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Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN 
, and IL-2 were sequentially acquired on
recognition of Ag, but surprisingly, only around half of the
CMV-specific CD8+ T cells responded to antigenic stimuli
with production of any cytokine measured. A dominant population
coexpressed TNF- and IFN-, and cells expressing TNF- only,
IFN- only, or all three cytokines together also occurred at lower
but clearly detectable frequencies. Interestingly, perforin expression
and production of IFN- and TNF- in CD8+ T cells
responding to staphylococcal enterotoxin B appeared to be largely
segregated, and no IL-2 was detected in perforin-positive cells.
Together, these data indicate that human CD8+ T cells can
be functionally segregated in vivo and have implications for the
understanding of human CD8+ T cell differentiation and
specialization and regulation of effector
mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytokines play important and diverse roles in controlling many viral
infections (1, 3, 4, 5). For example, IFN- and TNF- can
abolish replication of hepatitis B virus and lymphocytic
choriomeningitis virus
(LCMV)3 in hepatocytes
and clear the infection without destruction of infected cells (6, 7). Although important for immune defense, TNF- and IFN-
also are the primary mediators of septic shock caused by bacterial
toxins (8). However, during LCMV infection, effector T
cells in mice can rapidly turn on cytokine production on recognition of
specific Ag, and rapidly turn off production when Ag stimulation ceases
(9). This Ag-specific regulation of cytokine release
provides a means to localize the effects of cytokines in the vicinity
of infection while limiting the dangerous systemic effects
(10).
Another major attribute of CD8+ T cells is their ability to kill cells expressing specific peptide epitopes in complex with MHC class I through the release of cytotoxic granules containing perforin and granzymes A and B (2). Work with mice deficient in perforin have shown that this effector molecule is necessary for clearance of acute LCMV infection and for defense against Theiler’s virus and ectromelia virus (11, 12, 13), and it is of less importance for defense against other viruses including cowpox, coxsackievirus, and Semliki Forest virus (1, 2, 11, 13, 14). Perforin facilitates the entry of the granzymes A and B into target cells, where they induce apoptosis (15). The relative role of perforin and granzymes in inducing target cell death is uncertain, although recent evidence indicates that the importance of granzymes may be greater than previously thought (16, 17). Most interestingly, mice lacking granzyme A and B are equally incapable of controlling ectromelia infection as the perforin-deficient mice (18).
The CD45RA, CD27, and CD57 markers have been used to distinguish subsets of human CD8+ T cells (19). According to this classification, naive cells are believed to be CD27+45RA+57- and effector cells CD27-45RA+57+, whereas memory cells express CD27 and are negative for CD45RA and CD57, based on functional characteristics on polyclonal stimulation. In contrast, the CD45RO isoform replaces CD45RA in T cells after activation and has thus been used as a memory CD8+ T cell marker (20). Because CD45RO+ cells can reacquire expression of the RA isoform, it has been suggested that long-lived memory cells may be found in the CD45RA/RO double-positive population (21). More recently, Ag-experienced circulating T cells were grouped into effector memory and central memory cells, based on expression of the lymph node homing receptors chemokine receptor 7 and CD62L (22). The corresponding populations also have been described and carefully investigated in mice with CD62L and CD44 (23).
Although it is known that human CD8+ T cells do
express cytokines and cytolytic effector molecules, less is known about
the phenotype of CD8+ T cells responding with
cytokine production and cytolysis on recognition of specific Ag.
Furthermore, whether different cytokines and cytolytic molecules are
uniformly coexpressed on activation with Ag, or form discrete
subpopulations, is unknown. In this paper, we have studied human
CD8+ T cells specific for the HLA-A2-restricted
CMV pp65495–503 epitope and cells responding to
the superantigen staphylococcal enterotoxin B (SEB). CMV is a
ubiquitous human pathogen that persists indefinitely under efficient
control by the immune system. However, reactivation of the virus can
cause severe disease in immunosuppressed individuals, and strong
CD8+ T cell responses to CMV are commonly
detected in HIV-infected subjects (24, 25, 26). SEB is
produced by the common human pathogen Staphylococcus aureus
and activates T cells bearing TCR with specific V
regions
(27). We find that CD8+ T cells
responding to these Ags are functionally more diverse than previously
thought. We hypothesize that the CD8+ T cell
compartment may be able to choose preferential effector mechanisms
depending on site of infection, type of infectious agent, and severity
of infection. The results are further discussed in relation to the
identity of effector and memory T cell populations, and the
immunoregulatory role of perforin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC from healthy donors were isolated from heparinized whole blood by Ficoll-Paque PLUS density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden), and washed three times in RPMI 1640 before use in functional assays or cryopreserved. The study was approved by the institutional review board. B cell lines (BCL) were obtained by EBV transformation and were maintained at 37°C and 5% CO2 in complete tissue culture medium; RPMI 1640 tissue culture medium supplemented with 15% FCS, 50 µg/ml streptomycin, 100 µg/ml penicillin, and 2 mM L-glutamine. The peptide corresponding to the HLA-A2-restricted CMV pp65495–503 NLVPMVATV epitope was synthesized using solid phase F-moc chemistry (28, 29). SEB was purchased from Sigma (St. Louis, MO).
