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IMMUNOBIOLOGY
From the Bone Marrow Transplantation Program, School of
Medicine, University of Alabama at Birmingham.
Ex vivo expanded Epstein-Barr virus (EBV)-specific T
cells have been successfully applied clinically for adoptive
immunotherapy. However, the role of CD4+ T cells in the
therapeutic T-cell culture has not been established for the
reconstitution of EBV-specific immunity. We isolated and characterized
CD4+ T-cell lines from the ex vivo T-cell cultures.
Monoclonal line PD-F4 and oligoclonal lines ND-R4 and TD-B4 were
CD3+CD4+CD8 T-cell-mediated immunity is the major mechanism
providing specific protection against microbial infections, including
those by viruses.1 Virus-infected cells process viral
polypeptides and present antigenic epitopes on the cell surface. T
cells mount immune responses upon recognizing the antigen epitope in
the context of the major histocompatibility complex (MHC) molecules and
receiving various signals from the antigen-presenting cells. It has
been well established that CD8+ T cells are mostly
cytotoxic T lymphocytes (CTLs) that directly destroy virus-infected
cells,2 while CD4+ T cells serve primarily as
helpers by secreting various cytokines to regulate and coordinate
functions of T cells, B cells, and other immune
cells.3
Viruses have evolved different strategies to escape T-cell-mediated
immunity.4 Some viruses maintain a state of latent infection in immunocompetent individuals. As exemplified by the Epstein-Barr virus (EBV) and cytomegalovirus (CMV), viruses may disrupt
the host cell mechanisms of antigen presentation and/or adapt viral
replication programs to minimize the expression of viral targets
recognizable by immune surveillance. In individuals with compromised
immunity, such as recipients of stem cell transplants (SCTs), latently
infecting EBV and CMV may reactivate and cause morbidity and
mortality.5,6 Guided by the increased understanding of
mechanisms of cellular immunity against viral pathogens, strategies of
adoptive immunotherapy have been developed7 and
successfully applied to patients following SCT to prevent and treat
these viral complications.8,9 Adoptive immunotherapy
involves infusing ex vivo expanded, virus-specific T cells into
susceptible patients. Therapeutic CMV- or EBV-specific CTLs have been
prepared from ex vivo T-cell cultures stimulated with autologous
CMV-infected fibroblasts,10 or B-lymphoblastoid cell lines
(BLCLs),9 respectively.
In SCT patients, the infusion of polyclonal, BLCL-primed T-cell
preparations reconstitutes long-term cellular immunity against EBV,11 but CMV-specific CD8+ clones were found
to provide only short-term protection.12 It has been
suggested that a deficiency in CD4+ helpers may be
responsible for the failed long-term survival of the infused
CMV-specific CD8+ CTL, as the persistence of the
CD8+ CTL is correlated with CD4+ helper
functions in recipients of T-cell infusons.12 Thus, the
long-term efficacy of the BLCL-primed T cells may result from the
presence of a minor component of EBV-specific CD4+ cells in
the polyclonal T-cell culture. This is consistent with the findings
that BLCLs express both HLA class I and HLA class II and have the
capacity to present endogenously derived antigens to CD8+,
as well as CD4+ T cells.13,14 Indeed, we were
able to isolate specific CD4+ T cells that recognize
autologous BLCLs from T-cell cultures primed with BLCLs, and we showed
that they are cytolytic via a pathway independent of granzyme
B.15 We are interested in further studying the
CD4+ T cells derived from the BLCL-primed T-cell cultures,
because the information obtained from these cells may provide insight into their functions and into the mechanism by which the
CD4+ T cells contribute to adoptive immunotherapy.
On the other hand, although CD4+ T cells may facilitate
long-term reconstitution of specific immunity, they could also carry
undesirable side effects. It has been documented that CD4+
T cells possess nonspecific "bystander" cytotoxicity via Fas ligant
(FasL) and other tumor necrosis factor (TNF)- We report here that the CD4+ T cells in ex vivo
expanded, BLCL-primed T-cell cultures (1) produced TH1
and TH2 cytokines in response to antigenic stimulation and
(2) exerted MHC class II-restricted cytotoxicity through a mechanism
that was dependent on exocytosis and possibly involved granulysin.
