The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhou, H.
Right arrow Articles by Wilson, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhou, H.
Right arrow Articles by Wilson, M.
The Journal of Immunology, 2001, 167: 1325-1332.
Copyright © 2001 by The American Association of Immunologists

Heterogeneity of Channel Catfish CTL with Respect to Target Recognition and Cytotoxic Mechanisms Employed1

He Zhou*, Tor B. Stuge2,*, Norman W. Miller*, Eva Bengten*, John P. Naftel{dagger}, Jayne M. Bernanke{dagger}, V. Gregory Chinchar*, L. William Clem* and Melanie Wilson3,*

Departments of * Microbiology and {dagger} Anatomy, University of Mississippi Medical Center, Jackson, MS 39216


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. "Cold" target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In previous studies, catfish cell lines were cloned from alloantigen-stimulated PBL using catfish allogeneic long-term leukocyte lines, such as the B cell line 3B11, as stimulators. These clones were placed in five different groups based on their allospecificities and TCR{alpha}{beta} gene expression (1). Group I and II clones include cells that selectively express and rearrange TCR V{alpha} and V{beta} genes and can be considered bona fide CTL (1). Group I clones exhibit a narrow specificity, i.e., they specifically proliferate in response to and kill catfish 3B11 cells. In contrast, group II clones do not fit the typical mammalian CTL phenotype because they appear broadly specific in their target cell recognition, i.e., they kill and proliferate in response not only to 3B11, but also to 1G8, a B cell line derived from an unrelated outbred catfish. Group III is represented by a 3B11-specific noncytotoxic TCR{alpha}{beta}+ clone that is presumed to be a Th-like cell line. Group IV clones are proposed to be equivalents to NK cells because they kill targets nonspecifically and are TCR{alpha}{beta} negative. The group V clones are more difficult to define. Although they are TCR{alpha}{beta} negative, they show allospecificity in terms of responsiveness and cytotoxicity (1). Although it is possible that group IV and V clones represent {gamma}{delta} T cells, the failure to identify the TCR {gamma}{delta} genes in catfish leaves their cell type unknown.

Mammalian CTL recognize target cells presenting processed antigenic peptides via their TCR. CTL recognition is peptide specific and MHC restricted (2, 3, 4). Killing is mediated by two major pathways, the secretory perforin-dependent exocytic pathway and the ligand-based Fas ligand (FasL)4/Fas pathway, both of which are initiated by CTL-target adhesion and Ag cross-linking of TCR (5, 6). In the secretory exocytosis pathway, perforin and granzymes are released from granule cores into the extracellular space between the CTL and target cell. This pathway is Ca2+ dependent, and elevated levels of free Ca2+ in the extracellular space induce conformational changes in the perforin molecules, enabling them to insert into target cell membranes and form polyperforin pores (7, 8, 9). Once pores are formed, target lysis can result from increases in plasma membrane permeability and/or the effects of granzymes entering the target cell. Granzymes are serine proteases that proteolytically cleave and activate caspases, which in turn induce target cell apoptosis (10, 11). Alternatively, the relatively Ca2+-independent FasL/Fas cytolytic pathway is mediated by the interaction of membrane molecules; FasL is expressed on CTL membranes following Ag recognition/conjugate formation. The binding of FasL with target cell Fas activates initiator caspases (e.g., procaspase 8) via the adapter protein, Fas-associated death domain. Activated caspase 8 cleaves downstream caspases that in turn cleave key cellular structural and regulatory proteins and result in apoptosis. (12, 13).

It is now clear that fish possess cytotoxic cells (14, 15, 16). Recently, Hogan et al. (17) demonstrated by Ca2+ chelation experiments that catfish PBL NK-like effectors likely use a calcium-dependent perforin-based pathway. However, very little is currently known concerning the killing mechanisms used by fish cytotoxic T cells. To resolve this issue, a panel of catfish CTL clones that offer a unique system not currently available in any other fish species were used for studying the mechanisms of recognition and killing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

Catfish cell lines were grown at 27°C in medium consisting of equal parts AIM-V and L-15 (Life Technologies, Gaithersburg, MD) adjusted to catfish tonicity with 10% (v/v) deionized water and supplemented with 1 mg/ml NaHCO3, 50 U/ml penicillin, 50 µg/ml streptomycin, 20 µg/ml gentamicin, 50 µM 2-ME, and 2% heat-inactivated, pooled, normal catfish serum (18). This serum supplemented medium is designated AL-2 and nonserum supplemented medium is designated AL. The 3B11 and 1G8 cells are cloned autonomous B cells generated from two different outbred catfish by mitogen stimulation (18). 42TA is a macrophage cell line (19), and 28S.3 and G14D are cloned autonomous T cells. 42TA and 28S.3 are from two different outbred fish (20), whereas G14D was generated from an isogenic gynogenetic fish (21). Alloantigen-dependent cell lines TS32.15 (a representative of clone group I) and TS32.17 and TS32.43 (representatives of clone group II) were derived from alloantigen-stimulated cultures initiated with PBL from an outbred catfish (designated catfish 32) immunized in vivo with 3B11 cells. These nonautonomous T cells were restimulated weekly with irradiated 3B11 cells in conditioned medium consisting of AL-2 supplemented with 5% culture supernatant from PMA-calcium ionophore-stimulated PBL or culture supernatant of autonomous T cell lines (1).

LPS stimulation of catfish PBL was performed as previously described (18). Briefly, 107 PBL were cultured in AL-2 medium containing 500 µg/ml LPS (from Salmonella typhimurium; Sigma, St. Louis, MO) in 24-well tissue culture plates (Corning Glass, Cambridge, MA). Cell suspensions were cultured for 72–96 h at 27°C, washed in RPMI 1640, and resuspended in fresh AL-2 medium before being used as targets.

