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Intramural Research Support Program, Science Applications International Corporation-Frederick,
Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute, and
Veterinary and Tumor Pathology Section, Office of Laboratory Animal Resources, Division of Basic Sciences, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702; and
Cellular Cytotoxicity Laboratory, The Austin Research Institute, Heidelberg, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells. A histological staining
procedure was developed that demonstrated a granular staining pattern
for Gzm M and a cellular distribution similar to that observed by
Western blotting. These data indicate that the expression of Gzm M does
not always correlate with the lytic activity of cytotoxic cells.
However, expression of Gzm M in NK cells, CD3+,
CD56+ T cells, and T cells suggests that this enzyme
may play some role in innate immune responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Target cells lysed by granule-mediated killing exhibit all the morphological and biochemical characteristics of apoptotic cell death. Gzm B has asp-ase activity (in common with the caspase family of enzymes) and has been shown to play a critical role in triggering apoptosis in target cells either directly (9) or via the activation of cellular caspases (10, 11). Gzm A and K have also been proposed to play a role in target cell death (12, 13); however the extent to which Gzm A is involved in target cell lysis remains controversial (14, 15). The expression of Gzm B in effector cells is consistent with this enzyme being involved in target cell death and disintegration (16). However, expression of other Gzm is not so tightly linked to the lytic activity of effector cells (17). Indeed, by Northern blotting, Gzm M did not appear to be highly expressed in activated human, rat, or mouse T cells (6, 18). Furthermore, transfection of constructs of the mouse Gzm M 5'-flanking region coupled to a chloramphenicol acetyltransferase reporter gene into a small panel of mouse and rat cell lines indicated that transcription of chloramphenicol acetyltransferase occurred in NK, but not T, cell lines (7).
In an effort to more thoroughly characterize the expression of Gzm M at the protein level, we have used a mouse mAb known to be specific for Gzm M (8). We isolated highly purified leukocyte subsets by FACS sorting and analyzed the presence of Gzm M in these cells by Western blotting. Gzm M expression was compared with that of other Gzm associated with the lytic phenotype (Gzm A and B) as well as with that of the pore-forming protein perforin. An immunohistochemical procedure was also developed using this Ab, and the expression of Gzm M by individual cells in various highly purified cell fractions was assessed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-CD3, -CD4, -CD8, -CD56, -CD14, -CD45RO, -TCR-
, and
-TCR- conjugated to either FITC or PE were purchased from Becton
Dickinson (San Jose, CA). Mouse monoclonal anti-human Gzm M Ab
(4H10) was prepared as previously described (8). Mouse
mAbs to human Gzm A (GA9) and Gzm B (GB9) were provided by Dr. C. Erik
Hack, Central Laboratory of The Netherlands Red Cross Blood Transfusion
Service, University of Amsterdam (Amsterdam, The Netherlands). In some
experiments the mouse monoclonal 2C5 to human Gzm B was used. This Ab
was provided by Dr. Joseph Trapani (Austin Research Institute,
Melbourne, Australia). The rat mAb to perforin (P1-8) was purchased
from Kamiya Biomedical (Seattle, WA).
Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication 86-23, 1985).
Cell lines
The NK3.3 cell line was provided by Dr. Jackie Kornbluth (St. Louis University, St. Louis, MO). NK92 was provided by Dr. Howard Young (Laboratory of Experimental Immunology, National Cancer Institute-Fred-erick Cancer Research and Development Center, Frederick, MD). The monoclonal-like cell lines THP-1 and U937 as well as the megakaryoblastoid-like cell line M07e were provided by Dr. Dan McVicar (Laboratory of Experimental Immunology, National Cancer Institute-Frederick Cancer Research and Development Center).
Granule purification
NK92 cells (4 x 108) were prepared using Percoll gradient fractionation as previously described (6). Granules were concentrated from high density Percoll fractions (fractions 2–9) by centrifugation at 100,000 x g for 3 h. Concentrated granules and gradient fractions were analyzed for Gzm and perforin using Western blotting.
