| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ß genes by RT-PCR and for target cell specificity by
51Cr-release assay. Based on the above criteria, the
following five different cell types were identified among the 19 clones
analyzed: 1) TCR ß+ allospecific cytotoxic cells, 2)
TCR ß+ nonspecific cytotoxic cells, 3) allospecific
TCR ß+ noncytotoxic cells, 4) TCR ß-
nonspecific cytotoxic cells, and 5) TCR ß-
allospecific cytotoxic cells. The demonstration of cloned, TCR
ß+, allospecific cytotoxic effectors provides the
strongest evidence to date for the existence of cytotoxic T cells in
fish. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous work showed that several fish species possess nonspecific
cytotoxic cells (NCC)4
that lyse xenogeneic targets (reviewed in Ref. 2).
However, these effectors, suggested to be homologues of the NK cells of
"higher" animals, have not been well characterized at the molecular
level. In the best characterized of these systems, Evans and his
coworkers (2) identified a 34-kDa cell-surface receptor
protein, termed NCCRP-1, on channel catfish NCC that is thought to bind
target cells. In addition, a previously generated mAb, 5C6, was shown
to bind NCCRP-1 and to provided a facile way to identify NCC. However,
mAb 5C6, which reacts with head kidney-derived catfish NCC, fails to
react with catfish cytotoxic effectors derived from peripheral blood
leukocytes (PBL), suggesting that NCC and PBL-derived cytotoxic
effectors represent distinct populations (3). In our
laboratory, several recent developments within the channel catfish
model have greatly facilitated progress in characterizing PBL-derived
cytotoxic cells. First, the development of clonal long-term autonomous
(i.e., able to proliferate continuously in culture without the need for
restimulation or exogenous growth factors) lines of B cells, T cells,
and macrophages (4, 5, 6, 7) makes available well-defined
homogeneous populations of allotargets, which express both MHC class I
and II genes (8, 9). Second, the finding of greatly
enhanced cytotoxicity after one-way mixed leukocyte culture of catfish
PBL with x-irradiated allotargets provides a useful in vitro system for
generating large numbers of teleost cytotoxic cells (10).
Finally, as reported in this paper, it is now possible to clone mixed
leukocyte culture (MLC)-derived effectors and determine their phenotype
by monitoring their ability to express the TCR and ß genes and to
recognize and lyse specific allogeneic targets. As detailed below, the
findings in the current study demonstrate that catfish possess at least
four different types of cytotoxic cells that can be quantitatively
expanded in vitro by MLC. In addition, a fifth population of
noncytotoxic, allospecific TCR ß+ cells that
may correspond to mammalian Th cells was also isolated.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Channel catfish (Ictalurus punctatus; 1–2 kg) were obtained and maintained in individual tanks as described previously (11). Blood was drawn from the caudal vein of anesthetized (tricane methansulfonate) fish into heparinized vacutainers, and PBL were isolated by centrifugation over Lymphoprep (Accurate Chemicals, Westbury, NY) as described previously (6).
Cell lines and mAbs
1G8 and 3B11 cells are cloned autonomous B cells generated from
two different catfish by mitogen stimulation (5). 28S.1
(12) and 75C cells represent cloned and uncloned
autonomous T cells, respectively; both express TCR - and ß-chains.
Catfish cells were grown at 27°C in AL-5 medium, which consists of
equal parts AIM-V and L-15 media (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, 5 x
10-5 M 2-ME, and 5% heat-inactivated catfish
serum (6).
Alloantigen immunization
Channel catfish #32 was hyperimmunized i.p. with 3B11 cells, a clonal allogeneic B cell line (2–4 x 107 cells in 2 ml PBS). The fish was boosted after 14 days and then weekly for an additional 6 wk. Two weeks after the last boost, 10–15 ml blood was collected using Vacutainer blood collection tubes (Becton Dickinson, Franklin Lakes, NJ). The PBL were used to develop the alloreactive lymphocyte clones described below.