Cytotoxicity assays

Cytotoxicity assays were performed as described by Yoshida et al. (22). Briefly, the indicated numbers of effectors were mixed with 5 x 104 targets labeled with 51Cr (Amersham Pharmacia Biotech, Piscataway, NJ) in 200 µl AL-2 medium in round-bottom 96-well culture plates (Corning Glass). Contact between effectors and targets was induced by centrifugation for 1 min at 200 x g, and plates were incubated at 27°C for the indicated periods of time. At harvest, the cells were resuspended by pipetting and the plates centrifuged a second time for 3 min at 550 x g. One hundred microliters of cell-free supernatant was removed from each well, and the level of released 51Cr present in the supernatant was determined in a COBRA II auto gamma counter (Packard, Meriden, CT). E:T ratios ranging from 1:1 to 0.25:1 were used. Percentage of specific release was calculated using the following formula: % specific release = 100[(experimental cpm - minimum release cpm)/(maximum release cpm - minimum release cpm)].

Maximum-release wells received 100 µl 2% Nonidet P-40 (Sigma) instead of effector cells. Minimum-release wells received 100 µl AL-2 in the place of effector cells. All experiments were done in triplicate.

Experiments designed to measure the effects of potential inhibitors on target cell lysis involved preincubating effector cells with inhibitors such as EGTA, concanamycin A (CMA), brefeldin A (BFA), or PMSF (all purchased from Sigma) at 27°C for 2 h. Target cells, resuspended in medium containing the same concentrations of inhibitors, were then added. The plates were centrifuged as described above and incubated for 4 h at 27°C in the presence of the inhibitors.

Conjugate formation and apoptosis assays

Conjugate formation between effectors and targets was analyzed by two-color flow cytometry. Briefly, 107 effector cells were centrifuged, washed, and resuspended in 500 µl AL medium containing 2 µM 5-chloromethylfluorescein diacetate (CMFDA; green; Molecular Probes, Eugene, OR). The cells were incubated for 30 min at 27°C, washed twice in RPMI 1640, resuspended in 4 ml AL medium, and incubated again at 27°C for 30 min. Cells were then centrifuged and resuspended in 1 ml AL medium. Target cells (107) were treated as described above but were labeled in 500 µl AL medium containing 2 µg/ml 4-(4-(dihexadecylamino)styryl-N-methylquinolinium iodide (DiQ, red; Molecular Probes). To assay for conjugate formation, 100 µl CMFDA-labeled effectors were mixed with 100 µl DiQ-labeled targets and centrifuged for 1 min at 200 x g to induce conjugate formation and then incubated at 27°C for 10 min. The pellets were gently resuspended by "finger vortexing," and an additional 500 µl AL medium was added before two-color analysis on a FACScan flow cytometer (BD Biosciences, Mountain View, CA).

Apoptosis induction in target cells was measured by both FITC-annexin V fluorescence and [3H]thymidine DNA fragmentation assays. An annexin V-FITC apoptosis detection kit (BD PharMingen, San Diego, CA) was used in the flow cytometry assays. Briefly, 106 each effector and target cells were mixed, incubated for 30 min at 27°C, washed in PBS, and resuspended in 100 µl 1x binding buffer (supplied by the manufacturer). Samples were labeled by adding 5 µl FITC-annexin V solution and 10 µl propidium iodide (PI) followed by incubation for 15 min in the dark at room temperature. An additional 400 µl 1x binding buffer was added before analysis with a FACScan flow cytometer.

The [3H]thymidine release assays were performed as previously described (23). Target cells were cultured in AL-2 medium with 5 µCi/ml [methyl-3H]thymidine (Amersham Pharmacia Biotech) at 27°C for ~10 h. The cells were then washed in RPMI 1640 medium and seeded at 5 x 104/well in round-bottom 96-well plates in 100 µl AL-2 medium. Effector cells in 100 µl AL-2 medium were subsequently added at various E:T ratios ranging from 1:1 to 0.25:1. Contact between effectors and targets was induced by centrifugation for 1 min at 200 x g. Plates were incubated at 27°C for various times and harvested using a MicroMate 196 cell harvester. The cpm were determined by water lysis using a Matrix Direct Beta Counter 96 (Packard). All experiments were done in triplicate. Percentage of specific release was calculated using the following formula: % specific release = 100[(control cpm - experimental cpm)/control cpm].

Transmission electron microscopy

Cells were pelleted by centrifugation and prefixed with 3% glutaraldehyde in AL-2 medium at 4°C for 2 h, washed seven times with 0.1 M phosphate buffer (pH 7.2), and postfixed with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) for 2 h at room temperature. The cells were dehydrated in increasing concentrations of acetone, vacuum infiltrated with graded eponate-araldite (Ted Pella, Redding, CA), embedded in eponate-araldite, and polymerized at 60°C for 24 h. Ultrathin sections (~80 nm) were mounted on copper grids and stained with uranyl acetate and lead citrate (Polysciences, Warrington, PA). Sections were examined and photographed using a LEO 906 transmission electron microscope (Zeiss, Oberkochen, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Target recognition by channel catfish CTL clones

To define specificity of catfish clonal CTL, 4-h 51Cr-release assays were performed with representative effectors from groups I and II CTL clones against a panel of allogeneic targets. The allospecific group I (TS32.15) and broadly specific group II (TS32.17 and TS32.43) effectors were used against two cloned B cell targets, 3B11 and 1G8 (18), two cloned T cell targets, 28S.3 (20) and G14D (21), a cloned macrophage target, 42TA (19), and LPS blasts generated from PBL of seven different catfish (Fig. 1Go). In each experiment, TS32.15 effectors only killed 3B11 cells. In fact, 20-h assays at 60:1 E:T ratios did not reveal killing of 1G8, G14D, or 28S.3 targets by TS32.15 effectors (data not shown). In contrast, TS32.17 and TS32.43 killed both 3B11 and 1G8 cells. In addition, group II CTL killed LPS blasts from two isogenic gynogenetic catfish (G6 and G17) as well as some, but not all, outbred catfish. For example, TS32.17 killed LPS blasts from catfish 23, but not from catfish 42 or 57. TS32.43 lysed LPS blasts from catfish 42 and 57, but not from 23 (Fig. 1Go). Neither of the group II effectors lysed LPS blasts derived from catfish 8, 70B, or catfish long-term cell lines 28S.3 and 42TA (data not shown). However, they did lyse the autonomous T cell line G14D (data not shown). This result was expected because G14D was derived from the same gynogenetic catfish family as the G6 and G17 LPS blasts used above, a family shown previously to be MHC matched (21).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1. Cytotoxic responses of representative catfish alloantigen-dependent CTL. TS32.15 ({circ}), TS32.17 ({blacktriangledown}), and TS32.43 ({blacksquare}) were used as effectors in 4-h 51Cr-release assays against various allogeneic targets. SD of the triplicates never exceeded 11%.

