrophils were then magnetically enriched in a second separation step

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along with B-cells and monocytes. Since this cell fraction did not exhibit any cytotoxicity it may be concluded that low density neutrophils apparently do not kill allogeneic cells in a spontaneous manner. In contrast to mature mammalian red blood cells, the nucleated erythrocytes of rainbow trout [56], the amphibian Xenopus sp. [57], and chickens [58] express MHC class I molecules. MHC class I molecules are critical in the rejection of mammalian allogeneic cells by TCRþ CD8þ lymphocytes [59] and in cartilaginous fish [60] the rejection of allografts has been shown to be closely linked to the MHC class I genotype. MHC class I molecules have been detected in rainbow trout erythrocytes and also in RTG-2 cells, the second target cell used in this study [56]. Another study with rainbow trout indicated that CMC against virus-infected RTG-2 cells was MHC class I restricted [61]. The role of TCR and CD8 expression by effector cells, however, was not examined in that study. Collectively these results strongly indicate that cytotoxic T cells are important effector cells in CMC against allogeneic targets in rainbow trout, since CMC was executed by PBL subpopulations with the same phenotype and gene expression pattern as cytotoxic T cells of higher vertebrates. The cytotoxicity against allogeneic cells was executed by sIgM2 lymphocytes which expressed TCR and CD8 mRNA. Other PBL subpopulations neither killed allogeneic cells nor expressed TCR or CD8. Re-stimulation of sensitized sIgM2 lymphocytes but not of unsensitized sIgM2 lymphocytes with allogeneic cells resulted in enhanced expression of CD8. Additional studies are needed to clarify the extent to which NK-like cell activity added to the observed CMC. Acknowledgements This work was partly supported by a grant from the Deutsche Forschungsgemeinschaft (FI 604/3-1) and by ‘The Promotion of Basic Research Activities for Innovative Biosciences’ funded by Bio-oriented Technology Research Advancement Institution (BRAIN), Japan. The authors wish to thank Prof. T. Nakanishi and Dr J. Moore for critical reading of the manuscript, Dr G. Warr for supplying us with the anti-IgM mab 1.14, Dr A. Kuroda for the anti-granulocyte/monocyte mab Mm, and Mrs Noack, Mrs Weber and Mrs Schulz for excellent technical assistance.

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