mAbs and HLA-A2 tetramers
The following mAbs were purchased from BD Biosciences (San Jose,
CA): anti-CD3 PE, anti-CD3 APC, anti-CD8 PerCp,
anti-CD27 FITC, anti-CD28 FITC, anti-CD45RA FITC,
anti-CD45RO FITC, anti-CD57 FITC, anti-CD62L FITC,
anti-perforin FITC, anti-perforin PE, anti-TNF- APC,
anti-IFN- PE, anti-IFN- FITC, and anti-IL-2 PE.
Anti-granzyme B FITC and PE conjugates were obtained from Hoelzel
Diagnostika (Koeln, Germany). Anti-HLA-A2 FITC-conjugated Ab was
obtained from One Lambda (Canoga Park, CA). HLA-A2 tetrameric complexes
were a gift from Dr. G. S. Ogg and were produced as described
previously (30). Briefly, recombinant HLA-A2 H chain with
the transmembrane domain replaced with a BirA biotinylation substrate
sequence and 2-microglobulin were expressed in
Escherichia coli as inclusion bodies and purified. Complexes
of HLA-A2 H chains, 2-microglobulin, and CMV
pp65 peptide were refolded and biotinylated with purified BirA enzyme.
The biotinylated MHC-peptide complexes were recovered by FPLC
purification and ion exchange chromatography. Multivalent complexes
were prepared by mixing biotinylated protein complex with
streptavidin-PE (Sigma) at a molar ratio of 4:1.
Intracellular staining and flow cytometry
Measurement of surface marker expression, cytokine production, and perforin and granzyme B content in CD8+ T cells was performed by combined surface and intracellular staining with mAbs and subsequent four-color flow cytometric analysis (31). PBMC were either fresh or thawed and cultured overnight before being used in experiments. Phenotyping of CMV pp65-specific cells with HLA-A2/pp65 tetramers, mAbs against surface markers, intracellular perforin, and granzyme B was performed in PBMC ex vivo without prior stimulation. After incubation with tetramer and Abs for 30 min at 4°C, cells were washed three times and analyzed on a FACSCalibur (BD Biosciences) instrument with CellQuest software. Data presented on HLA-A2/pp65 tetramer staining cells was gated on the CD3 and CD8 double-positive population to avoid nonspecific background staining. Background levels of tetramer staining in nonresponding individuals was below detection (<0.01%). For detection of cytokine responses, 3 x 106 PBMC were stimulated with either SEB (5 µg/ml) or 0.1 µM antigenic peptide in the presence of purified anti-CD28 (3 µg/ml; BD Biosciences) and brefeldin A (15 µg/ml; Sigma) in 1 ml of complete tissue culture medium. For detection of cytokines, PBMC were stimulated for a standard 6 h, or in kinetics experiments, for 1–15 h. Anti-CD28 was included to provide additional costimulation. After an initial wash in PBS supplemented with 5% FCS, stimulated and unstimulated control PBMC were stained with mAbs against cell surface markers for 30 min at 4°C and subsequently washed once. For intracellular staining, cells were permeabilized with FACS permeabilizing solution (BD Biosciences) for 15 min at room temperature. Cells then were washed twice and incubated with Abs against intracellular markers for 30 min at 4°C, washed three times, and analyzed on a FACSCalibur (BD Biosciences) instrument with CellQuest software. Between 1 x 105 and 2.5 x 105 events were collected in tetramer and intracellular staining experiments depending on sample availability. Unstimulated control samples showed a low background staining that was subtracted in calculating the frequency of cytokine-producing cells. Staining with isotype control Abs was consistently negative.