 
Because group II effectors clearly killed both 3B11 and 1G8 cells (1), cold target-inhibition assays were performed as an initial step to define target-recognition structures. It was reasoned that if TS32.17 and TS32.43 recognize the different targets using different receptors, an excess of one susceptible target cell type should not inhibit the killing of another susceptible target. For example, group II CTL could use their TCR to recognize a specific target structure on 3B11 and use a second previously undescribed receptor, perhaps similar to those used by mammalian NK cells (24), to kill 1G8. In a representative experiment (Fig. 2Go), group II effectors were used against labeled 3B11 targets in 4-h 51Cr-release assays in the presence or absence of 10-fold excesses of unlabeled 3B11, 1G8, or 28S.3 (as a nonsusceptible target control) cells. Reciprocal assays were performed using 51Cr-labeled 1G8 cells. The 10-fold excesses of unlabeled 3B11 or 1G8 cells inhibited killing by TS32.17 and TS32.43 effectors equally well. As above, strict allospecificity was observed in the control experiments with TS32.15 effectors (i.e., killing of 3B11 targets was only inhibited by excess 3B11). In comparison, excess 28S.3 cells, a cell line resistant to killing, inhibited lysis marginally in both groups I and II, as did 1G8 for group I. These results indicate that groups I and II CTL each likely use a single mechanism for target cell recognition, although group II clones have the ability to recognize different targets. It seems probable that both groups are using their TCR.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 2. Representative cold target inhibition of catfish cytotoxic responses. The 4-h 51Cr-release assays were performed in the presence of 10-fold excess of unlabeled inhibitors: 3B11 ({circ}), 1G8 ({triangledown}), and 28S.3 ({blacktriangledown}). 51Cr-release assays without inhibitors (•) were the controls. SD of the triplicates never exceeded 10%.

 
Mammalian CTL must form conjugates with their targets to deliver a "lethal hit" (25, 26). Because previous studies using flow cytometry (27) and electron microscopy (17) showed that catfish PBL effector cells formed conjugates with allogeneic targets, it was deemed likely that catfish CTL clones also have that ability. Results from conjugation assays for both groups I and II CTL clones are shown in Fig. 3Go. Flow cytometry performed 10 min after mixing detected effector/target conjugates as dual-positive events. Approximately 12% dual-positive events were recorded when TS32.15 cells (group I) were mixed with their specific target, 3B11, indicating conjugate formation. However, only 1.3% dual-positive events were observed when TS32.15 cells were mixed with 1G8 cells (Fig. 3Go, A and B). In contrast, group II clones TS32.17 and TS32.43 formed conjugates with both 3B11 and 1G8 cells with a frequency of dual-positive events ranging from 30–49% (Fig. 3Go, C–F). These results clearly indicate that the inability of group I CTL to lyse 1G8 targets is due to their inability to bind 1G8 target cells.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 3. Conjugate formation between catfish alloantigen-dependent CTL and their targets. Effectors (TS32.15, TS32.17, and TS32.43) were labeled with the dye CMFDA (green), and targets (3B11 and 1G8) were labeled with DiQ (red). Flow cytometry was performed 10 min after the cells were mixed at an E:T ratio of 1:1. A and B, TS32.15 cells mixed with 3B11 and 1G8, respectively. C and D, TS32.17 cells mixed with 3B11 and 1G8, respectively. E and F, TS32.43 cells mixed with 3B11 and 1G8, respectively.

 
Cytolytic mechanisms used by the catfish CTL clones

The importance of both the secretory (perforin/granzyme) and ligand-based (FasL/Fas) pathways in mammalian cell-mediated cytotoxicity is well established. In mammalian CTL, these different mechanisms have been elucidated by selectively blocking particular effector molecules in each pathway (12, 28, 29, 30, 31). For example, the secretory pathway is absolutely Ca2+ dependent, whereas the FasL/Fas pathway is not (7, 8, 12). Recently, it was demonstrated that target cell lysis by catfish PBL-derived NK-like effectors was inhibited by Ca2+ chelation, indicating that killing was most likely mediated by the secretory perforin/granzyme pathway (17). To determine whether extracellular free Ca2+ is important for target cell killing by catfish groups I and II CTL clones, inhibitors that block this pathway in mammals were used in conjunction with cytotoxicity assays. Both group I and group II effectors were incubated with 51Cr-labeled 3B11 cells at a 1:1 E:T ratio in the presence of varying concentrations (0.01–10 mM) of EGTA-Mg2+ and were then assayed for killing. The addition of 1 mM EGTA-Mg2+ almost completely inhibited killing of 3B11 cells by TS32.15 cells, whereas lysis of 3B11 by group II clones (TS32.17 and TS32.43) was only partially inhibited (~40%; Fig. 4GoA). To insure that the concentrations of EGTA-Mg2+ were not directly toxic to catfish effector cells, "Ca2+ pulse" assays were performed. Briefly, catfish effector cells were preincubated in the presence of 2 mM EGTA-Mg2+ for 2 h and then mixed with 51Cr-labeled targets at three different E:T ratios in medium supplemented with either 5 mM CaCl2 or 5 mM MgCl2. In all cases, killing of 3B11 was restored by adding Ca2+ but not by adding Mg2+ (Fig. 4GoB).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of EGTA-Mg2+ on the cytotoxic activity of different catfish alloantigen-dependent CTL. A, TS32.15 ({circ}), TS32.17 ({blacktriangledown}), and TS32.43 ({blacksquare}) cells were preincubated in the presence of various concentrations of EGTA-Mg2+ for 2 h and then incubated with 51Cr-labeled 3B11 at a 1:1 E:T ratio for 4 h, also in the presence of the same concentrations of EGTA-Mg2+. As a control for spontaneous release, 51Cr-labeled 3B11 cells were also incubated with EGTA-Mg2+ ({diamond}). B, Effect of the addition of Ca2+ following pretreatment with EGTA-Mg2+ on cytotoxic activity. Catfish effectors were pretreated for 2 h in the presence of 2 mM EGTA-Mg2+ and then incubated with 51Cr-labeled 3B11 cells at three different E:T ratios for 4 h in medium containing either CaCl2 or MgCl2 (final concentration of 5 mM). ({circ}), Effectors with no added Ca2+ or Mg2+; ({blacktriangledown}), effectors with Mg2+; ({triangledown}), effectors with Ca2+; and (•), effectors not treated with EGTA-Mg2+ or added Ca2+ or Mg2+. Assays performed with effectors not pretreated but with added Ca2+ or Mg2+ showed similar results as effectors not treated with EGTA-Mg2+ or added Ca2+ or Mg2+ (data not shown). SD of the triplicates never exceeded 8%.