MLC-derived catfish lymphoid cell lines
MLC-generated cytotoxic effector cells were obtained as described previously (10). Briefly, 5 x 106 catfish PBL from a 3B11-immunized fish (fish #32) or nonimmunized fish (fish #10 and #75) were incubated with 2 x 106 irradiated 3B11 cells in 1 ml AL-5 medium/well using 24-well tissue culture plates (Corning Glass, Corning, NY) and incubated at 27°C in a humidified atmosphere containing 5% CO2/95% air. The cultures were passaged when cell density increased and culture medium became acidified. Typically, 1 ml AL-5 medium/well was added after day 5 of culture, and on day 6 the pooled contents of two wells were transferred to a 25-cm2 tissue culture flask (Corning) with an additional 4 ml of AL-5 medium. On day 8, 8 ml AL-5 medium was added, and on day 10 the contents (16 ml) of the 25-cm2 flask were transferred to a 75-cm2 T-flask (Corning) along with an additional 16 ml of medium. The cells were harvested on or after day 12 and cloned by limiting dilution (see below) or continuously cultured by weekly restimulation with irradiated 3B11 cells. In the later case, 106 MLC cells were cultured with 2 x 106 irradiated 3B11 cells in 1 ml complete medium containing 10% conditioned medium from 75C T cells (as a presumed source of growth factors). After 2 days, an additional 1 ml of 10% conditioned medium was added to the wells, and after 4 days, the pooled contents of two wells were transferred to a 25-cm2 flask together with 4 ml of 10% conditioned medium.
Cloning and propagation of MLC cells
Cloning of alloantigen-responsive cytotoxic cells was performed by limiting dilution in 10% conditioned medium using freshly isolated PBL- or MLC-activated leukocytes from both 3B11 immune and nonimmune fish. Briefly, 50-µl suspensions of PBL (20 cells/ml) or MLC-activated cells (6 cells/ml) were transferred to wells in 96-well round-bottom tissue culture plates together with 50 µl irradiated (4000–5000 rad) catfish allogeneic B cells (3B11) suspended at 2 x 105 cells/ml in conditioned medium. The cells were incubated at 27°C in 5% CO2/95% air. After 8–15 days in culture, proliferating responder cells were restimulated. Briefly, half of the culture supernatant in each well containing proliferating cells was removed and the cells were resuspended and completely transferred to a flat-bottom 96-well plate. Irradiated 3B11 cells (3 x 106 cells/ml) were suspended in 10% conditioned medium, and 100-µl aliquots were transferred to each well containing responder cells. The clones were cultured for 6 days and then transferred to the wells of 24-well tissue culture plates together with 2 x 106 irradiated 3B11 cells in 1.5 ml 10% conditioned medium and cultured 6 more days. From this point on, the cloned cells were passaged in wells of 24-well tissue culture plates at 6-day intervals by transferring 5 x 105 responder cells and 2 x 106 irradiated 3B11 cells in a final volume of 1 ml 10% conditioned medium per well. Optimal expansion of cloned cells during a 6-day culture period was achieved by adding 1 ml 10% conditioned medium to each well after 4 days. Alternatively, the pooled contents of three wells were transferred to 25-cm2 tissue culture flasks together with 3 ml 10% conditioned medium on day 4 and cultured in flasks for 2 more days. The specificity of alloantigen stimulation was assayed by culturing 105 cells from the various clonal MLC-derived lymphocyte lines with 2 x 105 irradiated 3B11 cells (as specific Ag) or 1G8 cells (as nonspecific Ag) in 200 µl of AL-5 medium with or without 10% conditioned medium in 96-well round-bottom tissue culture plates. Triplicate cultures were incubated at 27°C in a humidified atmosphere containing 5% CO2 and 95% air for 4 days. The cultures were pulsed with 0.5 µCi [methyl-3H]-thymidine (Amersham, Arlington Heights, IL) 18 h before harvesting by use of an automated cell harvester (Micromate 196 harvester, Packard, Meriden, CT). Incorporation of radionucleotide was measured using a direct beta-counter (Matrix 96, Packard).
Cytotoxic assays
51Cr-release assays were performed as previously described (13). Briefly, effector cells in 100 µl AL medium were mixed with 5 x 104 51Cr-labeled target cells in 100 µl AL medium in round-bottom 96-well tissue culture plates (Corning). The plates were centrifuged for 1 min at 200 x g to induce contact between effector and target cells and were incubated for 4 h at 27°C. The cells were resuspended by pipetting, and the plates were centrifuged again for 3 min at 550 x g. One hundred microliters of cell-free supernatant was removed from each well and cpm were determined in a COBRA II auto gamma-counter (Packard). E:T ratios were as indicated in the text and figure legends. All experiments were done in triplicate. Percent specific release was calculated using the following formula: % specific release = {100 x [cpm (experimental) - cpm (minimum release)] / [cpm (maximum release) - cpm (minimum release)]}. Maximum release wells received 100 µl 2% Nonidet P-40 (Sigma, St. Louis, MO) instead of effector cells to lyse all target cells. Minimum release wells received 100 µl AL instead of effector cells.
