| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Laboratory of Clinical and Experimental Immunology and Endocrinology, Wadsworth Center, and
Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY 12201
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2
h) to peptide programmed the cells to proliferate independently of Ag.
Although not required for cell division, Ag increased the survival of
proliferating cells and increased the total number of cell divisions in
the expansion process. These results indicate that Ag exposure begins a
program of cell division that does not require but is modified by
further TCR stimulation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we have investigated the relationship between clonal expansion and continued T cell stimulation by peptide Ag. A model system was used in which Ag could be removed from the responding T cells after the initial stimulation and cell proliferation could be monitored. Furthermore, the initial responding T cells were physically separated from their subsequent daughter cells and the critical periods for TCR engagement were determined. Identification of T cells at different points in clonal expansion was accomplished using the fluorescent dye, CFSE (9, 10), which we have previously used to examine Ag-driven CD4 T cell proliferation and to identify effector/memory marker expression during clonal expansion (11). We have extended these studies to determine the effects of Ag stimulation on the duration of clonal expansion. This information will be important for determining and modifying pathways of T cell differentiation and memory development.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The BALB/c ByJ and DO11.10 (12) mice used in these
experiments were bred and maintained at the Wadsworth Center Animal
Core Facility (Albany, NY) under specific pathogen-free conditions. The
majority of T cells in the DO11.10 mice are CD4+
cells which bear a TCR that recognizes a chicken OVA-derived peptide,
OVA323–339 (hereafter referred to as OVA),
presented by I-Ad (12). This TCR is
encoded by transgenes encoding V
8.2/V
13.1 chains and can be
identified by the anticlonotypic mAb, KJ1-26 (13). The
DO11.10 mice were originally obtained from Dr. D. Loh (Kamakura,
Japan). Unless otherwise indicated, the experiments were
performed using 6- to 8-wk-old mice. Both male and female mice were
used in different experiments with no discernible differences in the
results. All mice used in these studies were bred and maintained in
accordance with the guidelines of the Committee on Care and Use of
Laboratory Animals of the Institute of Laboratory Resources (National
Research Council, Washington, DC).
Reagents and Abs
Chicken OVA peptide (OVA323–339) was
synthesized and supplied by the Wadsworth Center Peptide Synthesis Core
Facility. Polyclonal rabbit anti-mouse Ig was prepared by affinity
purification on mouse Ig-Sepharose columns. mAbs GK1.5
(14) and 2B6 (15) (anti-CD4), 3.155
(anti-Lyt-2 (CD8)) (16), M1/70.15 (anti-MAC-1)
(17), J11d.8 (anti-J11d) (18), HO13.4
(anti-Thy1.2) (19), Mar 18.5 and MARK-1 (anti-rat
-chain) (20), and KJ1-26 (anti-DO11.10 clonotype)
(13) were prepared from the supernatants of hybridoma cell
lines, as previously described (21). Biotinylated
anti-CD25 (mAb 3C7) and anti-CD69 (mAb H1.2F3) were purchased
from BD PharMingen (San Diego, CA). CFSE was purchased from Molecular
Probes (Eugene, OR). Mitomycin C was purchased from Sigma-Aldrich (St.
Louis, MO).
Preparation of cells
In all experiments, enriched populations of CD4+ T cells were prepared as previously described (22). Briefly, RBCs were removed by hypotonic lysis. B cells were depleted using rabbit anti-mouse Ig followed by goat anti-rabbit Ig-coated magnetic beads (Advanced Magnetics, Cambridge, MA) and adherence to a magnet. Residual B cells, macrophages, and CD8+ T cells were removed by Ab and complement depletion using anti-J11d, anti-MAC-1 plus MAR18.5, and anti-CD8, respectively. Baby rabbit serum (Wadsworth Center Animal Core Facility) was used as a source of complement. When these procedures were used, cells were 90–95% CD4+ and <3% surface Ig+ as determined by flow cytometric analyses. APCs were prepared by T cell depletion of splenocytes using anti-Thy1-1.2 and complement followed by anti-CD4 (mAb 2B6) and anti-CD8 plus complement. Unless otherwise indicated, APCs were treated with mitomycin C (25 µg/ml) for 20 min at 37°C.