 
A second specific inhibitor of the secretory exocytosis pathway in mammals is CMA (28). CMA induces morphological changes in lytic granules and inhibits perforin-based cytotoxicity by increasing pH within the lytic granule, thereby accelerating perforin degradation (29). To determine whether CMA has similar effects on catfish CTL cytotoxicity, both group I and II effectors were preincubated with various concentrations of CMA for 2 h at 27° C. 51Cr-labeled 3B11 targets were then added at a 1:1 E:T ratio and, after 4 h of incubation, the cultures were assayed for lysis. As shown in Fig. 5GoA, 1 nM CMA almost completely inhibited the killing of 3B11 by TS32.15. In contrast, this concentration (and concentrations up to 100 nM CMA) only partially inhibited killing of 3B11 by group II effectors, TS32.17 and TS32.43, suggesting that they may use an additional killing mechanism. To confirm effects of CMA on vacuolar acidification, catfish CTL were examined using fluorescent microscopy. Group I and II effectors and 3B11 targets were each separately preincubated for 2 h at 27°C in the presence or absence of 100 nM CMA. Acridine orange (5 µg/ml) was then added to each cell preparation and, after 30 min, the cells were washed and examined by fluorescence microscopy. In all experiments, fluorescent orange granules were only found in the untreated cells; granules were absent in the CMA-treated cells (data not shown). Subsequently, the morphology of the lytic granules in the catfish CTL after CMA treatment was examined by transmission electron microscopy (Fig. 6Go). Before CMA treatment, both types of catfish CTL showed characteristics of activated lymphocytes (i.e., they had dispersed nuclear heterochomatin and an extended cytoplasm). Likewise, group I and II clones contained numerous granules that were very similar to the three types of lytic granules described in mammalian CTL or NK cells (32). In Fig. 6GoA, type I granules (large arrows) are characterized by electron dense cores with a small cortical rim of multivesicular component. Arrowheads mark a granule characteristic of type II lytic granules; it contains only the multivesicular component with no cores. Small arrows identify potential intermediate granules that contain both components. After CMA treatment, cytoplasmic granules exhibited major changes. Cores appeared empty, and the number of the small peripheral vesicles decreased (Fig. 6GoB). All other cellular organelles appeared to remain normal. The change in appearance of catfish granules mimics the effects of CMA treatment on mammalian CTL (33).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5. Effect of (A) CMA and (B) PMSF on cytotoxic activity of different catfish alloantigen-dependent CTL. TS32.15 ({circ}), TS32.17({blacktriangledown}), and TS32.43({blacksquare}) cells were preincubated with various concentrations of CMA or PMSF for 2 h and then incubated with 51Cr-labeled 3B11 cells at a 1:1 E:T ratio for 4 h in the presence of CMA or PMSF. As a control for spontaneous release, 51Cr-labeled 3B11 cells ({diamond}) were also incubated with CMA or PMSF. SD of the triplicates never exceeded 8%.

 


View larger version (111K):
[in this window]
[in a new window]
  		FIGURE 6. Morphology of cytoplasmic granules in the group I CTL, TS32.15. Cells were prepared as described in Materials and Methods. TS32.15 cells were incubated in (A) the absence or (B) the presence of 100 nM CMA for 2 h. A, Large arrows indicate type I granules; arrowheads, a type II granule; and small arrows, intermediate type granules. B, Arrows indicate granules with major changes.

 
Granzymes are an essential component of the secretory perforin-dependent pathway that induce target cell apoptosis by activating caspases (10, 11). To elucidate the role of granzymes in catfish CTL cytotoxicity, CTL effectors were treated with various concentrations of the serine esterase inhibitor PMSF and monitored for cytotoxic activity. PMSF has been shown to completely inhibit serine esterase activity of murine CTL (34). Fig. 5GoB shows the effects of PMSF treatment on group I and II CTL. The group I effector, TS32.15, was sensitive to PMSF inhibition as shown by an ~80% reduction in killing in the presence of 2 mM PMSF. In contrast, group II clones, TS32.17 and TS32.43, were less sensitive to PMSF, as <20% inhibition was observed at PMSF concentrations that significantly inhibited group I CTL killing.