RNA isolation, RT-PCR, 5'-rapid amplification of cDNA ends (RACE), and sequence analysis
Total RNA was prepared from the cell lines by the method of
Chomczynski and Sacchi (14). To detect expression of
channel catfish Ig, TCR, and actin, 
1 µg of total RNA was
converted into first-strand cDNA using 50 ng of an oligo(dT) primer
with 200 U of reverse transcriptase (SuperScript II; Life Technologies)
according to the manufacturer’s recommended protocol. One percent of
the first-strand reaction was used as template in RT-PCR. The reaction
mixture (100 µl) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM
MgCl2, 150 mM dNTP, 40 mM tetramethylammonium
chloride, and 0.5 µg of specific forward and reverse primers. Two
units of Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ)
were added, and 30 cycles of amplification (94°C for 1 min, 65°C
for 2 min, 72°C for 3 min) were performed. Primer pairs for Ig
(15), TCR (12), and actin
(16) have been described. The TCR V regions of the
different cell lines were obtained either by RACE (17)
using the 5'-RACE System version 2.0 (Life Technologies) or by RT-PCR
using specific oligo primers. Primers for the first-strand RACE cDNA
synthesis were 5'-GCAGGCAAATGAAAGTAGAATT-3' for (designated G-902)
and 5'-AAAACCCTGTCTCCTAACGATGTA-3' for ß (designated G-997). The
specific reverse and constant region primers for PCR amplification with
the 5'-RACE abridged anchor primer were 5'-GTTTGTAAATTGACGGCT-3' for
(designated G-1085) and 5'-CACTTCCTTCTCTGAAACA-3' for ß
(designated G-1013). Reaction conditions were as recommended by the
manufacturer. The TCR -chains of clones TS.32.1, TS.32.5, TS.32.17,
and TS.32.34 were amplified using a TCR leader region
forward primer (5'-ATGGCTTCAATAGGTGAC-3') and reverse G-1085. The
5'-RACE products and RT-PCR fragments were cloned into pCR2.1
(Invitrogen, Carlsbad, CA) and sequenced using the dideoxynucleoside
triphosphate chain-termination method (18). Nucleotide and
amino acid sequences were initially aligned using the Megalign program
(DNAstar, Madison, WI) by the Clustal V method with the PAM250 residue
weight table and then were adjusted by visual inspection to maximize
homology.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous studies demonstrated that high levels of nonspecific
cytotoxic activity directed against allogeneic target cells were
generated in one-way MLC using catfish PBL as responders and irradiated
allogeneic cells as stimulators (10). Subsequently,
cytotoxic cultures were found to continuously proliferate after weekly
restimulation with alloantigen(s) in the presence of conditioned
medium. Each stimulation was characterized by a short proliferative
response resulting in a 3- to 4-fold expansion of cell numbers.
Conditioned medium was not required for cell expansion but was
important for survival, i.e., cells that were no longer proliferating
remained viable for several days in various conditioned media, whereas
cells suspended in nonconditioned medium died quickly. Because the
phenotype(s) of the MLC-expanded catfish cytotoxic cells was not known,
cells responsible for killing allogeneic targets were cloned and
subsequently characterized using molecular probes for the catfish TCR
and ß genes. PBL from two nonimmune and one 3B11-immunized
channel catfish were cloned by limiting dilution either with or without
prior stimulation and expansion in MLC. The number of wells containing
proliferating cells (cloning efficiency) was estimated by microscopy.
Expansion in MLC before cloning dramatically increased the cloning
efficiency from both sources. For example, only 0.7% of PBL from
nonimmune fish #75 could be cloned directly, whereas 47% of the
MLC-expanded cells yielded clones. Prior immunization with alloantigen
appeared to increase the cloning efficiency as PBL from a
3B11-immunized fish (#32) yielded significantly higher numbers of
clones after both primary and repeat stimulation, i.e., 6.3% and 83%,
respectively. These results showed the feasibility of cloning catfish
cytotoxic cells and demonstrated that both prior immunization and MLC
expansion lead to markedly enhanced cloning efficiency.
