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Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742
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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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Long-lived memory CD4 and CD8 T cells also exhibit phenotypic heterogeneity based on surface expression of the lymph node-homing receptor, CD62 ligand (CD62L).3 Memory CD8 T cells that persist following viral or bacterial infection are heterogeneous for CD62L expression (5, 6). Similarly, CD62L heterogeneity has been observed in memory CD8 T cells generated from in vivo activation of TCR-transgenic T cells in adoptive hosts (6, 7, 8, 9) and in persisting memory CD4 T cells generated by transfer of TCR-transgenic effector CD4 T cells into adoptive hosts (10, 11). Because CD62L expression (CD62Lhigh) is generally associated with a naive phenotype, and loss of CD62L expression (CD62Llow) is a hallmark of effector/memory subsets (12), CD62L heterogeneity among memory T cells remains unexplained at present. Whether CD62L expression delineates functional subsets within the memory T cell pool is also not known.
To characterize the cellular bases of heterogeneity within the memory T cell pool, it is essential to establish parameters that reliably distinguish effector from resting memory T cells, as it has been difficult to assess the life span and identity of these subsets in vivo due to similar functions and phenotypes. Functionally, effector and memory CD4 and CD8 T cells both mediate effector functions such as cytokine production and/or cytolysis, although effector T cells generally perform these functions with more rapid kinetics (13). Progress has recently been made in defining new cell surface phenotypes that distinguish effector and memory CD8 T cells (14); however, there is still no phenotypic marker that reliably distinguishes effector and memory CD4 T cells. We have been exploring alternate ways to distinguish effector and memory T cells and have recently shown that effector and memory CD4 T cells differ biochemically, as assessed by the tyrosine phosphorylation profile (10). This biochemical analysis requires a relatively large number of purified cells; therefore, a functional assay employing a small number of T cells to distinguish effector and memory CD4 T cell subsets would be highly advantageous.
In this study we demonstrate that effector and memory CD4 T cells can be clearly distinguished based on activation profile to different types of TCR-mediated stimuli, and we use these assays to analyze heterogeneity within the memory CD4 T cell pool. We previously found that effector CD4 T cells could maintain effector properties for several months in vivo based on biochemical analysis of the persisting population (10). However, we could not rule out that a resting memory population was generated and masked by a dominant effector profile. Here, we demonstrate that both conventional resting memory T cells and persisting effector CD4 T cells comprise the memory T cell pool and can be isolated based on CD62L expression. These two memory subsets mediate recall responses and produce effector cytokines, yet differ in overall proliferative capacity, kinetics of activation, activation profile, and spontaneous proliferation to MHC class II. These findings suggest that different subsets of memory CD4 T cells may play disparate roles in recall responses and may also have different requirements for maintenance in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were obtained from National Cancer Institute Biological Testing Branch and were used between 8 and 12 wk of age. Hemagglutinin (HA)-TCR-transgenic mice (15) were bred as heterozygotes and maintained in the Microbiology Animal Facility at the University of Maryland (College Park, MD). MHC class II-/- mice (16) and recombinase-activating gene 2 (RAG2)-/- mice (17) on a BALB/c genetic background were purchased as breeding pairs from Taconic Farms (Germantown, NY) and bred and maintained in the animal facility under specific pathogen-free conditions.
Abs and reagents
The following Abs were purified from culture supernatants from
hybridomas maintained in the laboratory: C363.29B (anti-CD3
)
(18), GK1.5 (anti-CD4) (19), anti-CD8
(Tib-105, American Type Culture Collection, Manassas, VA), 212.A1
(anti-I-Ad), anti-Fc-
R (clone 2.4G2),
anti-Thy1.2 (TIB 238), and anti-Mac-1
(TIB 128). The 6.5
anti-clonotype Ab (15) directed against the HA-TCR
(rat IgG) hybridoma was purified and FITC conjugated as described
previously (10). The following mAbs were purchased from
PharMingen (San Diego, CA): PE- and biotin-conjugated anti-CD45RB
(clone C363.16A) (20), PE-conjugated anti-CD62L (clone
MEL-14), PE-conjugated anti-CD25 (clone 7D4), FITC-conjugated
Thy-1.2 (clone 30-H12), purified anti-mouse Ly-6G (clone RB6-8C5),
and PE-conjugated anti-CD4. Quantum Red-conjugated anti-CD4
(clone H129.19) was purchased from Sigma (St. Louis, MO). The MACS
anti-CD90 (Thy1.2) microbeads were purchased from Miltenyi Biotec
(Auburn, CA). The HA peptide 110–119 of the sequence, SFERFEIFPK, was
synthesized by Biopolymer Laboratory, University of Maryland School of
Medicine.