CD8+ T cell lines and cytotoxicity assay
Short-term CMV pp65-specific CD8+ T cell lines were established by culturing PBMC from healthy donors with circulating pp65-specific CD8+ T cells, as determined by HLA-A2/pp65 tetramer staining, for 6 days in complete tissue culture medium in the presence of 0.1 µM of pp65495–503 peptide. These T cell lines were subsequently analyzed for expression of surface markers, intracellular perforin, and granzyme B or used as effectors in a cytotoxicity assay. CTL activity was measured in a standard 51Cr-release assay. Briefly, peptide-coated BCL target cells were prepared by incubating cells with 10 µM peptide for 1 h at 37°C. Coated cells were labeled with 10 µl of 10 mCi/ml 51Cr for 1 h at 37°C. Titrated numbers of effector cells were incubated with 3 x 103 51Cr-labeled target cells for 4 h at 37°C, 5% CO2. After incubation, released radioactivity was measured and specific lysis was calculated according to the following formula: % specific release = ((experimental release - spontaneous release)/(maximum release - spontaneous release)) x 100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first were interested in the phenotype of human
CD8+ T cells specific for a well described MHC
class I-restricted peptide epitope, in terms of surface markers,
cytolytic effector molecules, and cytokines produced after recognition
of Ag. We addressed this by investigating circulating
CD8+ T cells specific for CMV with four-color
cytometry. Of 21 HLA-A2-positive healthy donors (H1-H21), PBMC from
donors H3, H7, and H18 had detectable numbers of
CD8+ T cells specific for the HLA-A2-restricted
CMV pp65495–503 epitope as determined by
tetramer staining. This CD8+ T cell population
has experienced Ag, is readily detectable with HLA-A2/pp65 tetramers,
and could be expected to have either a memory or an effector phenotype.
The frequency of pp65-specific cells ranged from 0.34 to 0.92% of
CD3+8+ cells in these three
donors (Table I
). The pp65-specific
CD8+ T cells displayed a mixed phenotype in terms
of surface markers, although there was a dominance of CD45RO over
CD45RA expression, and CD27 was more commonly expressed than
CD57.
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To assess the functional phenotype of circulating CMV
pp65-specific CD8+ T cells, the HLA-A2/pp65
tetramer was used together with intracellular staining with Abs against
perforin and granzyme B directly ex vivo. Surprisingly, most of
pp65-specific cells were low or negative in perforin expression,
whereas 43–67% of these cells were
positive for granzyme B (Table II and
Fig. 1). The relative lack of perforin
expression correlated with an absence of cytolytic capacity ex vivo
against BCL target cells incubated with the CMV
pp65495–503 peptide (Fig. 1).
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,
TNF-, and IL-2 in response to Ag as expected (Table II
). However,
only around half of pp65-specific
CD3+8+ cells produced
IFN- or TNF-. IL-2 expression could be detected in only <15% of
the specific CD8+ T cell population. Taken
together, these data show that expression of perforin and granzyme B is
not coordinately regulated in the CMV Ag-experienced
CD8+ T cell population. Furthermore, only around
half of all T cells carrying HLA-A2/pp65-specific TCRs respond with
production of any of the cytokines measured on recognition of CMV
peptide Ag. The phenotypic shift associated with Ag-driven differentiation into effector CTL
The CMV pp65-specific and HLA-A2-restricted
CD8+ T cells detected in circulation have
proliferated and differentiated in response to CMV infection in vivo. A
clear majority of CMV pp65-specific CD8+ T cells
in all three subjects do not express the lytic effector molecule
perforin, and no specific CTL activity was detected ex vivo. Thus,
these cells are most probably not lytic effector CTL, but rather some
kind of memory T cells. To investigate the response and phenotypic
changes in these T cells on recognition of Ag, PBMC from these donors
were cultured for 6 days in the presence of CMV pp65 peptide in vitro.
As expected, there was a strong expansion of the CMV pp65-specific
CD8+ T cell population. Furthermore, virtually
all of these cells acquired expression of both perforin and granzyme B
(Fig. 1). Acquisition of perforin correlated with lytic capacity
against pp65-peptide-coated BCL target cells (Fig. 1).
We next investigated the expression of surface markers before and after
the 6 day in vitro stimulation (Fig. 2).