To determine their phenotype, the resulting clones were cultured for
several weeks after which they were screened for expression of TCR ,
TCR ß, and Igµ genes by RT-PCR and for allospecific cytotoxic
activity in 51Cr-release assays. Based on
reactivity patterns, five groups of clones were identified. One group
(I) consisting of 10 clones, each derived from 3B11-immune fish #32,
contained cells that were TCR ß+ (Table I
). This group (represented by clone
TS.32.5 in Fig. 1) lysed 3B11 cells but
did not lyse any of three other allogeneic targets, each derived from
individual fish different from the one that gave rise to the 3B11 cell
line. A second group (II) contained three clones that were also TCR
ß+ but did not display strict specificity in
target cell lysis. Clone TS.32.32, which is representative of group II,
lysed 3B11 as well as 1G8 target cells with high efficiency (Table I &
Fig. 1). However, this clone failed to lyse two other allogeneic
targets (Fig. 1). The other two clones in group II were also capable of
lysing several different allogeneic targets but not others (data not
shown). Two other groups of clones (IV and V), isolated from both
immune and nonimmune fish, were TCR ß- but
differed in cytotoxic activity. The three clones in group IV lacked
allospecificity, i.e., they lysed 1G8 target cells as well as 3B11
cells (Table I). A representative member of this group, TS.75.2, lysed
three allogeneic target cells including 3B11 cells with no apparent
specificity (Fig. 1). However, TS.75.2 did not lyse autologous target
cells (75C) derived from the same fish (Fig. 1). The second group (V)
of TCR ß- cytotoxic cells consisting of 3
clones displayed allospecificity in target cell recognition, i.e., 3B11
cells were lysed more efficiently than 1G8 allogeneic target cells
(Table I). A representative clone from this group, TS.10.1, lysed four
different allogeneic target cells including 3B11 cells. However,
although 3B11 cells were lysed at high efficiency, lysis of three other
allogeneic target cells occurred at relatively low efficiencies (Fig. 1). Finally, a single clone, TS.32.4, was TCR
ß+ but was unable to lyse 3B11 or 1G8 target
cells in 4-h 51Cr-release assays. However,
TS.32.4 cells proliferated specifically to stimulation with irradiated
3B11 cells but not to stimulation with irradiated 1G8 cells (data not
shown).
|
|
ß+ clones
derived from fish #32 involved the same or different precursors,
TCR and ß cDNAs were sequenced for each clone. All of the
clones expressed members of the V 1a family (12). Two
clones, TS.32.12 and TS.32.44, exhibited identical VJ rearrangements
and two others, TS.32.15 and TS.32.49, rearranged identical V to
different J (Fig. 2A). Each
Vß rearrangement was unique, with clones TS.32.5, TS.32.12, and
TS.32.44 expressing different members from the same Vß family (Fig. 2B). These findings indicate that each of these cell lines
are clonal and likely derived from independent precursors.
|
ß+ cytotoxic cells proliferate in response to
stimulation with specific alloantigen
Irradiated stimulator cells were crucial for propagation of both
TCR ß+ and TCR
ß- cells during the process of cloning. To
test whether or not the various cytotoxic clones required specific
allostimulation for in vitro expansion (or if any allogeneic stimulator
would suffice) an allospecific TCR ß+ clone
(TS.32.5) and an allospecific TCR ß- clone
(TS.10.1) were stimulated with specific alloantigen (3B11 cells) or
with a different alloantigen (1G8 cells) in the presence or absence of
conditioned medium. As shown in Fig. 3,
TS.32.5 responded only to 3B11 cells, whereas clone TS.10.1
proliferated in response to both 3B11 and 1G8 cells. Furthermore,
TS.32.5 cells proliferated equally well after stimulation with 3B11
cells whether or not conditioned medium was included in the culture. In
contrast, a mixture of conditioned medium and irradiated allogeneic
cells appeared to synergistically enhance proliferation of TS.10.1
cells (Fig. 3).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß genes. One group of effectors (group IV) was nonspecific,
TCR ß-, and was considered to be equivalent
to mammalian NK cells, whereas two other groups of effectors (groups I
and V) demonstrated allospecific cytotoxic activity. Clones in group I
expressed messages for both catfish TCR and ß and may be
considered bona fide CTL. The second group of allospecific effector
clones (group V) lacked TCR ß expression. Therefore, it is not
clear whether these cells are CTL or a second type of NK-like cell.