Because the above cytotoxic studies strongly suggest that group I clones use solely the secretory pathway and group II clones use this and an additional pathway, the effect(s) of an inhibitor of the FasL/Fas pathway, BFA, was examined. In mice, BFA has been shown to completely block Fas-based cytotoxicity in the CD4+ CTL clone BK-1 and to only marginally decrease perforin/granzyme-based cytotoxicity in the CD8+ CTL clone OE4 (28). BFA also completely inhibits the cytotoxicity of perforin-deficient CTL on Fas-bearing targets (35). Moreover, a combination of CMA and BFA completely blocked all cytolytic activity of mouse alloantigen-specific CTL (28). Consequently, members of the two different catfish CTL groups were treated with BFA alone or with BFA and CMA. Fig. 7Go shows the results of BFA treatment on the group I and II CTL clones. In all cases, only partial inhibition could be observed at concentrations of 1–10 µM BFA. Moreover, addition of 100 nM CMA to 10 mM BFA failed to completely inhibit the cytotoxicity of the group II clones, TS32.17 and TS32.43 (Fig. 7GoB).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 7. A, Effect of brefeldin A on cytotoxic activity. TS32.15 ({circ}), TS32.17 ({blacktriangledown}), and TS32.43 ({blacksquare}) cells were preincubated with various concentrations of BFA for 2 h and then incubated with 51Cr-labeled 3B11 targets at a 1:1 E T ratio for 4 h also in the presence of BFA. B, Effect of combined CMA and brefeldin A treatment. Catfish effectors were preincubated with either 100 nM CMA (), 10 µM BFA (), a combination of both (), or with medium alone ({square}) for 2 h and then incubated with 51Cr-labeled 3B11 targets as described above. SD of the triplicates never exceeded 8%.

 
Previously, Hogan et al. (17) showed that catfish PBL NK-like effectors killed allogeneic targets by an apoptotic mechanism. Therefore, studies involving two different assays were undertaken to determine whether catfish CTL also kill via apoptosis. An early marker of apoptosis is the appearance of phosphatidylserine on the outer surface of the plasma membrane (36, 37). Because annexin V binds the negatively charged phosphatidylserine residues on the membrane with high affinity (38, 39), annexin V binding is considered to be a sensitive indicator of apoptotic membrane changes. Fig. 8GoA shows a representative binding experiment using group I (TS32.15) CTL and targets. When the TS32.15 cells were incubated with their specific target, 3B11, 36% of the cells were positive with FITC-annexin V, whereas only 4% were double-positive with both annexin V and the viability control, PI. In contrast, when TS32.15 cells were incubated with 1G8, very few cells (4%) stained with FITC-annexin V. Similar or higher levels of annexin V-positive staining were also observed when 3B11 or 1G8 were incubated with the group II clones, TS32.17 and TS32.43 (data not shown). Again, only a very low percentage of cells, <5%, stained with PI, indicating that the plasma membranes of the targets were intact. The second apoptotic assay was [3H]thymidine release, which detects cleaved DNA in target cells (23). In these experiments, [3H]thymidine-labeled targets were incubated at various E:T ratios with group I and II effectors, and specific release was measured. Each of the catfish CTL clones exhibited high levels of cytolytic activity toward 3B11 cells (Fig. 8GoB) as indicated by [3H]thymidine release >91%. In addition, time-course studies showed that DNA fragmentation occurred quickly (Fig. 8GoC); i.e., ~30% of the [3H]thymidine release occurred within 15 min, and release levels plateaued at 2 h.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 8. Apoptosis assays. A, Flow cytometry. The group I (TS32.15) CTL and 3B11 or 1G8 cells were mixed at a 1:1 E:T ratio, incubated for 1 h at 27°C, and stained with FITC-annexin V and PI according to the manufacturer’s protocol. Approximately 106 cells were analyzed by FACScan. Similar results (data not shown) were also observed at incubation times of 30 min and 4 h. B, [3H]Thymidine DNA fragmentation assays. The 3B11 targets were labeled overnight with [3H]thymidine, washed, and incubated with TS32.15 ({circ}), TS32.17 ({blacktriangledown}), and TS32.43 ({blacksquare}) at the indicated E:T ratios. C, Kinetics of [3H]thymidine release. The 3B11 targets were labeled as described in Materials and Methods and incubated with TS32.15 cells at an E:T ratio of 0.5:1 for various times. Specific [3H]thymidine release for this experiment was: 15 min, 28%; 30 min, 42%; 1 h, 60%; 2 h, 76%; 4 h, 88%; and overnight (time point not shown), 97%. SD of the triplicates never exceeded 40%.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Catfish group I and II clones are the first bona fide CTL described in ectothermic vertebrates, and the experiments conducted here represent the initial assessment as to how they recognize and kill their targets. The two groups of catfish CTL differ in their cytotoxic profiles. The group I representative, TS32.15, only killed 3B11 targets. In contrast, TS32.17 and TS32.43, the group II CTL representatives, killed not only 3B11 and 1G8, but also LPS blasts from different catfish. Furthermore, group II CTL showed differences in killing when compared with each other. This broader specificity of recognition makes TS32.17 and TS32.43 difficult to classify. Although they express unique TCR{alpha} and {beta} rearrangements (1) and form conjugates with their targets (Fig. 3Go), they do not match the phenotype and function of known mammalian CTL. Previously, it was speculated that these group II clones may represent catfish NK T cells. It was also thought that they possibly use two different receptors for target cell recognition. For example, it was speculated that group II CTL recognized 3B11 cells via their TCR because they were generated from alloantigen-stimulated cultures initiated with PBL from a catfish immunized with 3B11 and restimulated in vitro with irradiated 3B11 (1). It is also possible that they recognize 1G8 cells via a different mechanism similar to the mechanism used by mammalian NK cells (24). However, the results of cold target-inhibition assays demonstrated that excess unlabeled 3B11 or 1G8 cells inhibit the killing of either 3B11 or 1G8 by group II CTL, implying that identical recognition mechanisms are used. Although these findings do not formally prove that groups I and II CTL use TCR molecules to recognize targets cells, it seems likely. It is possible that each of the three clones could recognize a different alloantigen on the different target cells, with TS32.17 and TS32.43 recognizing common alloantigens, presumably MHC Ags that are shared on each of the different target cell types. TS32.15, TS32.17, and TS32.43 each express different members of the TCR V{alpha}1a family, and all three express V{beta}s from different families (1). Likewise, each of the allogeneic targets express both class I and II MHC genes (Refs. 40, 41 and unpublished data). Definitive proof awaits the complete sequencing of the different MHC loci in catfish and/or the determination of expressed MHC on 3B11 and 1G8 targets and the development of immune reagents such as Abs against catfish TCR and alleles of MHC molecules.