Human NK and T cell clones
NK and T cell clones NK and T cell clones were established from PBMC as previously described (19). For our experiments, cells were used after >4 wk of in vitro passage after cloning.
Cell separation
Mononuclear cells from leukocyte-enriched human buffy coats were
separated by Ficoll-Hypaque gradient centrifugation. The cells of the
monocyte-macrophage lineage as well as B cells were depleted by
sequentially incubating mononuclear cells on plastic flasks (60 min at
37°C) and in nylon wool columns. Nonadherent cells were then
fractionated on discontinuous Percoll density gradients as previously
described (20). NK cells were highly enriched in low
density gradient fractions (fractions 1 and 2), whereas the majority of
T cells were localized to high density gradient fractions 5 and 6. The
phenotypes of cells in each gradient fraction were routinely analyzed
by staining with anti-CD3 FITC and anti-CD56 PE followed by
analysis on a Becton Dickinson FACSort using LYSYS software. For
further purification of cell subsets, cells from Percoll fraction 2
were stained with anti-CD56 PE at the concentrations recommended by
the manufacturer and then sorted. In a similar manner, highly purified
T cells were sorted from Percoll fractions 5 or 6 using anti-CD3
FITC or anti-TCR- FITC. Human CD3+,
CD56+ T cells (usually enriched in Percoll
fractions 2 or 3) were sorted as cells positive for both CD3 and CD56.
T cells activated in vitro were stained and sorted using anti-CD4
PE and anti-CD8 FITC. The T cells are relatively rare cells
and were isolated by sorting from Percoll fraction 6 from selected
donors using anti-TCR- FITC.
Flow cytometric cell sorting
Cells stained with appropriate Abs were adjusted to 3 x
106 cells/ml, then filtered through a 30-µm
pore size nylon mesh to remove lumps. This cell suspension was
introduced into the MoFlo cell sorter (Cytomation, Ft. Collins, CO) for
phenotypic isolation. Electrostatic sorting proceeded at about 15,000
cells/s until a sufficient quantity of purified cells was isolated.
Purified cells were isolated into sterile RPMI 1640 containing
penicillin-streptomycin and 10% FBS. Single-laser excitation was
sufficient to excite PE- and FITC-labeled Abs. Fluorochrome emissions
were separated with a 550-nm DC mirror for photomultiplier tube
detection. Pulses were log amplified to produce two parameter dot plots
for bit map analysis. To purify or CD3+,
CD56+ T cells we used the high speed
electrostatic cells sorter, MoFlo (Cytomation). Special care was
necessary to minimize cell adhesion and maintain a monodisperse
solution of 5.0 x 107 cells/ml necessary to
exploit our fast sorting electronics and delivery system. The cells
were kept cold and filtered through a 30-µm pore size nylon mesh
before sorting. We found it necessary to pass the cells through the
flow cytometer twice. First, a preenrichment sort was performed at
25,000 cells/s without coincidence circuitry engaged. This process
enriched the cells from 4% to approximately 50% without losing any
or CD3+, CD56+ T
cells. Then we sorted this product for purity with the coincidence
circuit engaged. Our sort rate was 13,000 cells/s, and a final purity
of 95% or CD3+,
CD56+ T cells was achieved.
Neutrophils and monocytes
Human peripheral blood neutrophils were isolated from the pellet after Ficoll-Hypaque centrifugation. The light upper layer of neutrophils was collected with as few red cells as possible. Cells were suspended in 50 ml of PBS containing 3% dextran and left to stand for 30 min at room temperature. Red cells settled out. Supernatants enriched for neutrophils were then pelleted at 500 x g for 5 min. Pellets were resuspended in 20 ml of 0.2% NaCl for 90–120 s for hypotonic lysis of red cells, then adjusted to 0.9% NaCl. Cells were pelleted at 500 x g for 5 min, resuspended, and counted. Cells were routinely >95% neutrophils as estimated by Giemsa stain. Monocytes were prepared by staining cells in Percoll fraction 2 with anti-CD14 FITC followed by sorting as previously described. Monocyte purity was approximately 82%.