Donors and cell lines
Ex vivo T-cell culture
Cloning of CTLs CTLs were isolated by limiting dilution in 96-well, U-bottom tissue-culture plates. Cells were plated in serial dilutions, with 2.5 × 104 irradiated (30 Gy) allogeneic pooled PBMCs from healthy donors as feeders and 2.5 × 104 irradiated (100 Gy) autologous BLCLs as stimulators in a final volume of 200 µL. The cloning medium was supplemented with 200 U/mL IL-2 (Chiron, Emeryville, CA).Cytotoxicity assay by chromium release assays and cytotoxicity blocking Chromium release assays were performed as previously described.13,18 In brief, target cells were labeled with 51Cr (New England Nuclear, Boston, MA) for 1 hour (300 µCi/106 cells [11.1 × 106 Bq/106 cells]), harvested by centrifugation, washed in phosphate-buffered saline (PBS), and dispensed into 96-well V-bottom plates (ICN, Costa Mesa, CA) at 4 × 103 cells per well. Effector cells were added to indicated effector-to-target (E-to-T) ratios in equal volumes. After the cells were pelleted with centrifugation at 1000g and incubated for 4 hours at 37°C in 5% CO2, supernatant was harvested and counted in a gamma counter. Spontaneous release and total release for each target were used to calculate the percentage of specific release as:
Reverse transcriptase-polymerase chain reaction for T-cell
receptor V  amplification were a common 3'
primer and one of the twenty-four 5' primers according to
Genevee et al.19 A pair of T-cell receptor (TCR)
C primers were included in each reaction as an internal
control. The reaction cycles were as follows: 94°C for 1 minute,
60°C for 1 minute, and 72°C for 1 minute, for a total 30 cycles,
followed by 72°C for 5 minutes. The polymerase chain reaction (PCR)
products were separated by agarose gel electrophoresis, stained with
ethidium bromide, and evaluated visually. For sequencing of the
PCR-amplified fragments, aliquots of reverse transcriptase (RT)-PCR
reaction were used for sequencing from both directions with the PCR
primers in an ABI Prism automatic sequencer (Applied Biosystems, Foster
City, CA).
Flow cytometric determination of cell surface and intracellular markers Flow cytometry was performed on a FACScan (BD PharMingen). Surface markers were determined as described previously15 by staining with directly conjugated monoclonal antibodies specific for CD3, CD4, and CD8 (BD PharMingen). Multiple color staining of immunophenotypic markers, for both surface and intracellular antigens, was performed as described previously with modifications.15 In brief, T cells were incubated with stimulators at indicated ratios in a concentration of 1 × 106 cells/mL at 37°C with 5% CO2. For experiments in which stimulation lasted for up to 5 hours, BFA was added at 10 µg/mL at the beginning of the cocultivation, and fractions of the culture were harvested at indicated intervals. For experiments with stimulation longer than 5 hours, BFA was added 5 hours prior to cell harvest. After stimulation, EDTA was added to a final concentration of 2.5 mM, and the cells were incubated at room temperature for 10 minutes. Then, 10 vol Lysing Solution (BD PharMingen) was added and incubated for 10 minutes. The cells were either stained immediately or stored at 80°C.
For direct staining, the cells were washed with 3% fetal calf serum
and 0.1% NaN3 in PBS, incubated with
permeabilization buffer (BD PharMingen) for 10 minutes, aliquoted, and
stained with the following labeled antibodies (BD PharMingen):
CD3-peridinin chlorophyll protein (CD3-PerCP) or
CD3-allophycocyanin (CD3-APC); CD4-fluorescein
isothiocyanate (CD4-FITC) or CD4-PerCP; CD8-FITC or CD8-PerCP;
CD69-FITC; perforin-phycoerythrin (perforin-PE); interferon-
CD4+ T-cell lines with MHC class II-restricted specific cytotoxicity T-cell lines were cloned by limiting dilution from CTL cultures primed with autologous BLCLs. Several lines displayed an immunophenotype of CD3+CD4+CD8
(data not shown). PD-F4 showed specific cytotoxicity against autologous
BLCLs (DQ0602/DQ0609), which was sensitive to a monoclonal antibody against HLA-DQ, but not to the antibodies against DR or DP
(Figure 1A). This line did not exhibit
cytotoxicity against allogeneic BLCLs with fully mismatched HLA alleles
(Figure 1A; Allo C). While PD-F4 did not kill the allogeneic BLCLs
sharing a single MHC allele DQ0609 (Allo B), it lysed the partially
mismatched allogeneic BLCLs sharing the single MHC DQ0602 (Allo A), and
this cytotoxicity was blocked again by the anti-DQ antibody. Two more CD3+CD4+CD8 lines, ND-R4 and
TD-B4, displayed a similar pattern of specific cytotoxicity against
autologous BLCLs, but not allogeneic BLCLs with fully mismatched HLA
alleles (Figure 1B). While the specific cytotoxicity of TD-B4 was
inhibited by the HLA-DR-specific antibody, ND-F4 was sensitive to
antibodies against HLA-DR and HLA-DQ. The results from the above
experiments of antibody blocking and HLA-mismatched targets suggested
that the cytotoxicity of all 3 CD4 T-cell lines was restricted by MHC
II alleles. A restriction of functions by MHC II is consistent with the
characteristics of CD4+ T cells.