Isolation of naive and memory HA-TCR subsets
The detailed procedure for isolation of mouse CD4 T cells and subsequent separation into CD45RBlow (memory) and CD45RBhigh (naive) subsets was detailed previously (21). Briefly, CD4 cells (>90% pure) were isolated from HA-TCR spleen using immunomagnetic depletion with anti-CD8 and anti-I-Ad mAbs followed by anti-rat IgG-, anti-mouse IgG-, and anti-mouse IgM-coupled magnetic beads (Perseptive Biosystems, Cambridge, MA). CD4 T cells were fractionated into naive and memory subsets by positive and negative selection by MACS separation using biotin-conjugated anti-CD45RB (C363.16A, PharMingen) and streptavidin MACS magnetic beads (Miltenyi Biotec). The resultant CD45RBlow (memory) and CD45RBhigh (naive) populations were >95% CD4.
In vitro generation of effector cells
Ag-activated effector CD4 T cells were generated from HA-TCR CD4 T cells (1 x 106 cells/ml) incubated with 5 µg/ml HA peptide and 3 x 106 cells/ml T-depleted BALB/c splenocytes as APC in 24-well plates for 3–5 days at 37°C (10). For in vitro functional analyses, effector cells were centrifuged through Ficoll after 5 days to remove dead cells and contaminating accessory cells, washed in PBS, and resuspended in complete Clicks medium (10). Effectors generated in this way were >95% pure and had no residual APC.
Proliferation and cytokine assays
CD45RBhigh,
CD45RBlow, and in vitro generated effector
subsets (50,000 cells/well) were incubated in flat-bottom 96-well
plates with APC (150,000/well) in complete Clicks medium. Titrated
amounts of either HA peptide or anti-CD3 Ab were added. Cells
were incubated at 37°C, proliferation was assessed after 24 and
48 h by the addition of 1 µCi
[3H]thymidine (6.7 Ci/mmol)/well, and cells
were harvested after 18 h using a Tomtec 96-well plate harvester
(Wallac, Gaithersburg, MD). Radioactivity was quantitated using a
Microbeta Tri-luxe plate scintillation counter (Wallac), and
proliferation was expressed as the average of triplicates in which
errors were consistently <10%. Supernatants from duplicate cultures
set up for proliferation were collected after 24 and 48 h. IFN-
in supernatants was measured by specific ELISA (Endogen, Cambridge,
MA). Color reactions were read at 450 absorbance in an ELISA reader
(Bio-Rad), and units per milliliter of IFN- were calculated by
comparison to a known IFN- standard. For analysis of CD4 T cells and
CD62L subsets from adoptive transfer recipient mice, assays were set up
as described above, except that 37,500 T cells were added per well, and
proliferation was assessed after 24–72 h in culture.
Adoptive transfers and cell purification
Effector CD4 T cells were purified through Ficoll (LSM, ICN, Costa Mesa, CA) after 3 days in culture, washed three or four times in PBS, and resuspended in 107cells/0.5 ml of PBS. Effector cells or equal numbers of purified resting HA-TCR CD4 T cells from naive mice were injected in 0.5 ml into the tail vein of RAG2-/- mice. Adoptive transfer recipient mice were sacrificed 8–13 wk post-transfer, and splenic CD4 T cells were isolated using MACS magnetic sorting. Briefly, splenocytes were first incubated at 37°C for 1 h to remove adherent cells. Nonadherent cells were collected and incubated with FcR blocking reagent (Miltenyi) for 15 min followed by anti-Thy 1.2 microbeads (Miltenyi) for 10 min on ice. Cells were washed, resuspended in PBS/1% FCS, and positively selected over a MACS magnetic column.
FACS sorting and analysis
CD4 T cells from adoptive transfer recipient mice or HA-TCR mice
were enriched by immunomagnetic depletion. Briefly, splenocytes were
incubated with an Ab cocktail containing anti-Mac-1,
anti-FcR, anti-Ly6G, and anti-MHC II followed by
anti-rat IgG-, anti-mouse IgG-, and anti-mouse IgM-coupled
magnetic beads. The cells were subsequently stained with
FITC-conjugated anti-Thy 1.2 and PE-conjugated anti-CD62L
before sorting on an EPICS Elite ESP flow cell sorter (Coulter, Miami,
FL). The resultant Thy
1.2+CD62Llow and Thy
1.2+CD62Lhigh sorted
populations were 90–97% pure. For staining, cells were washed and
resuspended in stain buffer (PBS, 5% FCS, and 0.05% sodium azide).