Cell labeling and culture
CD4+ T cells were labeled with CFSE using previously described procedures (9, 11). After enrichment for CD4 cells, the cells (5 x 107 cells/ml) were incubated with CFSE (1 µM) in PBS for 10 min at 37°C. After washing three times with tissue culture medium containing 10% FBS (Life Technologies, Grand Island, NY), the cells were placed into tissue culture. Unless otherwise indicated, the unfractionated CD4 cells were cultured either in 96-well plates (1 x 105/well; Falcon Labware, Oxnard, CA) with 2 x 105/well APCs in 0.2 ml of RPMI 1640 medium supplemented with 10% FBS, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine or were cultured in bulk at a concentration of 1 x 106/ml with 2 x 106/ml APCs. Where indicated, OVA323–339 was added into the cultures. In all experiments, unless otherwise indicated, the data depicted are representative results from a minimum of three separate experiments.
Immunofluorescence staining, analysis, and sorting
Where indicated, mAbs were directly labeled with Cy5 (Biological Detection Systems, Pittsburgh, PA). Alternatively, biotinylated mAbs were detected using Cy5-streptavidin (Biological Detection Systems). Fluorescence staining was performed at 4°C in 100 µl with a predetermined optimal amount of primary Ab in balanced salt solution containing 2% FBS, 20 mM HEPES, and 0.1% NaN3. Staining with the secondary reagent was performed in a similar manner after washing the cells. Flow cytometry analyses and sorting of stained cells were performed using either a FACSCalibur or a FACSVantage (BD Biosciences, Mountain View, CA). With the latter cytometer, the Cy5 was excited using a helium-neon laser (632.6 nm emission). For sorting experiments, power of the 488-nm argon laser was reduced to 75 mW. Flow cytometry data was analyzed using CellQuest software (BD Biosciences). Percentages of cells at specific rounds of division were calculated using Modfit LT software (Verity Software House, Topsham, ME).
Adoptive transfer of DO11.10 T cells
The procedure is done as described by Kearney et al. (23) with some modifications. CFSE-labeled, DO11.10 CD4+ T cells (2.5 x 106) were suspended in sterile PBS and injected i.v. into 6- to 8-wk-old BALB/c mice. For sorted cells, lymph node cells were analyzed using flow cytometry to determine CFSE fluorescence on KJ1-26+ cells 48 h after adoptive transfer. For sorted cells, 1 day after adoptive transfer, some of the host mice were immunized s.c. with OVA323–339 (150 µg) in CFA while others were not immunized. For both groups, 48 h after the immunization period the axillary, brachial, and cervical lymph nodes (draining nodes for the immunized mice) were removed, pooled, and analyzed by flow cytometry.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To measure proliferation in response to culture with specific
peptide Ag, CD4 T cells obtained from DO11.10 mice (12)
were labeled with CFSE before culture with unlabeled APCs and
OVA323–339, and at different times the cells
were collected and examined by flow cytometry. As previously reported,
50–80% of the CD4+ T cells in DO11.10 mice
express both the transgenic TCR and TCR chains and are stimulated
by OVA/I-Ad (24). Hence, mAb KJ1-26
(13) was used to identify these clonotype-bearing T cells.
These cells proliferated when exposed to OVA and cumulative cell
division was indicated by the appearance of cells with decreased
fluorescence intensity. Cell division was limited to the
KJ1-26+ cells (Fig. 1
, a and b), and in
the absence of peptide the cells remained undivided, exhibiting a
single peak of high fluorescence intensity (Fig. 1, c and
d). Culture with OVA induced early activation of the
KJ1-26+ cells, as evidenced by expression of CD69
(Fig. 1c) and CD25 (Fig. 1d). Expression of both
markers was largely limited to the KJ1-26+ cells
after stimulation (data not shown). Indeed, at higher peptide doses
(e.g., 200 ng/ml) the percentages of KJ-126+
cells and the percentages of CD25+ and
CD69+ cells after stimulation were similar. These
data indicated that with these higher doses virtually all of the
KJ1-26+ cells had responded to peptide within
24 h. However, even at the highest tested peptide dose (2000
ng/ml), little proliferation was observed before 36 h. After this
time rapid cell division occurred such that by 60 h most cells had
divided, with the majority of cells having undergone three to four cell
divisions. Cell division was asynchronous; cells were distributed among
multiple divisions, including small numbers of
KJ1-26+ cells that remained undivided through
60–66 h in culture. At lower peptide doses, activation markers were
expressed on smaller numbers of KJ1-26+ cells and
fewer cells ultimately proceeded to divide. In addition, at lower
doses, the beginning of cell division was slightly delayed. Comparison
of the cell distribution among rounds of division indicates that cells
stimulated with 20 ng/ml OVA were approximately one cycle behind cells
stimulated with 10-fold higher amounts of peptide (Fig. 1b).