Although mammalian NK cells typically lyse target cells in a
nonspecific fashion, it has been demonstrated that human NK cells can
cause specific lysis of allogeneic PHA-induced lymphoblasts after
stimulation in vitro with homologous lymphoblasts (24). By
analogy, a similar mechanism for target cell recognition may exist for
catfish allospecific NK-like lysis. It should be noted that although
the TCR ß- allospecific cytotoxic effector
cells described here are termed NK-like, the possibility of TCR 
expression in one or both of these groups of clones was not ruled out.
Putative channel catfish TCR genes have not yet been identified,
although TCR (as well as TCR ß) genes have been isolated in
cartilaginous fish. Thus, equivalent genes are possibly present in
teleosts because it seems likely that TCR genes originated in an
ancestor common to all jawed vertebrates (25). Catfish
cytolytic cells that were TCR ß+ but
nonspecific in target cell recognition (group II) were difficult to
categorize because they did not match the phenotype and function of
known mammalian cytotoxic cells. It is possible that these clones may
recognize a target structure that is similar on the two target cells.
Although possibly not applicable to the clones in question, it is
interesting to note that mammalian CTL clones may convert from
allospecific to nonspecific target cell recognition after prolonged
culture or after culture in high concentrations of IL-2 (26, 27). It is also possible that these particular fish cells may
represent a form of NK-like T cells (28). An additional
TCR ß+ clone (group III) proliferated
specifically in response to irradiated 3B11 cells but did not lyse 3B11
cells or other allogeneic targets. This clone may be a candidate for a
catfish homologue of mammalian T helper cells. Although most of the cytotoxic cells derived in this study were cloned after stimulation in MLC, some were cloned directly from freshly isolated PBL. It is noteworthy that the cloning efficiency of PBL from the nonimmune catfish tested ranged from 0.5 to 1.0%, indicating that the numbers of NK-like cytotoxic effectors and/or precursor cells in PBL were relatively low. This is consistent with previous observations of spontaneous cytotoxic responses in channel catfish, namely that E:T ratios ranging from 15:1 to 40:1 were usually required for 50% lysis of allogeneic target cells by freshly isolated PBL in 4-h 51Cr-release assays (3, 13, 29). These observations imply that the spontaneous effector cell population(s) involved in lysis of any given allogeneic target likely ranges from 1 to 4% of the total PBL in the catfish employed in these studies.
This study did not formally demonstrate that the cells designated as
catfish CTL employed TCR molecules to recognize target cells, nor were
MHC molecules proven to be the structures recognized on target cells
(30). However, this seems likely considering the strict
specificity in target cell lysis displayed by the catfish CTL clones
combined with the fact that these effector cells express TCR ß
genes and the allogeneic target cells express both class I and II MHC
genes (Refs. 8 and 9 and our unpublished data). Preliminary
observations that blocking of target cell killing by some but not all
alloantisera suggest that catfish CTL clones most likely recognize an
alloantigen(s) on target cells (data not shown); whether this
alloantigen is MHC class I, class II, or another molecule is not clear.
Immunoprecipitation studies with such alloantisera have been equivocal
and have not yet resolved this issue (31). Consequently,
identification of the molecules on target and effector cells involved
in target cell recognition by catfish CTL clones awaits the development
of immune reagents specific for catfish TCR and MHC molecules.
The target cell recognition mechanism(s) employed by catfish NK-like effector clones was not determined in this study. However, one of the NK-like clones did not lyse autologous target cells but efficiently lysed three different allogeneic targets. It is possible that catfish NK-like cells express killer inhibitory receptors analogous in function to those found on human and rodent NK cells (32); engagement of self-MHC molecules on autologous/syngeneic target cells by killer inhibitory receptors on mammalian NK cells protects these cells from lysis by effectors (33, 34, 35). The availability of autologous and allogeneic clones of effector and target cells from catfish provides a promising model with which to study target cell recognition by fish NK-like cytotoxic cells at both functional and molecular levels.
Channel catfish alloreactive cytotoxic cells were shown to be distinct from NCC by several criteria. First, an NCC-specific mAb, 5C6, failed to block lysis of allogeneic target cells by PBL effectors (3). Second, channel catfish PBL stimulated in primary MLC lysed xenogeneic target cells poorly at E:T ratios that gave maximum lysis of allogeneic target cells (10). Finally, all of the NK-like as well as CTL clones tested did not react with mAb 5C6 (data not shown). Taken together, these results indicate that the various cytotoxic cells described herein are distinct from NCC.