TS32.15 cells form conjugates with 3B11 targets but not with 1G8, indicating that failure to lyse 1G8 is due to their inability to recognize and bind 1G8 targets. Group II CTL, TS32.17 and TS32.43, formed conjugates with both 3B11 and 1G8. It was also observed that higher percentages of group II CTL formed conjugates with 3B11 than did group I. This finding suggests that group II CTL may express more adhesion molecules on their surface, a situation that may contribute to their broad specificity.

In the present study, both groups of catfish CTL induce apoptosis in their targets (Fig. 8Go). Electron microscopy showed that both types of catfish CTL have granules. These granules exhibit characteristics of mammalian lytic granules and can even be classified into the same three types as in mammals (32). It should also be noted that similar granules have been observed in catfish NK-like cells, but not among catfish B cell lines or noncytotoxic T cell lines (Refs. 1, 18, 20 and our unpublished data). These findings indicate that these granules might be catfish lytic granules and suggest a role for the secretory perforin/granzyme pathway. Cytotoxic mechanisms were then examined using selective inhibitors of the two major mammalian cytolytic pathways. EGTA and CMA were chosen as inhibitors of perforin, and as shown in Figs. 4Go and 5GoA, each reagent inhibited killing by group I CTL, with concentrations >1 mM EGTA-Mg2+ or 1 nM CMA causing complete loss of cytotoxic activity. This implies that perforin plays a dominant role in group I CTL killing responses. In contrast, both compounds, even at higher doses, only partially inhibited target killing by group II CTL. These findings suggest that group II CTL likely use a second cytotoxic mechanism in addition to perforin/granzyme. CMA was also shown to induce morphological changes in the putative catfish lytic granules. After treatment, these granules appeared empty, demonstrating that CMA affects catfish CTL in a fashion similar to that of mammalian CTL (Fig. 6GoB; 33).

The potential contribution of granzymes to catfish CTL cytotoxic activity was examined by preincubating group I and II effectors with various concentrations of PMSF before adding targets. As shown in Fig. 5GoB, group I CTL killing was very sensitive to PMSF, whereas killing by group II effectors was not. We also attempted to measure granzyme activity of the catfish CTL spectrophotometrically by N-{alpha}-benzyloxycarbonyl-L-lysine thiobenzyl assays (42). Unforturnately, measurement of esterase release by the CTL effectors in the presence of targets was difficult to interpret because esterase activity could also be detected in lysates of target cells (data not shown). However, it should be noted that the results with PMSF are consistent with the EGTA- and CMA-inhibition studies, which strongly suggests that group II effectors use a second cytotoxic mechanism in addition to that involving perforin/granzyme.

A second cytolytic pathway found in mammals, the FasL/Fas pathway, has thus far proven difficult to study in catfish. The only inhibitor of the FasL/Fas pathway that was used with some success in the catfish system was BFA. Other approaches (e.g., the use of RNA synthesis inhibitors such as actinomycin D, protein synthesis inhibitors such as cycloheximide and emetine, as well as paraformaldehyde fixation) either gave ambiguous results or were toxic to catfish cells (our unpublished data). BFA, an inhibitor of intracellular glycoprotein transport, has been shown to be a selective inhibitor for Fas-based cytotoxicity in mammals (28, 35), presumably by preventing transport of FasL to the cell membrane (43, 44). BFA partially inhibited killing by both catfish group I and II CTL (Fig. 7GoA); killing was reduced by ~20–40% in each case. In another experiment, the combination of CMA and BFA further reduced target cell killing by group II CTL but did not completely abolish it (Fig. 7GoB). It may be that, in the catfish system, BFA is unable to block the transport of FasL, or alternatively, it is possible that catfish CTL already express surface FasL, and blocking the transport of newly synthesized FasL is insufficient to inhibit FasL-mediated killing. The answer to this issue can only be achieved with Abs reactive with catfish surface FasL. Despite the absence of direct molecular evidence for catfish Fas and FasL, several lines of evidence suggest the presence of a Fas/FasL pathway in fish: 1) a Fas homologue has been detected in a zebrafish EST library (GenBank accession number BE201956); 2) a homologue of Fas-associated death domain has been identified in catfish (unpublished observation); and 3) recently, a Fas homologue has been identified in a catfish EST library (M. Wilson, unpublished observation).

In addition to the above two lytic mechanisms, TNF may also contribute to cytotoxicity. In mammalian systems, TNF-mediated cytotoxicity does not require activation via the TCR and is monitored in 20-h assays at high E:T ratios (45). Based on this, TNF is probably not involved in the short-term (4-h) lysis of susceptible target cells by group II CTL unless fish cytotoxic cells are quite different from those in mammals. Currently, it is not possible to address the role of TNF in catfish cytotoxicity because of lack of catfish TNF-responsive cell lines and Abs to catfish TNF. Moreover, the failure to observe cytotoxicity of targets other than 3B11 by TS32.15 cells following 20-h assays suggests that the TNF mechanism may not contribute to killing by catfish group I effectors.