The clonality of the CD4+ lines was established by the
expression patterns of TCR
Profile of cytokine production by CD4+ CTLs CD4+ T cells regulate specific immunity mostly by producing cytokines in response to specific antigenic stimulation. To understand the profile and modulation of cytokine production by the CD4+ CTLs, we used flow cytometry to analyze cytokine production by simultaneous staining for intracellular antigens. We examined 3 cytokines in this study: IFN- , IL-2, and IL-4. The first
2 are TH1 cytokines that polarize the immune response to
CTL generation, and the third is a TH2 factor promoting
B-cell development and antibody production.21
Figure 3 shows that among the oligoclonal
ND-R4 cells, approximately 31.1% were positive for IFN-
Similarly to ND-R4, PD-F4 up-regulated the expression of cytokines in
response to stimulation by autologous BLCLs (Figure 4A-C; compare the staining with the
corresponding isotype antibodies in Figure 4D-F). There was no
detectable cytokine expression in PD-F4 7 days after priming without
further stimulation (Figure 4G-I) or with stimulation by allogeneic
BLCLs (Figure 4J-L). Unlike ND-R4, the monoclonal PD-F4 produced IL-2
(
Kinetics of cytokine production by CD4+ CTLs Recent evidence has shown that the on/off cycling of cytokine production in CD8+ T cells is tightly regulated in coordination with different phases of antigenic stimulation.23,24 The monoclonal PD-F4 cell line provided us an opportunity to study the on/off cycling for cytokine production in CD4+ T cells. We examined the expression of IFN- IL-2, and IL-4 in the PD-F4 cells over the course of 9 hours of
postantigenic stimulation. Figure 5 shows
the kinetics of cytokine expression by enumerating cells producing
IFN-, IL-2, or IL-4 alone (Figure 5A) and coexpressing other
cytokines (Figure 5B). Cells expressing cytokines became detectable at
the first hour after stimulation. At the seventh hour, the number of
cytokine-positive cells plateaued for all 3 cytokines. In this
particular experiment, the numbers of cells expressing IFN- and IL-4
were comparable, while IL-2-expressing cells were significantly fewer
(Figure 5A). During the seventh to ninth hours, the number of
cytokine-positive cells significantly decreased. IL-2- and
IL-4-expressing cells decreased most dramatically, almost to baseline
levels. In contrast, more than half of the IFN--expressing cells
remained positive at the ninth hour after stimulation. Figure 5B shows
the same PD-F4 sample analyzed for the kinetics of cells concurrently
expressing any 2 cytokines. Consistent with the pattern constructed by
enumerating cells positive for any one of the cytokines (Figure 5A),
the numbers of cells coexpressing cytokines reached plateau levels at
the seventh hour after stimulation. Similar percentages of cells
coexpressed IFN-/IL-2 and IFN-/IL-4, while fewer cells
coexpressed IL-2/IL-4. At the ninth hour after stimulation, all the
cells concurrently expressing 2 cytokines, including those coexpressing
IFN- (IFN-/IL-2 and IFN-/IL-4), declined to baseline levels
(Figure 5B). These results indicated that, while the CD4+ T
cells had on/off controls for cytokine expression similar to those seen
in the CD8+ CTLs, the offswitching for IL-2 and IL-4
expression appeared more prompt than for IFN-, or the
CD4+ T cells down-regulated the production of IFN- less
synchronously than that of IL-2 and IL-4.