Stained cells were analyzed using a FACSCalibur (Becton Dickinson, San
Jose, CA) with CellQuest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To characterize effector CD4 T cells relative to naive and memory
T cell counterparts, we first generated Ag-specific effector CD4 T
cells from HA-TCR transgenic mice in which a large proportion of
peripheral CD4 T cells express the transgene-encoded TCR specific for
peptide 110–119 of influenza HA and I-Ed
(15). We previously showed that activation of naive HA-TCR
CD4 T cells for 5 days with HA peptide in the presence of splenic MHC
class II+APC resulted in activated cells bearing
the functional and phenotypic attributes of differentiated effector CD4
T cells (10). We compared activation parameters of this
effector CD4 T cell population relative to ex vivo naive
(CD45RBhigh) HA-TCR CD4 T cell precursors by
stimulating with different doses of HA peptide in the presence of APC.
Overall, we found that effector cells exhibit peak proliferative
responses between 24–48 h, whereas naive CD4 T cells exhibit maximal
proliferation at 72 h (data not shown). As shown in Fig. 1
A, at 48 h effector CD4
T cells proliferate extensively at all Ag doses, whereas naive T cells
proliferate at a lower level than effectors only at higher Ag doses
(see inset), consistent with similar findings by Iezzi
(22). Effector CD4 T cells did not proliferate in response
to HA peptide alone, confirming the lack of contaminating APC (Fig. 1A). Restimulation of HA-TCR effectors with increasing doses
of Ag also resulted in increasing levels of IFN- production, whereas
naive HA-TCR CD4 T cells did not produce significant levels of IFN-
even at high peptide doses (Fig. 1B). These results
demonstrate that the in vitro-generated HA-TCR effector population is
functionally hyper-responsive to antigenic stimulation compared with
naive CD4 T cells, and that effector CD4 T cell proliferation, when
measured at early time points, correlates with cytokine secretion.
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Although naive and effector CD4 T cells can be clearly
distinguished by their response to antigenic stimulation (Fig. 1
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functional differences between memory and effector CD4 T cells are
not clearly defined. With the goal of defining functional
parameters that delineate effector and memory CD4 T cells, we
considered the striking differences in activation requirements to
anti-CD3-mediated stimulation exhibited by ex vivo naive and memory CD4
T cells (see Table I). We and others had
previously demonstrated that murine memory CD4 T cells are
hyporesponsive to soluble anti-CD3 Ab in the presence of MHC class
II+ splenic APC
(anti-CD3/II+APC), whereas naive CD4 T cells
are fully activated by this stimulus (21, 23) (see Table I). This memory CD4 T cell hyporesponsiveness is dependent on the
CD4-MHC class II interaction (21, 23), as memory CD4 T
cells can be activated by anti-CD3 presented by MHC class
II- APC
(anti-CD3/II-APC) (21). Given
this novel memory CD4 T cell-specific activation property, we asked
whether effector CD4 T cells would likewise exhibit hyporesponsiveness
to anti-CD3/II+APC.
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A. To ensure that these
subsets functionally represented naive and memory CD4 T cells, we
tested for the absence and the presence, respectively, of effector
cytokine production in response to HA peptide Ag. As shown in Fig. 2B, CD45RBlow (memory) HA-TCR CD4 T
cells produce high levels of IFN- in response to HA peptide, whereas
equivalent numbers of 6.5+
CD45RBhigh (naive) HA-TCR CD4 T cells produce
negligible levels of this effector cytokine. These results demonstrate
that the CD45RBlow subset of HA-TCR CD4 T cells
functionally represents memory T cells, thus enabling a novel
comparison of endogenous HA-specific memory to HA-specific
effectors.
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C, at all doses and time points effector CD4 T cells
exhibit the highest level of proliferation to
anti-CD3/II+ APC, compared with ex vivo
HA-TCR naive and memory subsets. Although effectors show maximal
proliferation after 24 h of stimulation and hyper-responsiveness
to anti-CD3/II+APC at the lowest doses of
anti-CD3 stimulation (0.01–0.1 µg/ml), naive CD4 T cells show
increased proliferation at 48 h only at higher doses of
anti-CD3 (1–5 µg/ml; Fig. 2C). By contrast, at all
time points and doses of anti-CD3 Ab, memory CD4 T cells fail to
exhibit a significant proliferative response. These results demonstrate
that when stimulated with anti-CD3/II+APC,
effector CD4 T cells are hyper-responsive, and memory CD4 T cells are
hyporesponsive.