The results described above provide the strongest evidence to date that channel catfish (and possibly other fish species) contain a variety of CTL and NK-like effector cells with different target cell preferences. The ability to clone various types of catfish cytotoxic cells along with the continued development of molecular probes and immunological reagents will likely facilitate study of both CTL and NK-like cells in fish as it has in mammals.
|
Footnotes |
|---|
2 The sequences presented in this article have been submitted to GenBank under accession numbers AF178012–AF178020 for TS.32.15, TS.32.49, TS.32.12, TS.32.44, TS.32.1, TS.32.5, TS.32.34, TS.32.17, and TS.32.43, respectively, and AF178021–AF178029 for TS.32.15ß, TS.32.49ß, TS.32.12ß, TS.32.44ß, TS.32.1ß, TS.32.5ß, TS.32.34ß, TS.32.17ß, and TS.32.43ß, respectively.
3 Address correspondence and reprint requests to Dr. L. William Clem, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505. E-mail address:
4 Abbreviations used in this paper: NCC, nonspecific cytotoxic cells; RACE, rapid amplification of cDNA ends; PBL, peripheral blood leukocyte; MLC, mixed leukocyte culture.
Received for publication September 15, 1999. Accepted for publication January 5, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ß genes. Mol. Immunol. 35:545.[Medline]
, ß, and T cell antigen receptor genes arose early in vertebrate phylogeny. Immunity 6:1.[Medline]
This article has been cited by other articles:
|
|
 |
|
  J. L. Stafford, M. Wilson, D. Nayak, S. M. Quiniou, L. W. Clem, N. W. Miller, and E. Bengten Identification and Characterization of a FcR Homolog in an Ectothermic Vertebrate, the Channel Catfish (Ictalurus punctatus) J. Immunol., August 15, 2006; 177(4): 2505 - 2517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Bernard, B. Riteau, J. D. Hansen, R. B. Phillips, F. Michel, P. Boudinot, and A. Benmansour Costimulatory Receptors in a Teleost Fish: Typical CD28, Elusive CTLA4 J. Immunol., April 1, 2006; 176(7): 4191 - 4200. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Boudinot, D. Bernard, S. Boubekeur, M.-I. Thoulouze, M. Bremont, and A. Benmansour The glycoprotein of a fish rhabdovirus profiles the virus-specific T-cell repertoire in rainbow trout J. Gen. Virol., October 1, 2004; 85(10): 3099 - 3108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
P. Boudinot, S. Boubekeur, and A. Benmansour Primary Structure and Complementarity-Determining Region (CDR) 3 Spectratyping of Rainbow Trout TCR{beta} Transcripts Identify Ten V{beta} Families with V{beta}6 Displaying Unusual CDR2 and Differently Spliced Forms J. Immunol., December 1, 2002; 169(11): 6244 - 6252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Robert, J. Gantress, L. Rau, A. Bell, and N. Cohen Minor Histocompatibility Antigen-Specific MHC-Restricted CD8 T Cell Responses Elicited by Heat Shock Proteins J. Immunol., February 15, 2002; 168(4): 1697 - 1703. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Boudinot, S. Boubekeur, and A. Benmansour Rhabdovirus Infection Induces Public and Private T Cell Responses in Teleost Fish J. Immunol., December 1, 2001; 167(11): 6202 - 6209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhou, T. B. Stuge, N. W. Miller, E. Bengten, J. P. Naftel, J. M. Bernanke, V. G. Chinchar, L. W. Clem, and M. Wilson Heterogeneity of Channel Catfish CTL with Respect to Target Recognition and Cytotoxic Mechanisms Employed J. Immunol., August 1, 2001; 167(3): 1325 - 1332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Khayat, T. B. Stuge, M. Wilson, E. Bengten, N. W. Miller, and L. W. Clem Thioredoxin Acts as a B Cell Growth Factor in Channel Catfish J. Immunol., March 1, 2001; 166(5): 2937 - 2943. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Hawke, J. A. Yoder, R. N. Haire, M. G. Mueller, R. T. Litman, A. L. Miracle, T. Stuge, L. Shen, N. Miller, and G. W. Litman Extraordinary variation in a diversified family of immune-type receptor genes PNAS, November 20, 2001; 98(24): 13832 - 13837. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, conserved position
of charged amino acids in the TCR 