Mechanisms of specific cytotoxicity by CD4+ CTLs Our interest in investigating killing mechanisms of the BLCL-primed CD4+ CTLs arose from reports that CD4+ T cells exerting cytotoxicity through FasL-mediated apoptosis may contribute to pathological bystander cytotoxicity.16,17 We have previously shown that the CD4+ lines established in our laboratory from BLCL-primed T-cell cultures do not express granzyme B, a dominant apoptosis-inducing molecule in the perforin/granzyme-mediated cytotoxic pathway.15 Although a lack of granzyme B expression is consistent with killing mechanisms independent of perforin/granzyme pathways, it was possible that granzymes other than type B were involved in the killing. To address this question, we examined the expression of perforin in the CD4+ T-cell lines isolated from BLCL-primed cultures. While flow cytometric staining detected perforin in the control CD8+ CTL line PD-F8 in the activated state (5 hours after stimulation; data not shown) as well as in the resting state (7 days after stimulation, Figure 6), all 3 CD4+ lines were negative for this protein (Figure 6) under the same detection conditions. A lack of perforin expression in the CD4+ CTLs was consistent with the established observation that most CD4+ T cells exert their specific cytotoxicity predominantly via upregulating FasL and other TNF- family molecules
upon activation.25 To establish that these
CD4+ lines indeed exerted specific cytotoxicity via
FasL/TNF- family molecules, we attempted to block the cytotoxicity
with antibodies or receptor-immunoglobulin fusion proteins that are
known to inhibit cytotoxicity by FasL (Nok1, Nok2, Fas-Ig) or TRAIL
(anti-hTRAIL, DR5-Ig). To our surprise, none of the above specific
biological reagents significantly inhibited the cytotoxicity of the
CD4+ lines (data not shown).
A resistance to blocking antibodies and receptor-immunoglobulin fusion
proteins suggested to us that FasL and TRAIL might not contribute
significantly to the specific cytotoxicity of the CD4+
CTLs. To confirm this possibility, we then examined the
CD4+ CTL lines with chemical reagents that are known to
block cytolytic pathways with a wider spectrum of specificity. CMA
acidifies intracellular vacuolar granules and is thought to inhibit
perforin/granzyme-mediated cytotoxicity by increasing degradation of
the content in the exocytotic granules.26 In contrast, BFA
selectively inhibits FasL and other TNF- In the presence of 0.1 nM CMA, the specific cytotoxicity of the CD4 T
cells showed a slight inhibition (Figure
7A). Within the concentrations of 0.3 to
0.9 nM, CMA inhibition of cytotoxicity reached its fullest extent. In
comparison with ND-R4 and TD-B4, PD-F4 appeared to have higher residual
CMA-resistant cytotoxicity. Figure 7B shows that ND-R4 and TD-B4
displayed an almost complete resistance to BFA when tested with
concentrations ranging from 5 to 40 µM. A small proportion of the
cytotoxicity by PD-F4 was sensitive to BFA (Figure 7B). This inhibition
was dose-dependent when BFA was within the concentration of 5 to 20 µM and appeared to reach the minimal level around 20 µM.
Nevertheless, the major portion of the cytotoxicity by PD-F4
was resistant to BFA, in correlation with a residual cytotoxicity
resistant to CMA (Figure 7A). EGTA completely blocked the cytotoxicity
of all the 3 lines at a concentration of 0.9 mM (Figure 7C). It should
be noted that the dose-dependent cytotoxicity inhibition to the
CD4+ lines by CMA and EGTA was very similar to a bulk CTL
culture (Figure 7A,C) that was composed mostly of BLCL-specific
CD8+ T cells (data not shown). The bulk culture provided a
control for cytotoxicity mediated by granule exocytosis, typically
found in CD8+ CTLs. We have shown before that the
cytotoxicity in the bulk T-cell culture primed with BLCL-based APC
is detectable only in the CD8+
fraction.13 These results suggest that the
CD4+ lines may exert their specific cytotoxicity through
granule exocytosis.
The above findings prompted us to examine the expression of cytolytic
granule-associated effector molecules other than perforin and
granzymes. Granulysin has recently been identified in the cytotoxic
granules of CTL and natural killer (NK) cells and is cytolytic against
tumor cells and microbes.30 With the monoclonal granulysin-specific antibody DH4, flow cytometric analysis revealed that granulysin was expressed in a low percentage of
phytohemagglutinin-activated T blasts, approximately 17% for
CD8+ and 10% for CD4+ cells (Figure
8D). In contrast, few CD4+ or
CD8+ cells in PBMCs expressed this protein (Figure 8E).