Although effector and memory CD4 T cells can be clearly distinguished
by their responses to anti-CD3/II+APC, we
wished to examine multiple functional parameters to obtain an
activation profile that clearly defines naive, effector, and memory CD4
T cell subsets. We thus analyzed proliferative responses of HA-TCR
naive, effector, and memory CD4 T cells to Ag/APC,
anti-CD3/II+APC and
anti-CD3/II-APC (memory CD4 T cell
hyporesponsiveness to anti-CD3/II+ APC is
overcome by anti-CD3/II-APC
(21); see Table I), to obtain an activation profile. As
shown in Fig. 2D, two themes emerge when examining
individual activation profiles of naive, effector, and memory CD4 T
cells. First, naive, effector, and memory CD4 T cells exhibit distinct
activation profiles to the three types of TCR stimuli. (Each activation
profile is depicted on a separate graph, so that relative responses to
Ag-vs anti-CD3 can be compared.) Naive CD4 T cells proliferate well
in response to Ag/APC and exhibit higher proliferative responses to
anti-CD3-mediated stimuli in the presence of
II+ or II-APC (probably
due to the activation of nonclonotype T cells). Effector CD4 T cells
exhibit high proliferative responses to Ag/APC and equally high
proliferative responses to anti-CD3/II+APC
and anti-CD3/II-APC (Fig. 2D,
middle graph). Although ex vivo-derived memory
(CD45RBlow) HA-TCR CD4 T cells proliferate in
response to Ag/APC and anti-CD3/II-APC, they
do not exhibit substantial proliferation in response to
anti-CD3/II+APC (Fig. 2D,
right-most graph). Thus, while both naive and effector CD4 T
cells exhibit antigenic responses lower or equivalent to
anti-CD3/II+APC responses, memory CD4 T cells
exhibit greater responses to Ag compared with
anti-CD3/II+APC.
The second theme that emerges from the activation assays in Fig. 2D is that naive, effector, and memory CD4 T cells exhibit
profound differences in proliferative capacity. Each subset responded
maximally to stimulation with
anti-CD3/II-APC, and comparison of these
responses reveals that effectors have the highest proliferative
capacity overall, followed by naive and then memory CD4 T cells. The
hyper-responsiveness of effectors is independent of stimulation by
residual Ag and/or APC, because effectors stimulated with either APC
alone or anti-CD3 or HA peptide alone exhibited negligible
proliferation (Fig. 2 and data not shown). These results indicate that
analysis of the activation profile to cognate and noncognate stimuli
and overall proliferative capacities can be used to unambiguously
distinguish effector and memory CD4 T cells (see Table I).
Analysis of memory generation in vivo
Given our ability to distinguish effector and memory CD4 T cells based on activation profile, we next applied this assay to analyze the generation of long-lived memory T cells from effector CD4 T cells in vivo. We had previously demonstrated that transfer of HA-TCR effector CD4 T cells into sublethally irradiated BALB/c adoptive hosts resulted in the persistence of Ag-specific T cells for many months that mediated recall responses to Ag (10), consistent with findings in other adoptive transfer systems (25, 26). Although these cells appeared smaller in size than effectors and were long-lived, this persisting population exhibited an effector-specific biochemical pattern of tyrosine phosphorylation. These results suggested that a proportion of transferred effectors were maintained in vivo as persisting effector CD4 T cells (10).
In this study, we asked whether these persisting cells likewise
exhibited an effector-specific or memory cell-specific activation
profile. For these analyses, we transferred HA-TCR effectors into
syngeneic RAG2-/- adoptive hosts to remove the
complication of endogenous T cells. We purified the persisting splenic
CD4 T cells 8–13 wk post-transfer and designated these cells as
CD4ET because they derived from mice that
received transferred effector cells (see Table II). For comparison, equal numbers of
resting HA-TCR CD4 T cells were transferred in parallel, and the cells
persisting 8–13 wk in vivo were designated CD4NT
(see Table II) because they derived from mice that received resting
HA-TCR CD4 T cells consisting predominantly of naive CD4 T cells.