However, once cell division had begun, the rate of division appeared
similar, regardless of peptide dose (see Fig. 1b; 20 ng/ml
at 66 h vs 200 ng/ml at 60 h). Peptide dose appeared to more
profoundly affect the percentage of cells that ultimately
divided (Fig. 1, a and b). These data indicate
that Ag dose regulates the percentage of cells that will proliferate
but has a more modest effect on the rate of clonal expansion.
Furthermore, cell division is preceded by activation marker (CD69 and
CD25) expression.
|
The use of CFSE to measure cell growth enabled us to identify
proliferating cells based upon the numbers of cell divisions that had
occurred after initial stimulation. Using this technique, we wished to
determine whether the requirements to activate resting cells and
progress to the first cell division are the same as those required to
progress into each subsequent round of cell division. In the current
study we have focused on the role of Ag in both inducing and sustaining
clonal expansion. While it is clear that Ag is essential for initial
CD4 cell activation, the requirement for Ag in ongoing proliferation is
less clear. To determine whether TCR signaling is required for each
round of cell division, we established a model in which Ag could be
removed after initial cell activation. DO11.10 CD4 T cells were labeled
with CFSE before culture with unlabeled APCs and OVA. After 60 h,
the dividing cells were collected and were fluorescence cell-sorted
based on CFSE fluorescence. Before sorting, the T cell population
contained a mixture of cells that had undergone up to five rounds of
cell division (Fig. 2a).
Furthermore, a portion of the cells remained undivided, although the
induction of activation markers indicated that they had been exposed to
OVA during the culture period (Fig. 1
and data not shown). The
undivided cells were isolated (Fig. 2b) and returned to
culture with fresh, unlabeled APCs but in the absence of OVA. At
different times, the cells were analyzed by flow cytometry. As
indicated in Fig. 2c, the cells continued to divide after
the removal of Ag. These data suggest that cell division is triggered
by interaction of the resting cell with Ag and that a proliferation
program begins such that clonal expansion can proceed in the absence of
further exposure to Ag.
|
). To
determine whether similar concentrations of Ag would program cells to
proliferate after Ag was withdrawn, CFSE-labeled DO11.10 T cells were
cultured for 36 h with varying amounts of OVA. The cells were then
sorted to collect only the undivided T cells, which were then returned
to culture with fresh APCs. In parallel, freshly prepared CFSE-labeled
DO11.10 CD4 T cells were similarly cultured with varying amounts of
OVA. Cell proliferation was assessed by flow cytometry after a 60-h
culture period. As indicated in Fig. 3, proliferation occurred when the cells were exposed to as little as 20
ng/ml OVA, regardless of whether OVA was removed before the first cell
division. As in Fig. 1, the Ag dose appeared to exert a greater effect
on the proportion of cells that participated in clonal expansion and
only a modest effect on the rate of proliferation. At low Ag doses most
cells did not divide even if Ag was maintained in the cultures. At high
Ag doses greater numbers of cells were stimulated to proliferate and
were programmed to proliferate in the absence of Ag. Hence,
Ag-independent proliferation does not require any more extensive TCR
clustering than proliferation that occurs in the continuous presence
of Ag.
|
a).
Immunization with OVA, as with culture with peptide, promoted specific
cell division in the KJ1-26+ population isolated
from the lymph nodes of the host mice (Fig. 4b). To
determine whether the host mice would support Ag-independent
proliferation, CFSE-labeled DO11.10 CD4 T cells were cultured with
unlabeled APCs and OVA before cell sorting and isolation of
peptide-exposed, undivided T cells. These cells were injected into
BALB/c mice. Lymph nodes were isolated from the host mice 48 h
after transfer and the KJ1-26+ cells were
identified by flow cytometry. As indicated in Fig. 4, c and
d, if the cells were exposed to peptide before transfer,
proliferation occurred in vivo.
|
, a and b, in the
absence of added OVA, the unlabeled CD4 cells did not express either
CD69 or CD25 (Fig. 5, a and b, boxed). However,
even in the absence of exogenous OVA the sorted cells proliferated in
the secondary culture (Fig. 5, a and b, circled).
If OVA was introduced into the cultures, CD69 and CD25 were readily
induced on the unlabeled indicator DO11.10 cells (Fig. 5, c
and d). Indeed, activation marker expression was observed at
concentrations as low as 5 ng/ml OVA, an amount less than that needed
to induce extensive cell proliferation (Fig. 1 and data not shown).
Taken together, these data indicate that continued cell division can
proceed in an Ag-independent fashion after initial stimulation.
|
A program for Ag-independent CD4 cell proliferation was induced
rapidly upon culture initiation. CFSE-labeled cells were cultured with
unlabeled APCs and peptide for various times (0–16 h). No cell
division was observed during these culture times. At the end of the
primary cultures, all of the cells populations, including noncultured
cells (Fig. 6a), were labeled
with Abs directed against MHC class II molecules
(I-Ad) to identify the APCs before sorting the
undivided T cells. The T cells were returned to culture for 3 days in
the absence of peptide before analysis by flow cytometry to determine
the extent of cell proliferation in the secondary cultures. As
indicated in Fig. 6b, as little as 2 h of exposure to
peptide was sufficient to induce proliferation in the secondary
culture. There was little difference between the cell division profiles
of cells cultured with OVA for 2 h as compared with cells cultured
for 16 h (data not shown), which suggested that signaling through
the TCR occurred on all cells shortly after the primary cultures were
begun.
|
The experiments described thus far showed that signaling through
the TCR on resting CD4 T cells was sufficient to stimulate multiple
rounds of cell division. However, it was unclear whether Ag was needed
solely to begin the begin the expansion process. We next wished to
examine the consequences of TCR ligation and the effects of continuous
Ag stimulation during clonal expansion. As shown in Fig. 1, TCR
expression remained elevated during peptide-induced proliferation. Such
expression might indicate that dividing T cells might still be capable
of responding to Ag. To address whether or not T cells become
refractory to Ag during clonal expansion, we stimulated DO11.10 CD4 T
cells with OVA and isolated daughter cells from each specific round of
cell division. The cells were then returned to culture in the presence
or absence of OVA. After 48 h the cells were collected and
analyzed by flow cytometry to determine the extent of proliferation in
secondary culture. As expected, cells from each round of cell division
could continue to divide in the absence of Ag (Fig. 7, upper panels).
Interestingly, cell division was not uniform, as cells early in the
expansion process (originally undivided cells) proliferated more in the
secondary culture than did cells derived from the latter portion of the
initial proliferation. Indeed, after division three cells generally did
not divide more than twice within the 48-h secondary culture period (W.
T. Lee, unpublished observations). This result suggests that as clonal
expansion proceeds, each successive division possesses a diminishing
capacity for further division. However, growth could be renewed by
again signaling through the TCR. At all cell divisions the addition of
Ag resulted in an increased cell division (Fig. 7, lower
panels). Hence, although Ag is not required for
proliferation, the TCR can still be stimulated to promote growth
throughout clonal expansion.
|
, for both undivided and dividing cells
a greater number was recovered at any time if Ag was present in the
culture. Analysis at later time points is complicated by the additional
cell division induced by Ag; however, within the first 25–36 h of
reculture the division profiles, as indicated by CFSE fluorescence,
were similar (data not shown). Thus, these data suggest that a
contribution toward increased cell numbers is made by positive effects
of Ag on cell viability. A further example of the effects of Ag on cell
vigor is indicated by increased cytokine secretion. The culture
supernatants were collected after 48 h of the secondary culture
and the amount of IL-2 secreted by the cells was determined using
ELISA. As shown in Fig. 8, higher levels of IL-2 were found when OVA
was present in the cultures. Hence, we conclude that although cell
division per se is Ag independent, cytokine secretion and cell
accumulation is maximal when Ag is continuously present.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As we and others have previously reported, after an initial lag period
stimulated CD4 cells rapidly divide (6, 11, 31, 32, 33).
Indeed, the observation of three to four cell divisions between 48 and
60 h after initiation of the cultures (see Fig. 1) suggests that
the initial doubling times are 4–6 h. However, not all cells are
stimulated to divide immediately, as cells are found at points of all
division numbers throughout the culture period, including cells that
had not divided by 66 h into the culture. Such nonuniform cell
division appeared to be characteristic of the T cells rather than due
to asynchronous contact with APCs, because increasing the numbers of
APCs as much as 10-fold did not alter the kinetics or percentages of
activated cells stimulated with any of the peptide concentrations (data
not shown). Furthermore, these proliferation properties were not due to
kinetic differences in activation, as within 24 h (and likely
sooner) all of the cells, including undivided cells, were stimulated to
express activation molecules. It is unclear as to why all cells are
activated early yet some cells begin proliferation early and some cells
begin proliferation very late. The presence of undivided cells after
exposure to Ag has previously been noted (34, 35). In
these previous studies, Ag stimulation in vitro or in vivo consistently
resulted in a small population of cells which had not appeared to
respond to Ag. In our study, the cells eventually did divide upon
reculture in the presence or absence of Ag. Indeed, those same cells
would divide in the initial culture and in the continuous presence of
Ag if the cultures were maintained for longer periods (data not
shown).
We have previously reported on the acquisition of memory markers on
CD4+ T cells and the relationships of these
markers to cell division (11). In that study we found a
varied relationship between marker expression and proliferation. For
example, up-regulation of CD4 and CD44 preceded cell division and was
limited to the dividing cells, while decreases in CD62L were
independent of cell division. Other reports have shown that CD25 is
expressed on activated T cells before cell division and is restricted
to the dividing cells (36). In the present study we have
extended these earlier findings to show that both CD69 and CD25 are
increased before cell division and may require less Ag for expression
than is required for proliferation. We cannot exclude that the small
numbers of cells which express the activation markers at low Ag doses
eventually divide but that the cells with the decreased CFSE
fluorescence were not detected in our analysis. It is also interesting
to note that increased expression of both of these activation markers
requires a continued presence of Ag, because marker expression is
rapidly lost in the absence of Ag (Fig. 5).
The expression of activation markers at different input doses of peptide offers insight into the relationship between TCR stimulation and cell division. At low peptide doses only small percentages of cells bear activation markers and little cell division is observed. In contrast, at high doses of peptide all of the KJ1-26+ cells express activation markers and all cells eventually divide. Increasing the peptide dose increases the percentage of activated cells, and more cells are found to proliferate. Examination of the division pattern indicates a slight difference in the numbers of cell divisions that are observed at each time with a modest shift in the onset of proliferation between suboptimal and optimal Ag doses. In contrast, there are large differences in the percentages of cells that divide with different Ag doses. These data suggest that under conditions where Ag is continuously present a threshold level of TCR clustering is needed to promote entry into cell division, and that once that level is achieved division proceeds at a fixed rate.
Once a threshold level of Ag is present, the initial triggering of the
TCR appears to be sufficient to induce cell division. Although it is
possible that reculturing the sorted cells separates them from an
inhibitory stimulus, such as signaling through CTLA-4, we find
that reculturing the cells with activated APCs does not diminish
subsequent proliferation and that cultures containing CTLA-4-Ig to
block interactions with B7 molecules do not affect the proliferative
outcome (W.T. Lee, unpublished observations). Thus, it is likely that
initial signaling induces gene expression and proteins involved in the
subsequent several rounds of cell division. In the absence of sustained
TCR signaling, the cells progressively lose the capacity for
proliferation and, within the same time frame, the peak cell numbers
appear to be at similar division points (approximately five to six
cycles). No round of cell division appears to have as great a
proliferative capacity as do activated, undivided cells (Fig. 6). This
may be because the cells are initially signaled and "programmed"
for a discrete number of cell divisions. However, it is also possible
that the cells produce growth promoting factors, such as cytokines,
that cells from subsequent divisions do not produce. Hence, separating
cells from individual division rounds from the initial undivided cells
might remove them from a source of a needed growth factor. We
also note that the addition of Ag to cells in all rounds of cell
division promotes further clonal expansion (Fig. 7). Hence, dividing
cells do not lose the capacity for TCR-mediated stimulation. The
additional cell growth in the presence of Ag suggests that either the
cells are induced to provide the central growth factor or that
stimulation of the TCR at any point during clonal expansion regenerates
division-promoting proteins.
Recent reports have indicated that CD8+ T cells
can proliferate independently of continued Ag stimulation
(31, 32, 33). In those studies exposure to Ag for as little as
2 h could program the cells to proliferate in the absence of Ag.
Likewise, we have shown that 2 h were sufficient to induce
proliferation of DO11.10 CD4+ T cells after
subsequent removal of OVA (Fig. 6). This time frame is similar to that
needed to form an immunological synapse and complete TCR-mediated
signal transduction (37, 38, 39). However, this is also a
shorter time than that previously indicated by other studies in which
12–24 h of TCR clustering were required to prime cells for
proliferation (4). It is unclear why our observations
using only a short exposure to Ag are different from these previous
studies. We believe that part of this discrepancy may be explained by
the nature of the ligand. We have observed that the superantigen
staphylococcal enterotoxin B
(SEB)3 stimulates
naive DO11.10 to proliferate even after SEB is removed (W.T. Lee,
unpublished observations). However, unlike OVA, the cells need to be
exposed to SEB for 8–12 h before superantigen-independent
proliferation occurs. An additional difference between our observations
and those from previous studies, including the work on CD8 cells, is
that we do not observe a decrease in TCR expression on OVA-treated
DO11.10 T cells after stimulation or at any point during clonal
expansion (see Fig. 1). It is unclear whether TCR down-regulation is
dependent on the nature of the ligand and whether this may be related
to the time required to program the cells for proliferation.
In this study we consistently recovered greater numbers of cells when Ag was present in the cultures, even at early time periods when the rate of cell division was similar to when Ag was absent. Furthermore, we find that cytokine secretion is minimal in the absence of Ag, even though the cells proliferate at the same rate as cells cultured in the presence of Ag. Although some of the IL-2 may have been used by the proliferating cells, preliminary experiments using neutralizing mAbs suggest that much of the secondary proliferation is IL-2 independent. Based upon our observations we propose that Ag plays two major roles in addition to initial activation of the resting naive T cell. We suggest that Ag extends the clonal expansion process by stimulating TCRs on dividing T cells and reinitiating synthesis of necessary cytokines or proteins required for continued cell division. We also suggest that Ag increases cell numbers early in the proliferative process, shifting the balance away from apoptosis and toward survival. However, our data in this aspect contrast with a previous study by Jelley-Gibbs et al. (6), which demonstrated that Ag induced cell death of effector cells in the midst of the culture. It is unclear as to why our results are different; however, we note some experimental differences such as the length of culture periods, addition of exogenous IL-2, and heterogeneity of the cell populations with respect to beginning cell division number (e.g., we began our cultures with stimulated cells all of a specific division number). A considerable difference may be that our examination focuses on early cell divisions and cells in the process of becoming effectors as opposed to effector cells themselves. Ongoing experiments will reconcile our data with this previous study.
An important observation in this study is that Ag-independent
proliferation can occur in vivo (Fig. 4). It is interesting to
speculate how this may contribute to normal immune responses.
Recent studies by Reinhardt et al. (40) have indicated
that shortly after activation responding T cells traffic to various
sites within the immunized host. Furthermore, proliferating T cells at
different rounds of cell division express different combinations of
adhesion and homing receptors (11). This suggests that
circulating cells at different division numbers may traffic to
different parts of the host and continue dividing in the absence of Ag.