Significantly, 65% of PD-F4, 55% of ND-R4, and 19% of TD-B4
expressed granulysin (Figure 8A-C). The CD8+ line PD-F8,
which expressed perforin (Figure 6), was also positive for granulysin
in 72% of the cells (Figure 8F). Granulysin expression was positive in
the T cells of the activated state (5 hours after stimulation; data not
shown), as well as of the resting state (7 days after priming; Figure
8A-C). This pattern of expression is similar to that of perforin, but
different from the one for cytokines.23 The same
cell preparation used for granulysin staining was also tested for
indirect staining with the antibody against IFN-
CD4+ T cells have been extensively studied as helpers regulating the immune system via diffusable factors including cytokines.3 Although cytotoxic CD4+ T cells have been reported,17,31-35 little is known about their role in virus-specific immunity, especially the relationship between their specific cytotoxicity and helper functions. In this study, we characterized the antigen-specific CD4+ CTLs from T-cell cultures generated ex vivo with a protocol used in adoptive immunotherapy for EBV-related tumors.36 Although the specific cytotoxicity of these CD4+ T cells was clearly MHC class II restricted, as expected for classic CD4+ T cells, the CD4+ CTLs showed uncommon features in cytokine production and cytolytic mechanism in response to antigenic stimulation. As expected for CD4+ T cells, ND-R4 and PD-F4 expressed
immunoregulatory cytokines in response to antigenic stimulation. Single ND-R4 cells expressed either IL-4 or IFN- Results from our study suggested that the CD4+ CTLs
controlled the off cycling for IL-2 and IL-4 production in ways similar to those reported for IFN- We found that the CD4+ CTLs possessed specific cytotoxicity
that could not be attributed to classic cytolytic pathways mediated by
perforin/granzyme or FasL.25 For CTLs exerting
cytotoxicity via the perforin/granzyme pathway, interaction between T
cells and target cells results in release of cytolytic granules
containing perforin and granzymes, which permeabilize the plasma
membrane of the target cells and set off a chain of enzymatic reactions quickly leading to destruction of the plasma membrane and programmed cell death. In contrast, in response to specific antigenic stimulation, most CD4+ CTLs up-regulate the expression of FasL, which
binds to its cognate receptor Fas/CD95 on the surface of target cells
and induces apoptosis of the target cells. Other mechanisms of
cytolysis, which mainly implicate members of the TNF- We showed in this study that the CD4+ CTL lines did not
express the pore-forming protein perforin. This is in contrast to the CD8+ CTLs, which were found positive for perforin with the
same experimental approach and detection technique (Figure 6). We have
previously reported that granzyme B is not expressed in the
CD4+ CTL lines isolated from BLCL-primed
culture.15 Although this is in line with the mainstream
concept that CD4+ T cells generally exert specific
cytotoxicity by FasL, functional assays with specific blocking agents
could not establish a confident link between the specific cytotoxicity
and FasL/TRAIL. Further blocking experiments with chemical inhibitors
confirmed that the CD4+ CTL-mediated killing was
independent of FasL or TRAIL, as the major proportion of the
cytotoxicity by the CD4+ CTL lines was resistant to BFA.