T cell stimulation
Highly enriched T cells from Percoll fraction 6 were incubated in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1x nonessential amino acids, 1 mM sodium pyruvate, penicillin (100 U/ml), streptomycin (100 µg/ml), 10 mM HEPES, and 5 x 10-5 M 2-ME, pH 7.4. PHA (PHA-P, Sigma, St. Louis, MO) was added to a final concentration of 2 µg/ml, and IL-2 (Hoffman-La Roche, Nutley, NJ) was added at 100 U/ml. Cells were incubated at 37°C in 5% CO2 for 3 days. Cells were then either incubated 1 additional day in the above medium or were added to tissue culture flasks containing immobilized anti-CD3 Ab. T150 flasks were coated with anti-CD3 by covering the plastic with 15 ml of OKT3 Ab (Ortho Diagnostics, Raritan, NJ) at 10 µg/ml in PBS. Ab was allowed to adhere for 1 h at 37°C, excess PBS was removed, and T cells were added at 1 x 106 c/ml and left for 1 day at 37°C in 5% CO2. In some experiments T cells were further expanded in T150 flasks coated with anti-CD3 in medium containing 100 U/ml IL-2 for up to 18 days.
Western blotting
Cells from all sources were washed thoroughly in PBS, and then pellets containing 5 x 106 cells were stored at -70°C before use. Pellets were gently resuspended in an appropriate volume (250 µl) of extraction buffer containing 50 mM Tris-HCl (pH 8.0), 0.5% Triton X-100, 300 mM NaCl, 5 mM EDTA, and 1 complete mini-protease inhibitor cocktail tablet (Roche, Mannheim, Germany) per 7 ml of buffer. Cells were incubated in extraction buffer for 15 min on ice, then centrifuged at 14,000 rpm (20,000 x g) for 10 min at 4°C. The supernatants were gently removed, and the protein content was measured using the bicinchoninic acid protein assay (Pierce, Rockford, IL). Cell extracts were either used immediately or stored at -70°C. For Western blotting cell extracts at 2.5 or 5 µg protein/well in Novex running buffer (NOVEX, San Diego, CA) were loaded in 14% NOVEX Tris-glycine gels. Molecular weight markers (NOVEX) were always run on all gels. Gels were run for 2.5–3 h at 125 V. Gels were then removed, and proteins were transferred to a polyvinylidene difluoride membrane for 2 h at 8 W constant power using the NOVEX protocol. Blots were then removed and blocked in blocking buffer (20 mM Tris-HCl buffer (pH 7.4), 5% nonfat dried milk, 135 mM NaCl, and 0.5% Tween 20) at 4°C for at least 90 min with gentle shaking. Appropriate Abs diluted in blocking buffer were then added to the blots for overnight incubation at 4°C. Dilutions of Abs usually used were 4H10 (1/2000), GA9 (1/500), GB8 (1/500), 2C5 (1/1000), and P1–8 (1/5000). After incubation blots were washed, and appropriate secondary Abs coupled to HRP and diluted 1/2000 in blocking buffer were added. For 4H10, GA9, and GB8 a secondary goat anti-mouse HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) was used, whereas P1–8 required a goat anti-rat HRP (Jackson ImmunoResearch Laboratories). After 45-min incubation at 4°C with constant shaking, blots were thoroughly washed and developed using the ECL Plus kit (Amersham, Aylesbury, U.K.) according to the manufacturer’s instructions.