Overall, the above results provide the strongest evidence to date that channel catfish, and presumably other teleosts, possess CTL with the ability to recognize different targets. Group I clones exhibit strict allospecificity and appear to kill predominantly by a perforin-dependent secretory pathway. Thus, they closely resemble mammalian CD8+ CTL. Recently, CD8 {alpha} and {beta} cDNAs have identified in rainbow trout (46), and it is hoped that similar molecular approaches will identify catfish CD8 homologues. The identification of catfish CD8 and CD4 homologues should facilitate the classification of catfish CTL. At present, it is impossible to definitively classify the broadly specific group II CTL clones. Nevertheless, the ability to clone various types of catfish cytotoxic cells is an important advancement in understanding cell-mediated responses in ectothermic vertebrates. These results clearly demonstrate that catfish CTL clones kill through bona fide cytotoxic mechanisms, indicating that the perforin/granzyme pathway is evolutionarily conserved. Herein, for the first time in any fish species, the existence of two kinds of catfish CTL and two cytotoxic pathways was demonstrated. In addition, lytic granules were identified in ectothermic vertebrate cytotoxic cells. In the future, these channel catfish clonal cell lines, in conjugation with the development of specific probes and Abs, should facilitate functional studies of fish CTL and NK cells in ways not possible in any other fish species.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI-19530 and U.S. Department of Agriculture Grant 99-35204-7844. Back

2 Current address: Division of Hematology, Stanford University School of Medicine, Stanford, CA 94305. Back

3 Address correspondence and reprint requests to Dr. Melanie Wilson, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505. E-mail address: mwilson{at}microbio.umsmed.edu Back

4 Abbreviations used in this paper: FasL, Fas ligand; CMA, concanamycin A; BFA, brefeldin A; CMFDA, 5-chloromethylfluorescein diacetate; PI, propidium iodide; DiQ, 4-(4-(dihexadecylamino)styryl)-N-methylquinolinium iodide. Back