Since BFA is inhibitory to surface transportation of polypeptides from
the endoplasmic reticulum, a resistance to this substance would suggest
that not only FasL and TRAIL but also other members of the TNF- The CD4+ CTL lines appeared to exert specific cytotoxicity via mechanism(s) involving exocytosis, possibly with granulysin. Supporting evidence includes our findings that the cytotoxicity mediated by the CD4+ CTLs was sensitive to CMA and EGTA. The detection of granulysin, but absence of other known cytolytic effectors in these CD4 CTLs, is consistent with the possibility that CD4+ CTLs might kill their targets by this protein. Granulysin is a newly discovered cytolytic molecule usually coexpressed with perforin in the cytolytic granules of CTL and NK cells.43 It has been found to be bactericidal against a broad range of microbes, including the intracellular parasite Mycobacterium tuberculosis.44 This particular study44 showed that granulysin, together with perforin, is expressed in antigen-specific CD8+ CTLs. A very recent work further demonstrated that granulysin can be expressed in the CD4+ T cells specific to Mycobacterium leprae, but did not determine whether these M leprae-specific CD4+ T cells produce any cytokines.45 It is speculated that granulysin works synergistically with perforin, which allows granulysin to enter the cells and kill the intracellular microbes. Recently, it was also found that synthetic granulysin itself is cytolytic to tumor and virus-infected cells.46,47 Granulysin causes damage to cell membranes and disrupts the transmembrane potential in mitochondria, which leads to apoptosis.48 Results from our study further the significance of granulysin by directly showing that T cells expressing granulysin were cytolytic to EBV-infected B cells. These CTLs may have a CD8+ phenotype, as exemplified by PD-F8, which coexpressed perforin and granulysin (Figures 6 and 8). Of interest is our finding that CD4+ CTLs expressed granulysin without coexpressing detectable amounts of perforin, suggesting that granulysin alone could be sufficient as a physiologically relevant cytolytic effector. Since the same CD4+ lines coexpressed multiple immunoregulatory cytokines, the CD4+ CTLs described in our study have the potential to play important roles in specific immunity. It should be noted that although from our study the correlation between granulysin and CD4+ CTL-mediated cytotoxicity is strong, a definitive conclusion awaits technical means that would directly and specifically disrupt the function of granulysin. Moreover, a granulysin-mediated pathway does not necessarily exclude the use of other cytolytic pathways by CD4 T cells, especially under different physiological conditions or against specific pathogens. Although our data suggested that the major part of the CD4+ CTL-mediated cytolysis, which occurred during the initial 4 hours upon encountering antigen-bearing cells, was independent of FasL, one could not exclude the possibility that FasL might be induced and contribute cytolysis at a later phase of antigen stimulation. The coordination between the specific cytotoxicity and immunoregulation for the CD4+ T cells also needs further study. The results reported in this study are relevant to clinical application
of adoptive immunotherapy against EBV. Since the antigen-specific CD4+ T cells in the BLCL-primed T-cell culture possessed
helper functions to regulate both TH1 and TH2
differentiation, potentially these cells would contribute to the
efficacy of T-cell therapy by promoting a full spectrum of specific
immunity against a given target. Furthermore, because the specific
cytotoxicity of the CD4+ CTLs was largely independent of
FasL and possibly other TNF-
The authors thank Dr R. Lopez (UAB) for the use of a flow cytometer, Dr T. Zhou for Fas-Ig and DR5-Ig, Dr W. Britt for discussion of results, and Drs A. Krensky and S. Okada (Stanford University) for the antibody DH4.
Submitted July 18, 2001; accepted December 19, 2001.
Supported by grants from the American Cancer Society (CRTG 97-043-EDT), the National Institutes of Health (R01 CA75566-01 and R21 CA84398-01), and the Amy Stretzer-Manasevit Award from the National Marrow Donor Program.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kenneth G. Lucas, Bone Marrow Transplantation Program, University of Alabama at Birmingham, 1900 University Blvd, THT 513, Birmingham, AL 35294; e-mail klucas{at}uabmc.edu.
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  H. M. Long, J. Zuo, A. M. Leese, N. H. Gudgeon, H. Jia, G. S. Taylor, and A. B. Rickinson CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus-transformed B cells Blood, July 23, 2009; 114(4): 807 - 815. [Abstract] [Full Text] [PDF]
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J. J. Endsley, J. L. Furrer, M. A. Endsley, M. A. McIntosh, A. C. Maue, W. R. Waters, D. R. Lee, and D. M. Estes Characterization of Bovine Homologues of Granulysin and NK-lysin J. Immunol., August 15, 2004; 173(4): 2607 - 2614. [Abstract] [Full Text] [PDF]
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F. Yanai, E. Ishii, K. Kojima, A. Hasegawa, T. Azuma, S. Hirose, N. Suga, A. Mitsudome, M. Zaitsu, Y. Ishida, et al. Essential Roles of Perforin in Antigen-Specific Cytotoxicity Mediated by Human CD4+ T Lymphocytes: Analysis Using the Combination of Hereditary Perforin-Deficient Effector Cells and Fas-Deficient Target Cells J. Immunol., February 15, 2003; 170(4): 2205 - 2213. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
chains
(TCR-V
20%; Figure 