Immunohistochemistry
Pellets of 1–10 x 106 cells were prepared by centrifugation at 500 x g for 15 min. Pellets were fixed for at least 24 h in Bouin’s fixative and embedded in paraffin, and 4- to 6-µm sections were prepared for immunohistochemistry. The 4H10 mouse mAb was used at a dilution of 1/500 or 1/1000 with the Vectastain ABC Mouse Elite Kit (Vector, Burlingame, CA). Tissue sections were pretreated with microwaving (twice for 5 min each time, 800 W) with Ag retrieval solution (Citra solution at neutral pH, Biogenex, San Ramon, CA). Hematoxylin was used as a counterstain.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Western blotting clearly demonstrates that Gzm M is easily
detectable in freshly isolated NK cells (Fig. 1
A). In contrast, T cells even
after activation in vitro with PHA alone or in combination with
anti-CD3 had levels of Gzm M that could not be detected under the
conditions we used. Activated T cells sorted into
CD4+ and CD8+ subsets,
which were >90% pure by FACS analysis (data not shown), were also
negative for Gzm M expression on Western blotting. The faint band
observed around 32 kDa is a nonspecific band that is observed in many
cells, particularly on overexposure of the film. This pattern of Gzm M
expression contrasted dramatically with that of Gzm B, which was
usually present at very low or undetectable levels in freshly isolated
NK cells from most donors using the conditions we employed, yet was
dramatically increased in activated T cells. The low levels of Gzm B in
freshly isolated NK cells were surprising, but this was observed with
all cell donors tested, even on using two different anti-Gzm B
mAbs. Gzm B expression was predominantly associated with the
CD8+ T cell population after in vitro
stimulation. Perforin was present at high levels in freshly isolated NK
cells and was also detected in T cells on activation. As with Gzm B,
perforin was found predominantly in the CD8+ T
cell population. Gzm A showed a somewhat more promiscuous distribution
pattern, being detected in NK cells and stimulated T cells of both the
CD4 and CD8 phenotypes. However, the levels of Gzm A expression showed
more donor-to-donor variability than the other Gzm. Even after long
term stimulation of T cells (up to 18 days), Gzm M could not be
detected (Fig. 1B).
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C).
Other Gzm and perforin were also not detectable in these cells, with
the exception of weak reactivity of the THP-1 line with perforin
Abs. Granzyme expression in T and NK cell clones
Since Gzm M in freshly isolated cells and cells stimulated for a
short time in vitro seems to associate predominantly with the NK
phenotype, we examined the expression of Gzm and perforin in human T
and NK clones that had been propagated for >4 wk in vitro. In contrast
to freshly isolated cells, some T cell clones showed a rather weak
expression of Gzm M (Fig. 2). Most of
these T cell clones were of the CD8+ phenotype
(TCC 9, 29, and 47) and also expressed Gzm B and perforin. However one
CD4+ T cell clone (TCC 43) expressed easily
detectable levels of Gzm M, Gzm A, Gzm B, and perforin. Gzm A was
expressed at some level in most T cell clones regardless of phenotype.
NK clones (NK 1, NK 5, and NK 1.5) all strongly expressed Gzm M, Gzm A,
Gzm B, and perforin. Thus, the Gzm M expression pattern of T cell
clones passed for >1 mo in vitro contrasts with that of freshly
isolated T cells or T cells stimulated for shorter periods of time in
vitro. It is possible that some minor population of T cells may express
Gzm M, and these cells have some growth advantage in vitro.
Alternatively, cell-specific expression of Gzm might not be so tightly
maintained on continuous in vitro passage, and a more permissive
expression pattern may ensue.
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T cells
When analyzing small purified T cell populations isolated from
Percoll density gradients, it was often observed that these cells
sometimes showed low levels of Gzm M expression on Western blotting
even though the majority of these cells were
CD3+. Since Western blotting does not allow for
quantitation of protein at the single-cell level, we developed an
immunohistochemical staining procedure to allow determination of Gzm M
in individual cells. Preliminary studies were performed using various
cell lines. By Western blotting analysis the NK92 cell line was Gzm M
positive, whereas NK3.3 and Jurkat cell lines were Gzm M negative (Fig. 3A). Also by
immunohistochemistry, NK92 demonstrated a positive cytoplasmic staining
that appeared to be granular. In contrast NK3.3 and Jurkat cells showed
no convincing reactivity after staining with the Gzm M Ab (Fig. 3B). Cell fractionation of NK92 cells indicated that Gzm M
was enriched in high density Percoll fractions (fractions 2–9). Gzm M
distribution was similar to that of the Gzm and perforin, suggesting a
granule location for Gzm M (Fig. 4).
Immunohistochemical staining of Percoll gradient fractions demonstrated
that low density Percoll fraction 2 (47% CD56+
by FACS analysis) had a similar number of cells staining for Gzm M by
immunohistochemistry (Fig. 5A). In sorted NK cell
populations (92% CD56+, FACS) most cells were
Gzm M positive (Fig. 5C). However, immunohistochemical
staining of Percoll fractions containing small lymphocytes (fraction
6) indicated that most cells were
negative, yet there were a few individual cells that were Gzm
M+ (Fig. 5B). Although the majority of
the cells in this fraction were CD3+ (87%
CD3+, FACS), there were cells with a small
lymphocyte morphology that were CD3-
CD56+. These small lymphocytes exhibit the same
phenotype and lytic capacity as NK cells (21, 22).
Furthermore, lymphocyte Percoll gradient fractions could also contain
T cells and CD3+,
CD56+ T cells. To determine Gzm M expression in
these less abundant lymphocyte subsets, we used FACS sorting to obtain
highly purified cell populations for analysis by Western blotting.
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). As expected, sorted T
cells (85% TCR+) did not express Gzm M.
These T cells also did not express Gzm A or Gzm B. Gzm A
expression has been observed in about 25% of T cells in other studies
(23). However, the T cells used here were isolated
from Percoll fraction 6 and so were presumably small T cells that had
not been activated. Indeed, when stained with CD45RO, about 50% of T
cells from Percoll fraction 6 stained positively, whereas 75–80% of T
cells from Percoll fractions 2 and 3 were
CD45RO+. CD45RO is found on most memory or
activated T cells, but only on a proportion of resting T cells.
CD3+, CD56+ T cells (95%
CD3+, CD56+) positively
sorted from Percoll fraction 3 expressed Gzm M as well as Gzm A and
perforin (Fig. 7). However, the strength
of Gzm M expression by CD3+,
CD56+ T cells did show some donor-to-donor
variability. Therefore, in some donors the strength of Gzm M expression
by CD3+, CD56+ T cells was
equivalent to that of their NK cells, whereas in other donors
CD3+, CD56+ T cells did
express Gzm M, but with a reduced signal strength compared with their
NK cells (data not shown). Interestingly, immunohistochemical staining
of CD3+, CD56+ T cells
indicated that most, but not all, cells were Gzm M positive (Fig. 5E). To determine whether all these cells or a specific
subset express Gzm M requires further investigation. Purified sorted
T cells (95% TCR +) also showed Gzm
M expression on Western blotting as well as Gzm A and perforin, and a
typical example is shown in Fig. 8. Also,
the majority of T cells were positively stained for Gzm M by
immunohistochemistry (Fig. 5D). As with
CD3+, CD56+ T cells, the
signal intensity for T cells showed some donor variability and
was usually less than that for an equivalent number of NK cells.
Therefore, Gzm M seems to be constitutively expressed at high levels in
NK cells (with both large and small lymphocyte morphology),
CD3+, CD56+ T cells, and
T cells, whereas it is not normally present in T cells
even after short term in vitro activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells, whereas
T cells do not express Gzm M even after short term in vitro
activation. Although five human granzymes have been identified, cloned, and sequenced for a number of years, their biological function(s) is still unclear. The association of these serine proteases together with perforin within the lytic granules of cytotoxic cells suggested an important role for these enzymes in target cell lysis. Indeed, it has been suggested that under physiological conditions the delivery of Gzm A and B is critical for target cells to undergo lysis (28). These authors therefore suggested that Gzm A and B probably account for all the death effector machinery delivered to target cells via perforin. However, other studies suggested that the role of Gzm A in target cell lysis was dispensable (14, 15). Therefore, the exact biological function of Gzm A remains unclear.
Furthermore, cytotoxic cells from Gzm B-/- mice and wild-type B6 mice demonstrate similar levels of lysis of target cells (although the kinetics of lysis may be delayed in Gzm B-/-). Therefore the major defect in Gzm B-/- cytotoxic cells is the induction of apoptosis in target cells (29, 30). Gzm A-/- and Gzm B-/- mice both have increased sensitivity to infection by the pox virus ectromelia (31, 32). Interestingly Gzm A x B-/- mice are extremely sensitive to ectromelia infection, being almost as sensitive as Pfp-/- mice. Furthermore, Gzm A x B -/- cytotoxic cells have no intrinsic defect in cytotoxic potential (32), suggesting the cytolytic process alone is insufficient to allow complete recovery from pox virus infection. Therefore, other synergistic anti-viral effects of these Gzm must occur in addition to lysis of target cells. One hypothesis is that Gzm may facilitate damage to viral DNA during target cell destruction, which, in turn, can reduce levels of infectious ectromelia virus. Viruses often encode for various protease inhibitors that presumably benefit viral replication by the inhibition of granzymes and caspases involved in target cell destruction. Therefore, it could be argued that the host may have evolved granzymes with a variety of enzymatic specificities to counteract the effects of viral protease inhibitors (4). Interestingly, during ectromelia infection increased viral titers were observed in Gzm A x B-/- mice as early as 3 days postinfection. This suggests that there is an early control of multiplication and/or spreading of virus by Gzm A and Gzm B before the appearance of immune CTL. Consequently, it is likely that Gzm A, Gzm B, and perforin from NK cells execute a critical first line of defense against ectromelia infection.
The preferential expression of Gzm M in NK, CD3+,
CD56+ T cells and T cells therefore may be
of significance. The recognition structures of these cells are thought
to be derived from a more limited repertoire than those of T
cells or B cells. This limited repertoire suggests recognition of a
highly restricted set of ligands. Indeed, recent data suggest that
T cells may recognize conserved molecular constituents of
infectious agents themselves or specific changes in surface proteins of
infected or damaged cells. Therefore, the V9-V2 subset of
T cells predominant in peripheral blood may recognize
nonpeptide-phosphorylated metabolite ligands found on infectious agents
or damaged cells (33, 34, 35). The predominant V1 T
cells of the intestine seem to recognize MIC (MHC class I
chain-related) proteins like MICA and MICB. These are the products of
divergent human class I genes regulated by promoter heat shock elements
(36). More recent data suggests that MICA and MICB may
also act as recognition structures for NK cells (37, 38).
NK T cells of the mouse recognize glycosylceramides in association with
the nonclassical class I molecule CDld (39) and the human
equivalents of NK T cells also seem to recognize glycolipid ligands in
association with CD1 molecules (40). It is likely that
most of the human NK T cells are present in our
CD3+, CD56+ T cell subset
(41); however, further studies using the appropriate
anti-V and anti-V reagents to define human NK T cells are
necessary.
The broader cross-reactivity of T cells, NK T cells, and NK
cells are features associated with cells of the innate immune system
and support the common assumption that these cells may have different
functions from T cells, which represent mediators of adaptive
immunity. NK cells and T cells are recruited very rapidly and
participate in early phases of immune responses. Prompt activation of
NK cells and T cells would provide a surveillance function of
infected and damaged cells whereby the well-documented lytic capacities
of these cells or other effector mechanisms could be of importance.
Alternatively, local release of cytokines by these cells may promote
the development of adaptive immune responses. Since Gzm M expression is
closely associated with NK cells and minor T cell subsets, it is
tempting to speculate that this enzyme may play some role either in the
effector phase of innate immune responses or, alternatively, in the
development of adaptive immune responses. Granulysin (a granule protein
of NK and T cells) has antimicrobial properties, and in combination
with perforin may control levels of intracellular bacteria
(42). Also, gene targeting of the neutrophil granule
protein elastase demonstrated that elastase-/-
mice were more susceptible to sepsis and death following i.p. injection
with Gram-negative bacteria (43). This effect was
apparently due to decreased intracellular killing of bacteria by
neutrophils. Therefore, these two granule proteins may play a role in
the effector phase of immune responses to microbes. The production of
recombinant Gzm M and the development of mouse strains in which Gzm M
has been functionally eliminated by gene targeting should provide some
information about the main physiological role of this unusual
granzyme.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas Sayers, Science Applications International Corporation-Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Building 560, Room 31-30, Frederick, MD 21702-1201.
3 Abbreviations used in this paper: Gzm, granzyme; TCC, T cell clone; MIC, MHC class I chain-related.
Received for publication March 22, 2000. Accepted for publication October 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the signaling chain of the T-cell receptor complex. Blood 91:585.9-V2 cells are stimulated in a cross-reactive fashion by a variety of phosphorylated metabolites. Eur. J. Immunol. 25:2052.[Medline]
T cells. Science 279:1737.14 NKT cells by glycosylceramides. Science 278:1626.24 and V11 coexpression defines a human NK1 T cell analog containing a unique Th0 subpopulation. J. Immunol. 159:5862.[Abstract]
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W. J. Grossman, J. W. Verbsky, B. L. Tollefsen, C. Kemper, J. P. Atkinson, and T. J. Ley Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells Blood, November 1, 2004; 104(9): 2840 - 2848. [Abstract] [Full Text] [PDF]
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S. S. Metkar and C. J. Froelich Human neutrophils lack granzyme A, granzyme B, and perforin Blood, August 1, 2004; 104(3): 905 - 906. [Full Text] [PDF]
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K. A. Sedelies, T. J. Sayers, K. M. Edwards, W. Chen, D. G. Pellicci, D. I. Godfrey, and J. A. Trapani Discordant Regulation of Granzyme H and Granzyme B Expression in Human Lymphocytes J. Biol. Chem., June 18, 2004; 279(25): 26581 - 26587. [Abstract] [Full Text] [PDF]
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K. Hochegger, P. Eller, and A. R. Rosenkranz Granzyme A: an additional weapon of human polymorphonuclear neutrophils (PMNs) in innate immunity? Blood, February 1, 2004; 103(3): 1176 - 1176. [Full Text] [PDF]
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T. J. Sayers, A. D. Brooks, C. Y. Koh, W. Ma, N. Seki, A. Raziuddin, B. R. Blazar, X. Zhang, P. J. Elliott, and W. J. Murphy The proteasome inhibitor PS-341 sensitizes neoplastic cells to TRAIL-mediated apoptosis by reducing levels of c-FLIP Blood, July 1, 2003; 102(1): 303 - 310. [Abstract] [Full Text] [PDF]
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L. Krenacs, M. J. Smyth, E. Bagdi, T. Krenacs, L. Kopper, T. Rudiger, A. Zettl, H. K. Muller-Hermelink, E. S. Jaffe, and M. Raffeld The serine protease granzyme M is preferentially expressed in NK-cell, gamma delta T-cell, and intestinal T-cell lymphomas: evidence of origin from lymphocytes involved in innate immunity Blood, May 1, 2003; 101(9): 3590 - 3593. [Abstract] [Full Text] [PDF]
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 N. Seki, Y. Hayakawa, A. D. Brooks, J. Wine, R. H. Wiltrout, H. Yagita, J. E. Tanner, M. J. Smyth, and T. J. Sayers Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated Apoptosis Is an Important Endogenous Mechanism for Resistance to Liver Metastases in Murine Renal Cancer Cancer Res., January 1, 2003; 63(1): 207 - 213. [Abstract] [Full Text] [PDF]
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A. J. da Silva, M. Brickelmaier, G. R. Majeau, Z. Li, L. Su, Y.-M. Hsu, and P. S. Hochman Alefacept, an Immunomodulatory Recombinant LFA-3/IgG1 Fusion Protein, Induces CD16 Signaling and CD2/CD16-Dependent Apoptosis of CD2+ Cells J. Immunol., May 1, 2002; 168(9): 4462 - 4471. [Abstract] [Full Text] [PDF]
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