CD45RA/RO isoform expression changed from a mixed pattern to
predominantly CD45RO+ and
CD45RA-. CD27, CD28, and CD57 expression
remained mixed. Thus, short-term in vitro stimulation of CMV
pp65-specific CD8+ T cells leads to uniform
expression of lytic effector molecules and acquisition of lytic
capacity. This functional shift is accompanied with up-regulation of
CD45RO and down-regulation of CD45RA.
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, and IFN- coexpression reveals
functional heterogeneity in CD8+ T lymphocytes
Although it is well-established that CD8+ T
cells produce diverse cytokines on antigenic stimulation, it is not
known whether these cells are homogeneous with regard to coexpression
of cytokines. Therefore, we investigated the expression of IL-2,
IFN-, and TNF- in CD8+ cells on direct ex
vivo 6-h stimulation of PBMC from donors H3, H7, and H18 with CMV pp65
peptide with four-color flow cytometry and simultaneous triple cytokine
staining. Patterns of coexpression of the three cytokines were analyzed
by gating and calculating the number of cells staining for one, two, or
three cytokines. Initial analysis showed that IFN- and TNF-
double-expressing cells were the most prevalent in the CMV
pp65-specific CD8+ T cell population (Fig. 3). CMV-specific cells producing either
IFN- only or TNF- only also could be detected as well as a small
population, 5–10% of responding cells, expressing all three
cytokines.
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,
and TNF- we used the superantigen SEB, which has the advantage of
stimulating a population of larger size while still activating T cells
via the TCR.
Initial analysis indicated CD8+ T cell
heterogeneity in cytokine expression 6 h after recognition of SEB
(Fig. 4A). Production of
IFN- and TNF- was readily detected after 1 h, and
significant IL-2 expression was detected first after 3 h (Fig. 5A). The number of
CD8+ T cells expressing at least one of the three
cytokines was close to maximal at 6 h, with only a marginal
increase at 15 h. The patterns of coexpression of the three
cytokines over time were analyzed by gating and calculating the number
of cells staining for one, two, or three cytokines. The dominant
population throughout the stimulation period coexpressed IFN- and
TNF- only (Fig. 5B). However, a population that expressed
all three cytokines emerged with delayed kinetics to reach a similar
frequency at later time points. Minor populations, which expressed
TNF- only, IFN- only, and IL-2 and TNF- together, also were
detectable. However, cells expressing IL-2 only or IL-2 together with
IFN- could not be detected in the response to SEB. Thus, we conclude
that CD8+ T cells are clearly heterogeneous with
regard to IL-2, IFN-, and TNF- expression after antigenic
stimulation.
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C). This analysis revealed that expression of TNF- alone
was the dominant phenotype at 1 h of stimulation. However,
coexpression of IFN- and TNF- rapidly became the most prevalent
phenotype representing up to 50% of cytokine-producing cells, followed
by IL-2, IFN-, and TNF- triple expression at later time points.
Approximately 30% of the cytokine-producing CD8+
T cells coexpressed IL-2, IFN-, and TNF- at later time points.
Taken together, these results demonstrate that single
CD8+ T cells may express one of several different
cytokine profiles in response to Ag. Furthermore, the data indicate the
sequential acquisition of TNF-, IFN-, and IL-2 expression in the
subpopulation that coexpresses these three cytokines. Cytokines and perforin segregates into subpopulations of CD8+ T cells that can be distinguished by surface markers
Next we examined whether circulating perforin-expressing
CD8+ T cells in healthy individuals were capable
of cytokine production on recognition of SEB. Virtually no IL-2
production was observed in perforin-expressing cells (Fig. 4B). Both IFN- and TNF- production could be detected
in perforin-positive cells, but at a significantly lower frequency. In
light of these data, we were next interested in defining the surface
marker phenotype associated with IL-2, IFN-, and TNF- production
in CD8+ T cells (Fig. 6). IL-2-secreting cells were almost
exclusively CD27+, CD28+,
and CD57-. The pattern of CD45 expression was
less distinct, although the majority were CD45RO+
and CD45RA-. IFN--expressing cells showed a
pattern of cell surface marker expression similar to IL-2-expressing
cells. However, although the majority (
80%) of IFN--positive
cells were CD27+, CD28+,
and CD57-, there also were some
IFN--expressing cells within the reciprocal subsets.
TNF--expressing CD3+8+
cells showed a similar distribution of markers as the
IFN--expressing cells. In contrast, perforin-positive cells were
almost exclusively CD57+ and CD45RA high, while
being largely negative for the CD27 and CD28 markers, as well as for
CD45RO (Fig. 6). These cells also were CD56+ and
CD161- (data not shown). Thus, perforin
expression in peripheral blood of healthy individuals is found in
CD8+ T cells with a surface phenotype largely
opposite that of IL-2-, IFN--, and TNF--producing cells. Taken
together these data indicate that expression of cytokines and perforin
segregates into subpopulations of CD8+ T cells
that can be distinguished by surface markers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that circulating CMV pp65-specific
CD8+ T cells express granzyme B much more
frequently than perforin indicates that these effector molecules are
differentially regulated (Fig. 1 and Table II). Tight control of
perforin expression seems rational given the cytotoxic potential of
this agent. Granzymes need to act together with perforin to induce
apoptosis in target cells, and regulating perforin may thus obliterate
the need for a tight control of the granzymes (18, 32). In
the mouse, memory CD8+ T cells established after
LCMV infection express perforin mRNA while not being cytotoxic directly
ex vivo (33). Taken together, these data suggest that when
T cells are not actively recognizing Ag, they down-regulate perforin
expression by a posttranscriptional mechanism but keep expressing the
granzymes.
Production of cytokines segregates into discrete subpopulations and
occurs with different kinetics. Detailed analysis indicates that
superantigen-stimulated CD8+ T cells first
initiate TNF- production, tightly followed by IFN-, and IL-2
production comes late (Fig. 5). Thus, the triple-expressing phenotype
is very rare after 1–2 h, although it becomes more prevalent later on.
However, this phenotype does not become dominant. Instead, TNF- and
IFN- double-expressing cells are dominant at 6 h of
stimulation. Minor populations comprising up to 10% of responding
CD8+ T cells include IL-2 plus TNF-
double-expressing cells and TNF- and IFN- single-expressing
cells. The combination of IL-2 plus IFN- and IL-2 single-expressing
cells are virtually absent. We observe similar dominance of TNF- and
IFN- double expression in analysis of CMV pp65-specific cells,
although detailed analysis is harder because of lower cell numbers
(Fig. 3). Our data suggest that cells expressing TNF- only or
IFN- only are more common and that cells producing all three
cytokines are less common in the CMV-specific population compared with
the more diverse SEB-reactive population. In support of the segregation
we observe in cytokine expression, single-cell analysis at the
molecular level of IL-2 gene expression in mouse models have recently
indicated that T cells are heterogeneous with regard to IL-2 expression
(34) and that IL-2 and IFN- are rarely coexpressed
after mitogenic stimulation (35).
The functional heterogeneity in CD8+ T cells that
we have observed may reflect an ability to adjust immune effector
mechanisms depending on the task. Experiments with gene knockout mice
have shown that perforin is necessary in defense against ectromelia
virus, whereas perforin deficiency had little impact on resistance to
cowpox virus (13). Also, IFN- together with TNF- can
clear hepatitis B virus and LCMV from hepatocytes noncytopathically
(6, 7). Our hypothesis is that the
CD8+ T cell compartment may be able to choose
preferential effector mechanisms depending on site of infection, type
of infectious agent, and severity of infection.
Circulating CMV pp65-specific CD8+ T cells are
heterogeneous with regard to the markers CD45RO, CD45RA, CD27, CD28,
CD57, and CD62L (Table I and Ref. 36). Because perforin
expression correlates with CMV pp65-specific CTL activity, expression
of perforin may be a reliable marker to distinguish a lytic effector T
cell population from a nonlytic memory cell population. The granzyme
B+ perforin- population
may fall within the effector memory population previously suggested by
others (22). Interestingly, immunohistochemical analysis
of lymphoid tissue from HIV-infected subjects have indicated previously
a relative lack of perforin compared with granzyme A (37).
CMV pp65-specific T cells in HIV-infected subjects were recently found
to express perforin more frequently than observed here in healthy
individuals (38). This difference may relate to
reactivation of CMV in HIV-infected subjects.
Perforin-expressing cells within the circulating polyclonal
CD8+ T cell population in healthy donors is
generally positive for CD45RA and CD57, while being mostly negative for
CD27, CD28, and CD45RO (Fig. 6). The finding that circulating
perforin-positive cells are generally CD27- and
CD45RA+ previously have been taken as evidence
that these two markers can be used to distinguish specific effector CTL
(19). However, we observe that differentiation of
circulating CMV pp65-specific T cells into lytic CTL in vitro is
associated with a down regulation of CD45RA. Furthermore, CD45RO is
up-regulated, and no clear pattern of CD27, CD28, or CD57 expression
can be detected (Fig. 2). Thus, by studying an epitope-specific CTL
population we get indications that active effector CTL are
characterized by high CD45RO expression and absent or low CD45RA
expression. This is supported by the findings in patients with
infectious mononucleosis by Callan et al. (30), indicating
that EBV-specific CD8+ T cells are largely
CD45RO+ and CD45RA- during
the acute phase of disease when Ag is abundant.
Our results indicate that neither circulating CMV-specific
CD8+ T cells nor activated CMV pp65-specific
effector CTL are contained within the
CD45RA+CD57+perforin+
triple-positive population of circulating CD8+ T
cells. Perforin generally is found in cells with a surface phenotype
opposite that of the cells producing cytokine in response to SEB, and
cytokine-producing cells are rarely found in the perforin-containing
population (Figs. 4B and 6). Thus, perforin expression and
cytokine production in response to SEB appears largely segregated in
healthy individuals. Increasing evidence indicates that perforin plays
an important role in down-regulating immune responses and autoimmunity
(39, 40, 41, 42, 43). Furthermore, CD57+ T cells
are expanded in HIV infection (44) and can suppress
generation of EBV-specific CTL in vitro (45). Oligoclonal
expansions of CD57+ CD8+ T
cells have been shown to occur in CMV infection (46).
However, only a minority of these cells were CMV-specific and
MHC-restricted, and the major activity displayed by these cells was
MHC-unrestricted proliferation (46). Taken together, these
results open the possibility that the
CD45RA+CD57+perforin+
population contains cells with an immunoregulatory role rather than
being CTL-engaged in immune defense against pathogens.
In summary, we have shown that the expression patterns of five different molecules that human CD8+ T cells use to respond against infection are unexpectedly complex. We have further analyzed the phenotype and function of an epitope-specific CD8+ T cell population and the identity of perforin-expressing cells ex vivo. These aspects of CD8+ T cell biology are of importance for our understanding of how these cells function to protect the organism against infectious disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Johan K. Sandberg, Gladstone Institute of Virology and Immunology. University of California, P.O. Box 419100, San Francisco, CA 94141-9100. E-mail address: jsandberg{at}gladstone.ucsf.edu
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; SEB, staphylococcal enterotoxin B; BCL, B cell line.
Received for publication February 6, 2001. Accepted for publication April 26, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T. Kozako, N. Arima, S. Toji, I. Masamoto, M. Akimoto, H. Hamada, X.-F. Che, H. Fujiwara, K. Matsushita, M. Tokunaga, et al. Reduced Frequency, Diversity, and Function of Human T Cell Leukemia Virus Type 1-Specific CD8+ T Cell in Adult T Cell Leukemia Patients J. Immunol., October 15, 2006; 177(8): 5718 - 5726. [Abstract] [Full Text] [PDF]
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J. J. Obar, S. Fuse, E. K. Leung, S. C. Bellfy, and E. J. Usherwood Gammaherpesvirus persistence alters key CD8 T-cell memory characteristics and enhances antiviral protection. J. Virol., September 1, 2006; 80(17): 8303 - 8315. [Abstract] [Full Text] [PDF]
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Y. Meng, H. Harlin, J. P. O'Keefe, and T. F. Gajewski Induction of Cytotoxic Granules in Human Memory CD8+ T Cell Subsets Requires Cell Cycle Progression J. Immunol., August 1, 2006; 177(3): 1981 - 1987. [Abstract] [Full Text] [PDF]
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H. S. Bashyam, S. Green, and A. L. Rothman Dengue Virus-Reactive CD8+ T Cells Display Quantitative and Qualitative Differences in Their Response to Variant Epitopes of Heterologous Viral Serotypes. J. Immunol., March 1, 2006; 176(5): 2817 - 2824. [Abstract] [Full Text] [PDF]
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O. A. Shlobin, E. E. West, N. Lechtzin, S. M. Miller, M. Borja, J. B. Orens, L. K. Dropulic, and J. F. McDyer Persistent Cytomegalovirus-Specific Memory Responses in the Lung Allograft and Blood following Primary Infection in Lung Transplant Recipients J. Immunol., February 15, 2006; 176(4): 2625 - 2634. [Abstract] [Full Text] [PDF]
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M. Fujiwara, H. Takata, S. Oka, H. Tomiyama, and M. Takiguchi Patterns of Cytokine Production in Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Human CD8+ T Cells after Stimulation with HIV-1-Infected CD4+ T Cells J. Virol., October 1, 2005; 79(19): 12536 - 12543. [Abstract] [Full Text] [PDF]
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M. T. Rock, S. M. Yoder, P. F. Wright, T. R. Talbot, K. M. Edwards, and J. E. Crowe Jr Differential Regulation of Granzyme and Perforin in Effector and Memory T Cells following Smallpox Immunization J. Immunol., March 15, 2005; 174(6): 3757 - 3764. [Abstract] [Full Text] [PDF]
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N. Anfossi, J.-M. Doisne, M.-A. Peyrat, S. Ugolini, O. Bonnaud, D. Bossy, V. Pitard, P. Merville, J.-F. Moreau, J.-F. Delfraissy, et al. Coordinated Expression of Ig-Like Inhibitory MHC Class I Receptors and Acquisition of Cytotoxic Function in Human CD8+ T Cells J. Immunol., December 15, 2004; 173(12): 7223 - 7229. [Abstract] [Full Text] [PDF]
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E. M. Aandahl, M. F. Quigley, W. J. Moretto, M. Moll, V. D. Gonzalez, A. Sonnerborg, S. Lindback, F. M. Hecht, S. G. Deeks, M. G. Rosenberg, et al. Expansion of CD7low and CD7negative CD8 T-cell effector subsets in HIV-1 infection: correlation with antigenic load and reversion by antiretroviral treatment Blood, December 1, 2004; 104(12): 3672 - 3678. [Abstract] [Full Text] [PDF]
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N. Anfossi, S. H. Robbins, S. Ugolini, P. Georgel, K. Hoebe, C. Bouneaud, C. Ronet, A. Kaser, C. B. DiCioccio, E. Tomasello, et al. Expansion and Function of CD8+ T Cells Expressing Ly49 Inhibitory Receptors Specific for MHC Class I Molecules J. Immunol., September 15, 2004; 173(6): 3773 - 3782. [Abstract] [Full Text] [PDF]
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Z. Wang, C. La Rosa, S. Mekhoubad, S. F. Lacey, M. C. Villacres, S. Markel, J. Longmate, J. D. I. Ellenhorn, R. F. Siliciano, C. Buck, et al. Attenuated poxviruses generate clinically relevant frequencies of CMV-specific T cells Blood, August 1, 2004; 104(3): 847 - 856. [Abstract] [Full Text] [PDF]
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B. L. Shacklett, C. A. Cox, M. F. Quigley, C. Kreis, N. H. Stollman, M. A. Jacobson, J. Andersson, J. K. Sandberg, and D. F. Nixon Abundant Expression of Granzyme A, but Not Perforin, in Granules of CD8+ T Cells in GALT: Implications for Immune Control of HIV-1 Infection J. Immunol., July 1, 2004; 173(1): 641 - 648. [Abstract] [Full Text] [PDF]
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E. J. Wherry and R. Ahmed Memory CD8 T-Cell Differentiation during Viral Infection J. Virol., June 1, 2004; 78(11): 5535 - 5545. [Full Text] [PDF]
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E. Mallard, F. Vernel-Pauillac, T. Velu, F. Lehmann, J.-P. Abastado, M. Salcedo, and N. Bercovici IL-2 Production by Virus- and Tumor-Specific Human CD8 T Cells Is Determined by Their Fine Specificity J. Immunol., March 15, 2004; 172(6): 3963 - 3970. [Abstract] [Full Text] [PDF]
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 R. Yamasaki, K. Yokota, H. Okada, S. Hayashi, M. Mizuno, T. Yoshino, Y. Hirai, D. Saitou, T. Akagi, and K. Oguma Immune response in Helicobacter pylori-induced low-grade gastric-mucosa-associated lymphoid tissue (MALT) lymphoma J. Med. Microbiol., January 1, 2004; 53(1): 21 - 29. [Abstract] [Full Text] [PDF]
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N. L. Alves, B. Hooibrink, F. A. Arosa, and R. A. W. van Lier IL-15 induces antigen-independent expansion and differentiation of human naive CD8+ T cells in vitro Blood, October 1, 2003; 102(7): 2541 - 2546. [Abstract] [Full Text] [PDF]
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J. K. Sandberg, N. M. Fast, K. A. Jordan, S. N. Furlan, J. D. Barbour, G. Fennelly, J. Dobroszycki, H. M. L. Spiegel, A. Wiznia, M. G. Rosenberg, et al. HIV-Specific CD8+ T Cell Function in Children with Vertically Acquired HIV-1 Infection Is Critically Influenced by Age and the State of the CD4+ T Cell Compartment J. Immunol., April 15, 2003; 170(8): 4403 - 4410. [Abstract] [Full Text] [PDF]
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K. Abel, L. Compton, T. Rourke, D. Montefiori, D. Lu, K. Rothaeusler, L. Fritts, K. Bost, and C. J. Miller Simian-Human Immunodeficiency Virus SHIV89.6-Induced Protection against Intravaginal Challenge with Pathogenic SIVmac239 Is Independent of the Route of Immunization and Is Associated with a Combination of Cytotoxic T-Lymphocyte and Alpha Interferon Responses J. Virol., March 1, 2003; 77(5): 3099 - 3118. [Abstract] [Full Text] [PDF]
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E. M. Aandahl, J. K. Sandberg, K. P. Beckerman, K. Tasken, W. J. Moretto, and D. F. Nixon CD7 Is a Differentiation Marker That Identifies Multiple CD8 T Cell Effector Subsets J. Immunol., March 1, 2003; 170(5): 2349 - 2355. [Abstract] [Full Text] [PDF]
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E. V. Ravkov, C. M. Myrick, and J. D. Altman Immediate Early Effector Functions of Virus-Specific CD8+CCR7+ Memory Cells in Humans Defined by HLA and CC Chemokine Ligand 19 Tetramers J. Immunol., March 1, 2003; 170(5): 2461 - 2468. [Abstract] [Full Text] [PDF]
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D. Zhang, P. Shankar, Z. Xu, B. Harnisch, G. Chen, C. Lange, S. J. Lee, H. Valdez, M. M. Lederman, and J. Lieberman Most antiviral CD8 T cells during chronic viral infection do not express high levels of perforin and are not directly cytotoxic Blood, January 1, 2003; 101(1): 226 - 235. [Abstract] [Full Text] [PDF]
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E. Ozdemir, L. S. St. John, G. Gillespie, S. Rowland-Jones, R. E. Champlin, J. J. Molldrem, and K. V. Komanduri Cytomegalovirus reactivation following allogeneic stem cell transplantation is associated with the presence of dysfunctional antigen-specific CD8+ T cells Blood, November 15, 2002; 100(10): 3690 - 3697. [Abstract] [Full Text] [PDF]
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F. Buseyne, D. Scott-Algara, F. Porrot, B. Corre, N. Bellal, M. Burgard, C. Rouzioux, S. Blanche, and Y. Riviere Frequencies of Ex Vivo-Activated Human Immunodeficiency Virus Type 1-Specific Gamma-Interferon-Producing CD8+ T Cells in Infected Children Correlate Positively with Plasma Viral Load J. Virol., November 13, 2002; 76(24): 12414 - 12422. [Abstract] [Full Text] [PDF]
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H. Tomiyama, T. Matsuda, and M. Takiguchi Differentiation of Human CD8+ T Cells from a Memory to Memory/Effector Phenotype J. Immunol., June 1, 2002; 168(11): 5538 - 5550. [Abstract] [Full Text] [PDF]
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D. C. Douek, M. R. Betts, J. M. Brenchley, B. J. Hill, D. R. Ambrozak, K.-L. Ngai, N. J. Karandikar, J. P. Casazza, and R. A. Koup A Novel Approach to the Analysis of Specificity, Clonality, and Frequency of HIV-Specific T Cell Responses Reveals a Potential Mechanism for Control of Viral Escape J. Immunol., March 15, 2002; 168(6): 3099 - 3104. [Abstract] [Full Text] [PDF]
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) in fresh PBMC (left) and after a 6-day in vitro
stimulation with pp65 peptide (right).