Received for publication February 23, 2001. Accepted for publication May 29, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stuge, T., M. Wilson, H. Zhou, K. Barker, E. Bengten, G. Chinchar, N. Miller, L. W. Clem. 2000. Development and analysis of various clonal alloantigen-dependent cytotoxic cell lines from channel catfish. J. Immunol. 164:2971.[Abstract/Free Full Text]
  2. Hedrick, S. M.. 1988. Specificity of the T cell receptor for antigen. Adv. Immunol. 43:193.[Medline]
  3. Marrack, P., C. Hannum, M. Harris, K. Haskins, R. Kubo, M. Pigeon, R. Shimonkevitz, J. White, J. Kappler. 1983. Antigen-specific, major histocompatibility complex-restricted T cell receptors. Immunol. Rev. 76:131.[Medline]
  4. Swain, S. L.. 1983. T cell subsets and the recognition of MHC class. Immunol. Rev. 74:129.[Medline]
  5. Nagata, S., P. Golstein. 1995. The Fas death factor. Science 267:1449.[Abstract/Free Full Text]
  6. Henkart, P. A.. 1994. Lymphocyte-mediated cytotoxicity: two pathways and multiple effector molecules. Immunity 1:343.[Medline]
  7. Henkart, P. A.. 1985. Mechanism of lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 3:31.[Medline]
  8. Podack, E. R., J. D. Young, Z. A. Cohn. 1985. Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules. Proc. Natl. Acad. Sci. USA 82:8629.[Abstract/Free Full Text]
  9. Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31.[Medline]
  10. Heusel, J. W., R. L. Wesselschmidt, S. Shesta, J. H. Russell, T. J. Ley. 1994. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 76:977.[Medline]
  11. Darmon, A. J., T. J. Ley, D. W. Nicholson, R. C. Bleackley. 1996. Cleavage of CPP32 by granzyme B represents a critical role for ganzyme B in the induction of target cell DNA fragmentation. J. Biol. Chem. 271:21709.[Abstract/Free Full Text]
  12. Rouvier, E., M-F. Luciani, P. golstein. 1993. Fas involvement in Ca2+-independent T cell-mediated cytotoxicity. J. Exp. Med. 177:195.[Abstract/Free Full Text]
  13. Boldin, M. P., T. M. Goncharov, Y. V. Gotsev, D. Wallach. 1996. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85:803.[Medline]
  14. Miller, N. W., M. R. Wilson, E. Bengten, T. B. Stuge, G. W. Warr, L. W. Clem. 1998. Functional and molecular characterization of teleost leukocytes. Immunol. Rev. 166:187.[Medline]
  15. Evans, D. L., L. Jaso-Friedmann. 1992. Nonspecific cytotoxic cells as effectors of immunity in fish. Annu. Rev. Fish Dis. 2:109.
  16. Fischer, U., M. Otatake, T. Nakanishi. 1998. In vitro cell-mediated cytotoxicity against allogeneic erythocytes in ginbuna crucian carp and goldfish using a non-radioactive assay. Dev. Comp. Immunol. 22:195.[Medline]
  17. Hogan, R., W. Taylor, M. Cuchens, J. Naftel, L. W. Clem, N. W. Miller, V. G. Chinchar. 1999. Induction of target cell apoptosis by channel catfish cytotoxic cells. Cell. Immunol. 195:110.[Medline]
  18. Miller, N. W., M. A. Rycyzyn, M. R. Wilson, G. W. Warr, J. P. Naftel, L. W. Clem. 1994. Development and characterization of channel catfish long-term B cell lines. J. Immunol. 152:2180.[Abstract]
  19. Miller, N. W., V. G. Chinchar, L. W. Clem. 1994. Development of leukocyte lines from the channel catfish (Ictalurus punctatus). J. Tissue Cult. Methods 16:117.
  20. Wilson, M. R., H. Zhou, E. Bengten, L. W. Clem, T. B. Stuge, G. W. Warr, N. W. Miller. 1998. T-cell receptors in channel catfish: structure and expression of TCR {alpha} and {beta} genes. Mol. Immunol. 35:545.[Medline]
  21. Hogan, R., G. Waldbieser, C. Goudie, A. Antao, U. Godwin, M. Wilson, N. W. Miller, L. W. Clem, T. McConnell, W. Wolters, V. G. Chinchar. 1999. Molecular and immunological characterization of gynogenetic channel catfish. Mol. Mar. Biol. Biotechnol. 1:317.
  22. Yoshida, S. H., T. B. Stuge, N. W. Miller, L. W. Clem. 1995. Phylogeny of lymphocyte heterogeneity: cytotoxic activity of channel catfish peripheral blood leukocytes directed against allogeneic targets. Dev. Comp. Immunol. 19:71.[Medline]
  23. Matzinger, P.. 1991. The JAM test: a simple assay for DNA fragmentation and cell death. J. Immunol. Methods 145:185.[Medline]
  24. Moretta, A., R. Biassoni, C. Bottino, D. Pende, M. Vitale, A. Poggi, M. C. Mingari, L. Moretta. 1997. Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes. Immunol. Rev. 155:105.[Medline]
  25. Perez, P., J. A. Bluestone, D. A. Stephany, D. M. Segal. 1985. Quantitative measurements of the specificity and kinetics of conjugate formation between cloned cytotoxic T-lymphocytes and splenic target cells by dual parameter flow cytometry. J. Immunol. 134:478.[Abstract]
  26. Martz, E.. 1980. Immune T lymphocyte to tumor cell adhesion: magnesium sufficient, calcium insufficient. J. Cell Biol. 84:584.[Abstract/Free Full Text]
  27. Stuge, T. B., S. H. Yoshida, V. G. Chinchar, N. W. Miller, L. W. Clem. 1997. Cytotoxic activity generated from channel catfish peripheral blood leukocytes in mixed leukocyte cultures. Cell. Immunol. 177:154.[Medline]
  28. Kataoka, T., N. Shinohara, H. Takayama, K. Takaku, S. Kondo, S. Yonehara, K. Nagai. 1996. Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J. Immunol. 156:3678.[Abstract]
  29. Kataoka, T., K. Togashi, H. Takayama, K. Takaku, K. Nagai. 1997. Inactivation and proteolytic degradation of perforin within lytic granules upon neutralization of acidic pH. Immunology 91:493.[Medline]
  30. Stenger, S., R. J. Mazzaccaro, K. Uyemura, S. Cho, P. F. Barnes, J. P. Rosat, A. Sette, M. B. Brenner, S. A. Porcelli, B. R. Bloom, R. L. Modlin. 1997. Differential effects of cytolytic T cell subsets on intracellular infection. Science 276:1684.[Abstract/Free Full Text]
  31. Lowin, B., C. Mattman, M. Hahne, J. Tschopp. 1996. Comparison of Fas(Apo-1/CD95)- and perforin-mediated cytotoxicity in primary T lymphocytes. Int. Immunol. 8:57.[Abstract/Free Full Text]
  32. Burkhardt, J. K., S. Hester, C. K. Lapham, Y. Argon. 1990. The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments. J. Cell Biol. 111:2327.[Abstract/Free Full Text]
  33. Kataoka, T., K. Takaku, J. Magae, N. Shinohara, H. Takayama, S. Kondo, K. Nagai. 1994. Acidification is essential for maintaining the structure and function of lytic granules of CTL: effect of concanamycin A, an inhibitor of vacuolar type H+-ATPase, on CTL-mediated cytotoxicity. J. Immunol. 153:3938.[Abstract]
  34. Pasternack, M. S., H. N. Eisen. 1985. A novel serine esterase expressed by cytotoxic T lymphocytes. Nature 314:743.[Medline]
  35. Simon, M. M., M. Waring, A. Lobigs, T. Nil, R. T. Thao, S. Chin Hla, A. Mullbacher. 2000. Cytotoxic T cells specifically induce Fas on target cells, thereby facilitating exocytosis-independent induction of apoptosis. J. Immunol. 165:3663.[Abstract/Free Full Text]
  36. Zwaal, R. F., P. Comfurius, E. M. Bevers. 1993. Mechanism and function of changes in membrane-phospholipid asymmetry in platelets and erythocytes. Biochem. Soc. Trans. 21:248.[Medline]
  37. Martin, S. J., C. P. Reutelingsperger, A. J. McGahon, J. A. Rader, R. C. Van Schie, D. M. LaFace, D. R. Green. 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Ab1. J. Exp. Med. 182:1545.[Abstract/Free Full Text]
  38. Tait, J. F., D. Gibson. 1994. Measurement of membrane phospholipid asymmetry in normal and sickle-cell erythocytes by means of annexin V binding. J. Lab. Clin. Med. 123:741.[Medline]
  39. Raynal, P., H. B. Pollard. 1994. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium and phospholipid-binding proteins. Biochim. Biophys. Acta. 1197:63.[Medline]
  40. Antao, A. B., V. G. Chinchar, T. J. McConnell, N. W. Miller, L. W. Clem, M. R. Wilson. 1999. MHC class I genes of the channel catfish: sequence analysis and expression. Immunogenetics 49:303.[Medline]
  41. Godwin, U. B., M. Flores, S. Quiniou, M. R. Wilson, N. W. Miller, L. W. Clem, T. J. McConnell. 2000. MHC class II A genes in the channel catfish (Ictalurus punctatus). Dev. Comp. Immunol. 24:609.[Medline]
  42. Green, G. D., E. Shaw. 1979. Thiobenzyl benzyloxycarbonyl-L-lysinate, substrate for a sensitive colorimetric assay for trypsin-like enzymes. Anal. Biochem. 93:223.[Medline]
  43. Klausner, R. D., J. G. Donaldson, J. Lippincott-Schwartz. 1992. Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Biol. 116:1071.[Free Full Text]
  44. Pelham, H. R.. 1991. Multiple targets for brefeldin A. Cell 67:449.[Medline]
  45. Ratner, A., W. R. Clark. 1993. Role of TNF-{alpha} in CD8+ cytotoxic T lymphocyte-mediated lysis. J. Immunol. 150:4303.[Abstract]
  46. Hansen, J. D., P. Strassburger. 2000. Description of an ectothermic TCR coreceptor, CD8{alpha}, in rainbow trout. J. Immunol. 164:3132.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Virol.Home page
H. D. Morales and J. Robert
Characterization of Primary and Memory CD8 T-Cell Responses against Ranavirus (FV3) in Xenopus laevis
J. Virol., March 1, 2007; 81(5): 2240 - 2248.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Zhou, E. Bengten, N. W. Miller, L. W. Clem, and M. Wilson
The T Cell Receptor {beta} Locus of the Channel Catfish, Ictalurus punctatus, Reveals Unique Features
J. Immunol., March 1, 2003; 170(5): 2573 - 2581.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhou, H.
Right arrow Articles by Wilson, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhou, H.
Right arrow Articles by Wilson, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS