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The Center for Immunology and the Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition, there is evidence that memory cells are more readily activated than naive cells, as evidenced by their ability to respond to lower doses of Ag (10, 11), by their lesser dependence on costimulatory receptor-ligand interactions (12, 13, 14), and by their enhanced responses to stimulation with anti-TCR Abs (15). The increased expression of adhesion receptors on memory cells promotes their adherence to APCs, leading to higher levels of engagement of TCR and other receptors that might be involved in activation; this is likely to contribute to their enhanced responses in comparison with those of naive cells.
Whether naive and memory cells might also differ with respect to their sensitivity to signaling through the TCR and CD4 or CD8 coreceptors, independently of contributions from other adhesion molecules, is less clear. One approach to addressing this question is to determine whether activation can occur when purified MHC Ag is presented to T cells on a surface that lacks ligands for adhesion molecules or other receptors and also whether this differs for naive vs memory cells. In previous studies using this approach, it was demonstrated that purified class I alloantigen (alloAg) incorporated onto cell-size microspheres could stimulate in vitro generation of a cytolytic response by CD8+ cells from previously primed mice, provided that exogenous cytokines were added, but could not stimulate a primary in vitro response by cells from naive mice (16). While this observation suggested the possibility that naive and memory cells might differ in their sensitivity to Ag-dependent stimulation, it could not be ruled out that the observed differences might have resulted from differing precursor frequencies and/or selective expansion of clones with higher affinity TCRs during the primary in vivo response.
The use of cells from mice expressing a transgenic TCR eliminates issues of precursor frequency and potential differences in TCR affinities for Ag in the populations being studied. Using this approach and taking advantage of the fact that OT-1 transgenic mice with a TCR that is specific for OVA257–264 (SIINFEKL) complexed to H-2Kb have an endogenous population of TCR transgene-positive memory cells, we have quantitatively compared the requirements for Ag-dependent activation of naive vs memory CD8+ T cells to respond to IL-2 in the absence of contributions from receptors other than TCR and CD8. The results demonstrate that memory cells are much more sensitive to stimulation and can be effectively activated at levels of Ag that do not activate naive cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 and AKR mice were purchased from Charles River Laboratory (Wilmington, MA). OT-1 mice (17), originally provided by Dr. Francis Carbone (Monash Medical School, Victoria, Australia), were bred under specific pathogen-free conditions at the University of Minnesota. Mice were between 2 and 6 mo of age when used. RDM4 (H-2k), an AKR lymphoma, and EL4 (H-2b), a C57BL/6 thymoma, were maintained in vitro in RPMI 1640 supplemented with 10% FCS, 4 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin and streptomycin, 10 mM HEPES, and 50 µM 2-ME (RP-10). E.G7 cells (EL4 cells transfected with OVA) were maintained in vitro in RP-10 supplemented with 250 µg/ml G418.
Preparation of Ag-bearing microspheres
H-2Kb, Db and Kk class
I MHC proteins were purified by mAb affinity chromatography from EL4
(H-2b) and RDM4 (H-2k) tumor cells, as
previously described (18, 19). Purified Ags were eluted from the
affinity columns in 0.5% deoxycholate and 0.15 M NaCl in 10 mM Tris
(pH 8.2) and stored at -20°C until used. Anti-V
2 mAb B20.1 was a
kind gift of Dr. Kristin Hogquist, Center for Immunology, University of
Minnesota, Minneapolis, MN. Class I Ags and anti-TCR mAb were
immobilized on 5-µm diameter sulfate polystyrene latex microspheres
(Interfacial Dynamics Corporation, Portland, OR) by adding protein to
beads in suspension at 107/ml in PBS and incubating for
1.5 h at 4°C with continuous mixing. Next, 0.5 ml of 1% BSA in
PBS per ml of bead suspension was added to block remaining sites on the
beads, and mixing was continued for an additional 0.5 h at 4°C.
Coimmobilization of both B20.1 mAb and H-2Db was completed
by sequential immobilization of first B20.1 and then H-2Db.
Following immobilization and blocking, beads were washed and suspended
in RP-10 for addition to cultures. Final bead preparations were
quantitated by counting using a hemacytometer, and levels of protein
incorporated onto the surface were determined by flow cytometry.
Flow cytometry analysis and cell sorting
Beads or cells (
2 x 105) were washed
1x in HBSS containing 2% newborn calf serum and 0.1%
NaN3 (flow buffer). Beads coated with class I protein were
incubated for 20 min on ice with mAb specific for H-2Kk
(11–4.1 mAb), H-2Kb (Y-3 mAb), or H-2Db
(28–14–8 mAb). All anti-class I Abs were purified from hybridoma
culture supernatants by passage over protein A-Sepharose columns. Beads
were washed once with flow buffer and incubated for an additional 20
min on ice with goat anti-mouse Ig-FITC (Jackson ImmunoResearch
Laboratories, West Grove, PA). Cells were stained for 20 min on ice
with directly conjugated Abs. Abs used were obtained from PharMingen
(San Diego, CA) and included 53–6.7-FITC or -phycoerythrin (PE)
(anti-CD8 mAb), H129.19-PE (anti-CD4 mAb), B20.1-PE
(anti-V2 TCR mAb), 7D4-FITC (anti-IL-2R mAb), IM7-FITC or
-PE (anti-CD44 mAb), and AL-21-FITC (anti-Ly-6C mAb). The
analysis was performed on a FACScan using CellQuest software (Becton
Dickinson, San Jose, CA). Mean channel fluorescence values for beads or
cells were converted to molecules of equivalent soluble fluorochrome
(MESF) by comparison with a calibration curve generated using Quantum
26 calibrated fluorescent standard microbeads (FCSC, San Juan, Puerto
Rico).
OT-1 lymph node cells (LNC) were column-enriched for CD8+ cells as described below, stained with anti-CD8-FITC and anti-CD44-PE mAbs, and sorted using a FACStarPlus flow cytometer. Cells were gated for size and viability by forward and side scatter and sorted into CD44lowCD8+ and CD44highCD8+ populations. For some experiments, CD44low and CD44high populations were obtained by sorting column-enriched CD8+ cells using only anti-CD44-FITC mAb.
In vitro formation of Kb/OVA257–264 Ag complexes
OVA257–264 peptide (SIINFEKL) was purchased from Chiron Mimotopes (Clayton, Victoria, Australia). Stimulator cells were prepared using irradiated C57BL/6 spleen cells suspended at 5 x 106/ml and incubated for 1 h at 37°C with OVA257–264 at a final concentration of 0.2 µM. Cells were then washed three times with RP-10 before their addition to cultures. Microspheres having H-2Kb or H-2Db immobilized on the surface were resuspended at 5 x 106/ml in RP-10, incubated for 2 h at 37°C with OVA257–264 at the concentrations indicated in figure legends, and then washed three times with RP-10 before they were added to cultures.
Cell culture
Spleen cells were obtained from naive or alloAg-primed C57BL/6 mice. In vivo priming was performed 6 to 12 wk before use by i.p. injection of 20 x 106 live RDM4 cells in a volume of 0.5 ml of PBS. Spleen cells or LNC were obtained from OT-1 TCR transgenic mice. In all cases, cells were enriched for CD8+ cells by passage over Cellect-plus mouse CD8-enrichment columns (Biotex Laboratories, Edmonton, Alberta, Canada) according to the procedure provided by the manufacturer. CD8+-enriched populations were cultured in RP-10 medium in flat-bottom microtiter wells with either Ag-coated microspheres or stimulator cells. Where indicated, cultures were supplemented with supernatant from Con A-stimulated rat spleen cells (ConASN) (T-STIM, Collaborative Biomedical Products, Bedford, MA) at a final concentration of 5% or with human rIL-2 (R&D Systems, Inc., Minneapolis, MN) at a final concentration of 5 U/ml. Proliferation was measured at the times indicated in figure legends (2–5 days) by the addition of 1 µCi of [3H]TdR per well for the last 8 h of culture. Cultures were harvested onto filters using a LKB-Wallac harvester and counted in a Betaplate liquid scintillation counter (Wallac, Turku, Finland).
Cytotoxicity assay
Lytic activity of CD8+ cells from in vitro cultures was determined in a standard 51Cr release assay. For allogeneic responses, RDM4 targets were used, with EL4 targets as a control for specificity. For OT-1 responses, E.G7 targets were used, with EL4 targets as a control for specificity. Briefly, varying numbers of effector cells were added to 1 x 104 51Cr-labeled target cells in a final volume of 0.2 ml in V-bottom microtiter wells. After a 4-h incubation at 37°C, plates were centrifuged, 0.1 ml of the supernatant was removed, and radioactivity was counted. Spontaneous release was determined by measuring 51Cr release from target cells incubated in medium alone for 4 h; total release was determined by measuring 51Cr release from target cells incubated in 5% Triton X-100. Percent specific lysis was calculated as follows: 100 x (experimental release - spontaneous release)/(total release - spontaneous release). Data are expressed as LU, where one LU is defined as the number of effector cells required to give 50% specific 51Cr release.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies demonstrated that spleen cells from mice primed with alloAg could be stimulated in vitro to develop cytolytic activity in response to purified class I alloAg on silica microspheres together with supernatant from ConASN (16). In contrast, no cytolytic activity developed in response to this minimal stimulus when cells from naive mice were used. More recent work has shown that purified class I proteins can be immobilized directly onto latex microspheres, and that a very high surface density of Ag can be achieved (20, 21). In vitro stimulation of naive and primed cells was reexamined using CD8+-enriched cells, to determine whether in vitro primary responses could be obtained at high Ag densities and whether the IL-2 cytokine was sufficient to support generation of a secondary response.
Spleen cells from C57BL/6 mice, either naive or primed 6 to 12 wk
previously with live RDM4 (H-2k) allogeneic tumor cells,
were enriched for CD8+ T cells as described in
Materials and Methods. CD8+ cells increased from
9% to 57% of total cells following enrichment, while CD4+
cells decreased from 8% to <1%. Stimulation of primed cells with
H-2Kk on microspheres resulted in the generation of
cytolytic activity within 5 days, provided that IL-2 was added to the
cultures (Fig. 1
A). The
response to H-2Kk-microspheres was completely dependent
upon exogenous IL-2, while irradiated spleen stimulator cells from AKR
(H-2k) were able to stimulate a response in its absence.
The response to purified class I proteins on the microspheres was
Ag-specific; H-2Db microspheres stimulated no greater
cytolytic activity than the low level generated in response to IL-2
alone. The response to H-2Kk was also dependent upon the
density of class I on the surface of the microspheres.
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B) despite the
development of potent cytolytic activity in these cultures (Fig. 1A). This might be due to the fact that the frequency
of Ag-specific responder cells was too low to detect in this assay,
although it is possible that proliferation is not required for the
generation of lytic activity by the memory cells, and that class I Ag
alone is not a sufficient stimulus to promote proliferation. Even using microspheres with a very high surface density of Ag as the stimulus along with either IL-2 or ConASN, CD8+ cells from naive mice generated no detectable proliferative or cytolytic response (data not shown). Although a relatively large number of precursor CD8+ cells in naive mice are responsive to a single allogeneic class I protein, it is still possible that a response by naive cells is not detected due to the possibility that the number of specific precursors is too low, while the expanded number of precursors in a memory population is sufficient to allow detection of a response. This might particularly be the case if class I Ag is not a sufficient stimulus to promote significant IL-2-dependent proliferation of the Ag-specific cells. Alternatively, naive and memory CD8+ T cells might have differing requirements for activation via the TCR and CD8, the only receptors involved in stimulating a response when class I Ag is the only ligand on the "APC" surface. To examine these possibilities further, experiments were performed using CD8+ cells from TCR transgenic mice in which precursor frequency was not an issue.
CD8+ cells from OT-1 mice proliferate and develop cytolytic activity in response to class I/peptide Ag complexes and IL-2
OT-1 mice are transgenic for the - and ß-chains of a TCR that
is specific for OVA257–264 peptide (SIINFEKL) bound
to H-2Kb (17); essentially all of the CD8+
cells express the transgenic V2+ TCR, and >90% of the
V2+ cells are CD8+. CD8+ cells
enriched from the lymph nodes of these mice, as described in
Materials and Methods, are about 90% CD8+ and
<0.5% CD4+. To determine whether class I Ag is sufficient
to stimulate proliferation, H-2Kb was immobilized on
microspheres and pulsed with varying concentrations of
OVA257–264 peptide to form Ag complexes. Then the
microspheres were washed to remove unbound peptide, placed in culture
with OT-1 CD8+ T cells, and IL-2 was added. The cells
proliferated in response to the Ag over days 3 through 5, with the
level of proliferation being both dependent upon the concentration of
peptide used to form complexes with Kb on the microspheres
and maximal at 1 to 2 µM peptide (Fig. 2A). The level of
response obtained was also dependent upon the density of Kb
on the surface of the beads (Fig. 2B). Microspheres
with a Kb density in the range of that found on normal
cells (made using 1 or 2 µg class I per 107 beads)
stimulated a strong response, and this was further increased at higher
Kb density, concomitant with peak proliferation occurring
earlier.
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, legend). Microspheres made using
Db, which binds OVA257–264 (22), and pulsed
with peptide also did not stimulate a response (Figs. 2 and 5, legends,
and data not shown), confirming a requirement for the presence of the
Kb restriction element on the bead and making it unlikely
that OT-1 cells were responding to peptide that had come off the beads
and was being presented by other CD8+ cells in the culture.
This possibility was further ruled out in experiments using transwells
(data not shown). Kb-microspheres that had been pulsed with
peptide only stimulated a response when they were added on the same
side of the transwell as the cells. No response was obtained when they
were placed on the other side of the membrane, even though free peptide
added to the other side could readily cross the membrane and stimulate
a response.
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The ability of CD8+ cells from OT-1 mice to
respond to class I/peptide complexes despite not having been immunized
with OVA raised the possibility that the inability to stimulate naive
allogeneic responses with class I alloAg might be accounted for by low
numbers of precursors in normal mice. However, some TCR transgenic mice
have a pool of memory cells that express the transgenic TCR despite
never having been exposed to the relevant Ag (23). There is
considerable evidence that these cells also express an endogenous TCR
-chain, and it is this additional TCR that stimulates memory cell
generation. Therefore, we examined this possibility in the OT-1 mice
and found that they do in fact have a population of CD8+,
V2+ cells with a memory surface phenotype.
The expression level of CD44 provides a good marker for murine
CD8+ cells that have previously responded to Ag, with naive
cells being CD44low and memory cells being
CD44high (24, 25, 26, 27). Lymph node (Fig. 3A) and spleen
CD8+ T cells (data not shown) from OT-1 mice are about 10%
CD44high. The CD44high cells also express Ly-6C
(Fig. 3D), another marker of memory CD8+
T cells (28). The CD44high cells appear to be resting
memory cells; they have the forward scatter profile of small, resting
lymphocytes (data not shown) and express low levels of CD25 (Fig. 3B) and CD69 (Fig. 3C). Essentially
all of the CD44high cells express the V2+
transgenic TCR (data not shown).
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, E and
F). Both populations were >99% CD8+.
Proliferation (day 3) of the two populations was identical in response
to irradiated C57BL/6 spleen cells pulsed with OVA257–264
(Fig. 4, legend). The two populations
were also stimulated with IL-2 and Kb-microspheres pulsed
with OVA257–264, varying either the
concentration of peptide used for the pulsing (Fig. 4A) or the density of Kb on the beads
(Fig. 4B). Under all conditions, CD44high
cells gave a strong response while CD44low cells responded
very weakly. The difference is even more dramatic when it is taken into
account that only about 32% of the cells in the CD44high
population are, in fact, CD44high. Assuming that the
CD44low cells present in this population respond in the
same way as the highly purified CD44low cells, then it can
be estimated that the CD44high cells had a >20-fold
greater response in this experiment. Responses of both the
CD44high and CD44low populations to
Kb/OVA257–264 microspheres were completely dependent upon
the addition of exogenous IL-2 (Fig. 4, legend).
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is representative of these results.
To determine whether differing response levels of naive and memory
cells might result from different kinetics of response, proliferation
was measured over days 2 through 5. Responses of CD44low
and CD44high cells to stimulation with irradiated C57BL/6
spleen cells pulsed with OVA257–264 were virtually
identical with respect to time course and magnitude (Fig. 5A). In contrast,
CD44high cells again responded well to IL-2 and
Kb microspheres pulsed with OVA257–264, while
CD44low cells responded only marginally at all times (Fig. 5B).
The differential ability of naive and memory cells to respond to a
minimal stimulus is quantitative, and CD44low cells do make
a weak proliferative response to a high surface density of Ag (Fig. 4).
To determine whether lytic effector function develops in the two cell
populations, CD44high and CD44low cells were
obtained by FACS and stimulated for 3 days with IL-2 and Kb
microspheres pulsed with OVA257–264. CD44high
cells developed potent Ag-specific cytotoxicity by day 3, and this was
dependent upon stimulation with Ag, since no cytotoxicity was obtained
in cultures stimulated with IL-2 alone (Fig. 6). In contrast, CD44low
cells did not develop significant cytotoxic activity when stimulated
with Ag and IL-2.
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Class I/peptide Ag is a ligand for both the TCR and CD8
coreceptor, and both are likely to contribute to signaling for
activation. The large quantitative difference in the ability of naive
vs memory cells to respond to class I Ag complexes (Figs. 4 and 5)
could result from CD44high cells expressing higher
levels of these receptors on their surfaces; this was examined by flow
cytometry. CD44high cells express slightly higher levels of
CD8 than do CD44low cells, while CD44low cells
actually have slightly higher levels of the transgenic V2 TCR. (Fig. 7). Somewhat lower expression of the
transgenic TCR on the CD44high population is consistent
with the suggestion that these cells also express a TCR having an
endogenous -chain. Thus, the slightly higher level of CD8 on the
memory cells could potentially contribute to a greater response to
class I Ag. However, differences in expression levels of the
Kb/OVA257–264-specific TCR would, if anything,
predict a lower sensitivity of the CD44high population to
antigenic stimulation, rather than the substantially higher sensitivity
that is observed.
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), but
the difference was not as dramatic as that seen when Ag was used as the
stimulus (Figs. 4 and 5). Although the differences are not great, the
response of CD44low cells to anti-TCR mAb was
consistently found to be lower than that of CD44high cells
in multiple experiments. Similar results were obtained when the surface
density of the immobilized anti-TCR mAb was varied; the differences
between CD44low and CD44high cells were not
increased when the level of stimulus was decreased (data not shown).
Potential differences in CD8 contributions were examined by including
non-Ag class I protein, a ligand for CD8 but not TCR, on the surface of
the microspheres along with the anti-TCR mAb. For both
CD44low and CD44high cells, non-Ag class I
caused about a 50% increase in the level of response over that
obtained with anti-TCR mAb alone (Fig. 8). This increase occurred
even when anti-TCR mAb was at a level where further increasing its
density did not increase responses. It is likely that the class I
protein contributes by supporting adhesion and/or signaling mediated by
CD8, but the naive and memory cells do not appear to differ
significantly in this respect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) might
be attributable to differences in precursor numbers or in vivo
selection for clones with higher affinity TCRs, these factors are
eliminated when cells from TCR transgenic mice are used. Despite having
the same TCR, OT-1 cells with a memory phenotype respond much more
readily to Ag than do cells with a naive phenotype (Figs. 4 and 5).
These differences are seen using purified class I Ag on microspheres as
the stimulus. Since these "artificial APCs" lack ligands for other
adhesion molecules or receptors, the results strongly suggest that
CD44low and CD44high cells differ at the level
of signaling via TCR and/or the CD8 coreceptor.
The population of CD8+ cells in OT-1 mice that have a
memory phenotype very likely results from cells that express both the
transgenic TCR and a second TCR that derives from endogenously
rearranged -chains and that have also responded to environmental Ag
via this second TCR. Lee et al. (23) have shown that DO11.10 mice,
transgenic for the - and ß-chains of a TCR specific for
OVA323–339 and I-Ad, have a population of
CD4+ T cells with a memory phenotype, and that these cells
have a second TCR as a result of expressing endogenous -chains. No
memory cells were observed in mice having the DO11.10 TCR transgenes
but lacking the recombinase activating gene that allows endogenous
-chains to be expressed. Similarly, the OT-1
CD8+CD44high population studied in this report
is not present in mice having the OT-1 TCR on a recombinase activating
gene-/- background (data not shown). Also consistent with
this memory population arising as a result of expressing dual TCRs is
the finding that the V2 transgene product is expressed at somewhat
lower levels on the CD44high population than on the
CD44low population (Fig. 7).
The CD44high population in OT-1 mice appears to consist of
resting memory cells rather than activated cells; they are small, do
not express CD69 or CD25 activation markers, and do not proliferate in
response to IL-2. Furthermore, no cytolytic activity can be detected
when specific killing of EL4 target cells expressing OVA is measured in
a 4-h 51Cr release assay using freshly isolated spleen or
lymph node CD8+ T cells (data not shown), while potent
lytic activity is present by day 3 after in vitro stimulation with Ag
(Fig. 6). As others have experienced with TCR transgenic mice, we have
been unable to substantially increase the pool of memory cells in OT-1
mice by in vivo priming with Ag. Although the CD44high OT-1
cells do not result from intentional priming, the results obtained
using cells from in vivo primed alloAg-specific cells confirm that
memory cells can respond to the minimal stimulus of class I Ag and IL-2
(Fig. 1).
The difference in the ability of CD44high and
CD44low cells to respond to class I is quantitative, not
qualitative; the CD44low cells can make a weak
proliferative response when the surface density of Ag is very high
(Fig. 4, A and B), although the cells that
respond do not develop lytic effector function (Fig. 6). Based on the
level of responses as a function of peptide dose and class I density
(Fig. 4), it appears that CD44high cells are at least
eightfold more sensitive to Ag than CD44low cells. This is
probably an underestimate of the difference, since the sorted
CD44high populations included a significant fraction of
CD44low cells. Furthermore, the somewhat lower expression
of the transgenic TCR on the CD44high population may
decrease their sensitivity to Ag. T cells having dual TCR have been
shown to have a decreased sensitivity to physiologic ligands due to
reduced surface density of each TCR (29).
In a comparison of the peptide dose responses for naive and memory CD8+ cells of F5 transgenic mice specific for an influenza nucleoprotein peptide, memory cells were found to have a 10- to 50-fold higher sensitivity for stimulation of IL-2-dependent proliferation (11). Ag presentation to the T cells was via spleen cell APCs in that study, and differential expression of adhesion molecules on the memory cells could therefore potentially contribute to an increased avidity of interaction with the APCs. The somewhat smaller difference between naive and memory cell activation requirements that we observe when only TCR and CD8 interactions are involved suggests that greater sensitivity of signaling via TCR and the CD8 coreceptor and increased avidity due to adhesion receptors binding their ligands may both contribute to the greater sensitivity of memory cells for activation.
A recently reported study (30) has shown that naive
CD8+CD44low cells from 2C mice with a
transgenic TCR specific for alloAg, Ld, and p2Ca peptide
(31) can be stimulated to proliferate in response to purified
Ld pulsed with peptide. However, responses were very weak
unless a variant peptide p2C-QY5 was used. Ld/p2C-Q is
bound by the 2C TCR with very high affinity (32), much higher than the
affinity of the OT-1 TCR for Kb/OVA257–264
(33), suggesting that naive cells can be stimulated to respond in the
absence of other receptor interactions if sufficient TCR engagement can
be achieved. This is consistent with our finding that naive OT-1 cells
do make a weak response at very high class I and peptide levels (Fig. 4). 2C mice have a population of CD8+ cells that express
high CD44 levels, and this population could respond effectively to
Ld and the p2Ca peptide (30). Although no quantitative
comparisons of CD44low and CD44high responses
were reported, this observation suggests that there may be a
differential sensitivity of naive and memory cells from 2C mice similar
to what we have demonstrated for OT-1 CD8+ cells.
Differential sensitivity of naive and resting memory cells at the level of TCR and/or CD8 or CD4 coreceptor signaling has not previously been demonstrated under conditions in which contributions of adhesion receptors to avidity differences can be ruled out. However, differences in the activation requirements for naive vs activated effector cells have been observed for CD4+ T cells using class II/peptide complexes immobilized in microtiter wells (34). Naive cells exhibited more stringent requirements for costimulation than did effector cells but also required higher peptide concentrations for activation when costimulation was provided. Thus, it may be the case that increased sensitivity to stimulation through the TCR and CD8/CD4 coreceptors is acquired upon differentiation to effector cells and is retained following reversion to a resting memory state.
Differences between naive and effector cells have also been observed
with respect to signal generation via the TCR. CD4+ T cells
that were primed in vivo and then rested exhibited a higher sensitivity
than did naive cells with regard to peptide-dependent activation of
phospholipase C-
1 and mitogen-activated protein kinase as well as
intracellular Ca2+ increase (35). Class II-transfected
fibroblasts were used as APCs in this study, and the observed
differences might therefore reflect either differences in contributions
by adhesion receptors or intrinsic differences in signaling via the
TCR. That there may be intrinsic differences in TCR-dependent signal
generation in CD8+ naive vs effector T cells is suggested
by a recent study demonstrating differences in tyrosine phosphorylation
of CD3 components and differences in the activation of CD8-bound
p56lck in response to stimulation with
anti-CD3 mAb (36).
CD44low and CD44high OT-1 cells exhibit a large
difference in sensitivity to Ag (Figs. 4 and 5). When anti-TCR mAb
is used as the stimulus, some difference in the levels of response is
still seen, but this difference is less dramatic than that observed
using Ag as the stimulus (Fig. 8). The high affinity interaction of the
Ab with the TCR may not accurately mimic the interaction with Ag and
may obscure differences in signal generation in naive vs memory cells.
Coimmobilizing anti-TCR mAb along with non-Ag class I as a ligand
for CD8 enhances the levels of response of both naive and memory cells,
and the differential in response between the two cell types remains
comparable with that seen in response to anti-TCR mAb alone (Fig. 8). These results raise the possibility that the differential
sensitivity of naive and memory cells may be predominantly at the level
of TCR signaling, and work is in progress to further examine
this.
The greater sensitivity of CD8+ memory T cells to stimulation by Ag through the TCR and/or CD8, as demonstrated here, together with increased expression of adhesion receptors (7), will act to insure that effector CTL can be generated rapidly in response to even very low levels of Ag, levels that may be too low to initiate a primary response by naive cells. In the experiments described here, costimulatory ligands are not present on the Ag-bearing surface, and responses therefore depend upon the addition of IL-2. This suggests that an effective in vivo memory CTL response could be generated even though the specific class I/peptide Ag complex is not being presented on professional APCs, as long as a CD4+ T helper response is occurring to provide a source of IL-2. Memory CD4+ T cells have less stringent requirements for costimulation than do naive cells (12, 13, 14), and this may also be the case for memory CD8+ T cells. The ability to construct artificial APCs by incorporating purified B7 ligands onto microspheres (37) will make it possible to examine this in more detail and to determine how costimulation requirements relate to the greater Ag sensitivity described here.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, University of Minnesota, Box 334 Mayo, 420 Delaware St. S.E., Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this paper: alloAg, alloantigen; PE, phycoerythrin; MESF, molecules of equivalent soluble fluorochrome; ConASN, supernatant from Con A-stimulated rat spleen cells; LNC, lymph node cells.
Received for publication August 4, 1997. Accepted for publication December 8, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 B. Zhu, A. L.J. Symonds, J. E. Martin, D. Kioussis, D. C. Wraith, S. Li, and P. Wang Early growth response gene 2 (Egr-2) controls the self-tolerance of T cells and prevents the development of lupuslike autoimmune disease J. Exp. Med., September 29, 2008; 205(10): 2295 - 2307. [Abstract] [Full Text] [PDF]
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 C. E. Rolle, R. Carrio, and T. R. Malek Modeling the CD8+ T Effector to Memory Transition in Adoptive T-Cell Antitumor Immunotherapy Cancer Res., April 15, 2008; 68(8): 2984 - 2992. [Abstract] [Full Text] [PDF]
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 A. Valujskikh and X. C. Li Frontiers in Nephrology: T Cell Memory as a Barrier to Transplant Tolerance J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2252 - 2261. [Full Text] [PDF]
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 L. H. Hogan, D. O. Co, J. Karman, E. Heninger, M. Suresh, and M. Sandor Virally Activated CD8 T Cells Home to Mycobacterium bovis BCG-Induced Granulomas but Enhance Antimycobacterial Protection Only in Immunodeficient Mice Infect. Immun., March 1, 2007; 75(3): 1154 - 1166. [Abstract] [Full Text] [PDF]
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K. D. Klonowski, A. L. Marzo, K. J. Williams, S.-J. Lee, Q.-M. Pham, and L. Lefrancois CD8 T Cell Recall Responses Are Regulated by the Tissue Tropism of the Memory Cell and Pathogen J. Immunol., November 15, 2006; 177(10): 6738 - 6746. [Abstract] [Full Text] [PDF]
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P. M. Gray, S. L. Reiner, D. F. Smith, P. M. Kaye, and P. Scott Antigen-Experienced T Cells Limit the Priming of Naive T Cells during Infection with Leishmania major J. Immunol., July 15, 2006; 177(2): 925 - 933. [Abstract] [Full Text] [PDF]
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C. Kao, M. M. Sandau, M. A. Daniels, and S. C. Jameson The Sialyltransferase ST3Gal-I Is Not Required for Regulation of CD8-Class I MHC Binding during T Cell Development. J. Immunol., June 15, 2006; 176(12): 7421 - 7430. [Abstract] [Full Text] [PDF]
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H. Choi, S.-Y. Cho, R. H. Schwartz, and K. Choi Dual Effects of Sprouty1 on TCR Signaling Depending on the Differentiation State of the T Cell J. Immunol., May 15, 2006; 176(10): 6034 - 6045. [Abstract] [Full Text] [PDF]
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F. J. Schnell and G. J. Kersh Control of Recent Thymic Emigrant Survival by Positive Selection Signals and Early Growth Response Gene 1 J. Immunol., August 15, 2005; 175(4): 2270 - 2277. [Abstract] [Full Text] [PDF]
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B. R. Winders, R. H. Schwartz, and D. Bruniquel A Distinct Region of the Murine IFN-{gamma} Promoter Is Hypomethylated from Early T Cell Development through Mature Naive and Th1 Cell Differentiation, but Is Hypermethylated in Th2 Cells J. Immunol., December 15, 2004; 173(12): 7377 - 7384. [Abstract] [Full Text] [PDF]
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R. Carrio, O. F. Bathe, and T. R. Malek Initial Antigen Encounter Programs CD8+ T Cells Competent to Develop into Memory Cells That Are Activated in an Antigen-Free, IL-7- and IL-15-Rich Environment J. Immunol., June 15, 2004; 172(12): 7315 - 7323. [Abstract] [Full Text] [PDF]
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 A. T. Larregina, A. E. Morelli, O. Tkacheva, G. Erdos, C. Donahue, S. C. Watkins, A. W. Thomson, and L. D. Falo Jr Highly efficient expression of transgenic proteins by naked DNA-transfected dendritic cells through terminal differentiation Blood, February 1, 2004; 103(3): 811 - 819. [Abstract] [Full Text] [PDF]
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F. G. Lakkis and M. H. Sayegh Memory T Cells: A Hurdle to Immunologic Tolerance J. Am. Soc. Nephrol., September 1, 2003; 14(9): 2402 - 2410. [Full Text] [PDF]
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 K. A. Mitchell and B. P. Lawrence Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Renders Influenza Virus-Specific CD8+ T Cells Hyporesponsive to Antigen Toxicol. Sci., July 1, 2003; 74(1): 74 - 84. [Abstract] [Full Text] [PDF]
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V. V. Parekh, D. V. R. Prasad, P. P. Banerjee, B. N. Joshi, A. Kumar, and G. C. Mishra B Cells Activated by Lipopolysaccharide, But Not By Anti-Ig and Anti-CD40 Antibody, Induce Anergy in CD8+ T Cells: Role of TGF-{beta}1 J. Immunol., June 15, 2003; 170(12): 5897 - 5911. [Abstract] [Full Text] [PDF]
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T. N. J. Bullock, D. W. Mullins, and V. H. Engelhard Antigen Density Presented By Dendritic Cells In Vivo Differentially Affects the Number and Avidity of Primary, Memory, and Recall CD8+ T Cells J. Immunol., February 15, 2003; 170(4): 1822 - 1829. [Abstract] [Full Text] [PDF]
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J. Valenzuela, C. Schmidt, and M. Mescher The Roles of IL-12 in Providing a Third Signal for Clonal Expansion of Naive CD8 T Cells J. Immunol., December 15, 2002; 169(12): 6842 - 6849. [Abstract] [Full Text] [PDF]
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 E. Schott, N. Bertho, Q. Ge, M. M. Maurice, and H. L. Ploegh Class I negative CD8 T cells reveal the confounding role of peptide-transfer onto CD8 T cells stimulated with soluble H2-Kb molecules PNAS, October 15, 2002; 99(21): 13735 - 13740. [Abstract] [Full Text] [PDF]
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P. J. Dunne, J. M. Faint, N. H. Gudgeon, J. M. Fletcher, F. J. Plunkett, M. V. D. Soares, A. D. Hislop, N. E. Annels, A. B. Rickinson, M. Salmon, et al. Epstein-Barr virus-specific CD8+ T cells that re-express CD45RA are apoptosis-resistant memory cells that retain replicative potential Blood, July 18, 2002; 100(3): 933 - 940. [Abstract] [Full Text] [PDF]
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C. S. Schmidt and M. F. Mescher Peptide Antigen Priming of Naive, But Not Memory, CD8 T Cells Requires a Third Signal That Can Be Provided by IL-12 J. Immunol., June 1, 2002; 168(11): 5521 - 5529. [Abstract] [Full Text] [PDF]
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T. Walzer, C. Arpin, L. Beloeil, and J. Marvel Differential In Vivo Persistence of Two Subsets of Memory Phenotype CD8 T Cells Defined by CD44 and CD122 Expression Levels J. Immunol., March 15, 2002; 168(6): 2704 - 2711. [Abstract] [Full Text] [PDF]
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O. F. Bathe, N. Dalyot-Herman, and T. R. Malek IL-2 During In Vitro Priming Promotes Subsequent Engraftment and Successful Adoptive Tumor Immunotherapy by Persistent Memory Phenotypic CD8+ T Cells J. Immunol., October 15, 2001; 167(8): 4511 - 4517. [Abstract] [Full Text] [PDF]
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S.-i. Fujii, K. Shimizu, T. Shimizu, and M. T. Lotze Interleukin-10 promotes the maintenance of antitumor CD8+ T-cell effector function in situ Blood, October 1, 2001; 98(7): 2143 - 2151. [Abstract] [Full Text] [PDF]
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 R. N. Germain The Art of the Probable: System Control in the Adaptive Immune System Science, July 13, 2001; 293(5528): 240 - 245. [Abstract] [Full Text]
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P. R. Rogers and M. Croft CD28, Ox-40, LFA-1, and CD4 Modulation of Th1/Th2 Differentiation Is Directly Dependent on the Dose of Antigen J. Immunol., March 15, 2000; 164(6): 2955 - 2963. [Abstract] [Full Text] [PDF]
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C. V. Hartig, G. W. Haller, D. H. Sachs, S. Kuhlenschmidt, and P. S. Heeger Naturally Developing Memory T Cell Xenoreactivity to Swine Antigens in Human Peripheral Blood Lymphocytes J. Immunol., March 1, 2000; 164(5): 2790 - 2796. [Abstract] [Full Text] [PDF]
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P. S. Heeger, N. S. Greenspan, S. Kuhlenschmidt, C. Dejelo, D. E. Hricik, J. A. Schulak, and M. Tary-Lehmann Pretransplant Frequency of Donor-Specific, IFN-{gamma}-Producing Lymphocytes Is a Manifestation of Immunologic Memory and Correlates with the Risk of Posttransplant Rejection Episodes J. Immunol., August 15, 1999; 163(4): 2267 - 2275. [Abstract] [Full Text] [PDF]
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M. J. Deeths, R. M. Kedl, and M. F. Mescher CD8+ T Cells Become Nonresponsive (Anergic) Following Activation in the Presence of Costimulation J. Immunol., July 1, 1999; 163(1): 102 - 110. [Abstract] [Full Text] [PDF]
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 M. Pihlgren, C. Arpin, T. Walzer, M. Tomkowiak, A. Thomas, J. Marvel, and P. M. Dubois Memory CD44int CD8 T cells show increased proliferative responses and IFN-{gamma} production following antigenic challenge in vitro Int. Immunol., May 1, 1999; 11(5): 699 - 706. [Abstract] [Full Text] [PDF]
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J. M. Curtsinger, C. S. Schmidt, A. Mondino, D. C. Lins, R. M. Kedl, M. K. Jenkins, and M. F. Mescher Inflammatory Cytokines Provide a Third Signal for Activation of Naive CD4+ and CD8+ T Cells J. Immunol., March 15, 1999; 162(6): 3256 - 3262. [Abstract] [Full Text] [PDF]
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P. E. Fields, R. J. Finch, G. S. Gray, R. Zollner, J. L. Thomas, K. Sturmhoefel, K. Lee, S. Wolf, T. F. Gajewski, and F. W. Fitch B7.1 Is a Quantitatively Stronger Costimulus Than B7.2 in the Activation of Naive CD8+ TCR-Transgenic T Cells J. Immunol., November 15, 1998; 161(10): 5268 - 5275. [Abstract] [Full Text] [PDF]
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