Division and, presumably, differentiation at these different sites
permit the cells to respond to unique and various microenvironments. We
suggest that this allows for different types of effector and memory
specialization. Additional experiments are under way to address this
hypothesis.
In summary, we have studied the role of Ag and, by extension, TCR signaling during clonal expansion within primary T cell responses. We have shown that continued division of activated cells occurs independently of the presence of Ag. Daughter cells from each round of cell division have the capacity to divide in the absence of TCR ligation. However, cells from each round of cell division retain the ability for additional TCR signaling, such that Ag may continuously influence and lengthen the expansion process. We propose first that continued Ag exposure may increase the immune response and, second, that cell division in the absence of Ag permits growth and differentiation to occur at secondary sites within the host. The latter may contribute to functional diversity of the Ag-specific T cell response.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. William T. Lee, Laboratory of Clinical and Experimental Immunology and Endocrinology, Wadsworth Center, P.O. Box 22002, Albany, NY 12201-2002. E-mail address: William.Lee{at}wadsworth.org
3 Abbreviation used in this paper: SEB, staphylococcal enterotoxin B.
Received for publication September 19, 2001. Accepted for publication December 6, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T cell receptors. Annu. Rev. Immunol. 16:523.[Medline]
during T-cell activation. Nature 385:83.[Medline]
This article has been cited by other articles:
|
|
 |
|
  K. S. Mathurin, G. W. Martens, H. Kornfeld, and R. M. Welsh CD4 T-Cell-Mediated Heterologous Immunity between Mycobacteria and Poxviruses J. Virol., April 15, 2009; 83(8): 3528 - 3539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chen, L. Shao, Z. Ali, J. Cai, and Z. W. Chen NSOM/QD-based nanoscale immunofluorescence imaging of antigen-specific T-cell receptor responses during an in vivo clonal V{gamma}2V{delta}2 T-cell expansion Blood, April 15, 2008; 111(8): 4220 - 4232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. L. Wissinger, W. W. Stevens, S. M. Varga, and T. J. Braciale Proliferative Expansion and Acquisition of Effector Activity by Memory CD4+ T Cells in the Lungs following Pulmonary Virus Infection J. Immunol., March 1, 2008; 180(5): 2957 - 2966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Yarke, S. L. Dalheimer, N. Zhang, D. M. Catron, M. K. Jenkins, and D. L. Mueller Proliferating CD4+ T Cells Undergo Immediate Growth Arrest upon Cessation of TCR Signaling In Vivo J. Immunol., January 1, 2008; 180(1): 156 - 162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.-H. Hovav, M. W. Panas, S. Rahman, P. Sircar, G. Gillard, M. J. Cayabyab, and N. L. Letvin Duration of Antigen Expression In Vivo following DNA Immunization Modifies the Magnitude, Contraction, and Secondary Responses of CD8+ T Lymphocytes J. Immunol., November 15, 2007; 179(10): 6725 - 6733. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Mueller, M. S. Thomas, B. J. Burbach, E. J. Peterson, and Y. Shimizu Adhesion and Degranulation-Promoting Adapter Protein (ADAP) Positively Regulates T Cell Sensitivity to Antigen and T Cell Survival J. Immunol., September 15, 2007; 179(6): 3559 - 3569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. De Riva, C. Bourgeois, G. Kassiotis, and B. Stockinger Noncognate Interaction with MHC Class II Molecules Is Essential for Maintenance of T Cell Metabolism to Establish Optimal Memory CD4 T Cell Function J. Immunol., May 1, 2007; 178(9): 5488 - 5495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Prlic, G. Hernandez-Hoyos, and M. J. Bevan Duration of the initial TCR stimulus controls the magnitude but not functionality of the CD8+ T cell response J. Exp. Med., September 4, 2006; 203(9): 2135 - 2143. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zhang, W. Kim, L. Zhou, N. Wang, L. H. Ly, D. N. McMurray, and R. S. Chapkin Dietary Fish Oil Inhibits Antigen-Specific Murine Th1 Cell Development by Suppression of Clonal Expansion J. Nutr., September 1, 2006; 136(9): 2391 - 2398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Lewandowski, M. Marquis, F. Aumont, A.-C. Lussier-Morin, M. Raymond, S. Senechal, Z. Hanna, P. Jolicoeur, and L. de Repentigny Altered CD4+ T Cell Phenotype and Function Determine the Susceptibility to Mucosal Candidiasis in Transgenic Mice Expressing HIV-1 J. Immunol., July 1, 2006; 177(1): 479 - 491. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Colombetti, V. Basso, D. L. Mueller, and A. Mondino Prolonged TCR/CD28 Engagement Drives IL-2-Independent T Cell Clonal Expansion through Signaling Mediated by the Mammalian Target of Rapamycin. J. Immunol., March 1, 2006; 176(5): 2730 - 2738. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. Whitmire, N. Benning, and J. L. Whitton Precursor Frequency, Nonlinear Proliferation, and Functional Maturation of Virus-Specific CD4+ T Cells. J. Immunol., March 1, 2006; 176(5): 3028 - 3036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E. Foulds and H. Shen Clonal Competition Inhibits the Proliferation and Differentiation of Adoptively Transferred TCR Transgenic CD4 T Cells in Response to Infection. J. Immunol., March 1, 2006; 176(5): 3037 - 3043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Obst, H.-M. van Santen, D. Mathis, and C. Benoist Antigen persistence is required throughout the expansion phase of a CD4+ T cell response J. Exp. Med., May 16, 2005; 201(10): 1555 - 1565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Munitic, P. E. Ryan, and J. D. Ashwell T Cells in G1 Provide a Memory-Like Response to Secondary Stimulation J. Immunol., April 1, 2005; 174(7): 4010 - 4018. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Williams and M. J. Bevan Shortening the Infectious Period Does Not Alter Expansion of CD8 T Cells but Diminishes Their Capacity to Differentiate into Memory Cells J. Immunol., December 1, 2004; 173(11): 6694 - 6702. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. A. Corbin and J. T. Harty Duration of Infection and Antigen Display Have Minimal Influence on the Kinetics of the CD4+ T Cell Response to Listeria monocytogenes Infection J. Immunol., November 1, 2004; 173(9): 5679 - 5687. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Chang, J.-H. Cho, S.-W. Lee, S.-Y. Choi, S.-J. Ha, and Y.-C. Sung IL-12 Priming during In Vitro Antigenic Stimulation Changes Properties of CD8 T Cells and Increases Generation of Effector and Memory Cells J. Immunol., March 1, 2004; 172(5): 2818 - 2826. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bajenoff and S. Guerder Homing to Nonlymphoid Tissues Is Not Necessary for Effector Th1 Cell Differentiation J. Immunol., December 15, 2003; 171(12): 6355 - 6362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. De Boer, D. Homann, and A. S. Perelson Different Dynamics of CD4+ and CD8+ T Cell Responses During and After Acute Lymphocytic Choriomeningitis Virus Infection J. Immunol., October 15, 2003; 171(8): 3928 - 3935. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Donahue and D. A. Fruman Proliferation and Survival of Activated B Cells Requires Sustained Antigen Receptor Engagement and Phosphoinositide 3-Kinase Activation J. Immunol., June 15, 2003; 170(12): 5851 - 5860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Bonnevier and D. L. Mueller Cutting Edge: B7/CD28 Interactions Regulate Cell Cycle Progression Independent of the Strength of TCR Signaling J. Immunol., December 15, 2002; 169(12): 6659 - 6663. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Pompos and K. L. Fritsche Antigen-Driven Murine CD4+ T Lymphocyte Proliferation and Interleukin-2 Production Are Diminished by Dietary (n-3) Polyunsaturated Fatty Acids J. Nutr., November 1, 2002; 132(11): 3293 - 3300. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
and •) or from the first cell division (
and 
).
Open symbols represent cultures without OVA; closed symbols
represent cultures with OVA (0.2 µg/ml). b, After
48 h culture supernatants from wells containing the indicated cell
types were collected and assayed for IL-2. Cells were cultured in the
presence (
) or absence (