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A) was down-regulated in the
CD4ET population, consistent with analogous
adoptive transfer systems (25, 26). In addition, while
naive HA-TCR CD4 T cells were primarily CD62Lhigh, both Ag-activated
effectors and CD4ET persisting 8–16 wk in adoptive hosts
were heterogeneous for CD62L expression, with two distinct populations
of CD62Llo and CD62Lhi clearly discernible in the
CD4ET population (Fig. 3A). To
determine whether CD4ET cells represented resting
memory CD4 T cells in terms of activation parameters, we asked whether
these persisting cells likewise exhibited hyporesponsiveness to
anti-CD3/II+APC. We thus assayed the ability
of CD4ET cells 10 wk post-transfer to respond to
different doses of anti-CD3 in the presence of
II+APC compared with CD4NT
that persisted 10 wk post-transfer. As shown in Fig. 3B,
CD4ET cells exhibit a high proliferative response
to low doses of anti-CD3 Ab (0.1 and 1 µg/ml) compared with
CD4NT cells, which respond substantially only at
the highest dose tested (1 µg/ml). These results indicate that the
CD4ET cells that persist in vivo resemble
effector CD4 T cells in their hyper-responsiveness to
anti-CD3/II+APC, similar to in vitro
generated effectors (Fig. 2C).
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D. The activation profile of
CD4ET cells in Fig. 3C demonstrates
that CD4ET cells proliferate at high levels in
response to Ag/APC, anti-CD3/II+ APC, and
anti-CD3/II- APC similar to in vitro
generated effector cells (Fig. 2D). Controls with APC, Ag,
or anti-CD3 Ab alone yielded negligible proliferative responses,
confirming the lack of contaminating Ag and APC in the purified
CD4ET population (Fig. 3B). From these
results we conclude that at least a fraction of the persisting
CD4ET cells exhibit effector-specific activation
profiles. This maintenance of effector-specific properties over time is
consistent with our previous findings that CD4ET
cells derived from BALB/c adoptive hosts exhibited effector-specific
biochemical profiles (10). Isolation and functional analysis of heterogeneous CD4ETsubsets
Although the persisting CD4ET cells
exhibited a high proliferative capacity and hyper-responsiveness to
anti-CD3/II+APC similar to effector T cells,
the presence of conventional resting memory T cells that exhibit lower
proliferative capacities and hyporesponsiveness to
anti-CD3/II+APC would be masked by dominant
effector functions. The phenotypic heterogeneity in CD62L expression
found in the CD4ET-persisting population (see
Fig. 3A) suggested that the long-lived memory T cell pool
may consist of more than one subset of CD4 T cells. To determine
whether differences in CD62L expression delineated subsets of
long-lived memory T cells that differed in function and/or activation
profile, we sorted the persisting CD4ET cells
into CD62LloET and CD62LhiET populations
(histogram plots in Fig. 4A)
for subsequent functional analysis. For comparison, we also sorted
CD62L T cell subsets directly from purified fresh HA-TCR CD4 T cells
that were predominantly naive (CD62LloN and
CD62LhiN, refer to Table II for nomenclature). Although the
CD62LhiN subset predominated in CD4 T cells derived from
naive HA-TCR mice as previously reported (10), the
proportion of the CD62LloET subset in
CD4ET cells ranged from equivalent (see Fig. 4A) to up to 3 times greater than the CD62LhiET
subset, when individual mice were examined (data not shown).
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secretion
by CD62LET and CD62LN
subsets in response to Ag. As demonstrated in Fig. 4B, both
CD62LET subsets secrete IFN- in response to
antigenic stimulation compared with the negligible level of IFN-
secreted by CD62LN subsets in response to
antigenic stimulation. Interestingly, the level of IFN- in culture
supernatants from CD62LloET cells is 2- to 3-fold higher
than that in supernatants from CD62LhiET cells, although
both express comparable numbers (
85%) of 6.5+
cells (data not shown). To further examine the cytokine properties of
the CD62LET subsets, we performed a dose and
kinetic analysis of Ag-specific cytokine production (Fig. 4C). As illustrated in Fig. 4C,
CD62LloET produces a higher level of IFN- with faster
kinetics than the CD62LhiET subset. After 30 h in
culture with Ag/APC, CD62LloET produces significant levels
of IFN-, while the CD62LhiET subset does not produce
appreciable IFN- levels. After 48 h in culture, however, the
CD62LhiET subset produces substantial levels of
IFN-, although this level is still one-third of that produced by
CD62LloET. These results establish that both
CD62LET subsets mediate recall responses, yet
differ in the level and kinetics of effector cytokine production, with
the CD62LloET subset exhibiting rapid kinetics and
higher levels of IFN- production compared with the
CD62LhiET subset.
Because the CD62LET subsets that persisted in
vivo differ in kinetics of activation similar to previous distinctions
between effector and resting memory T cells (27), we asked
whether these CD62LET subsets could likewise be
distinguished based on activation profile, demonstrated here to
delineate effector and memory T cells (see Fig. 2). We thus measured
the proliferative response of sorted CD62LET
subsets to Ag or anti-CD3 Ab in the presence of
II+ or II- APC. As shown
in Fig. 5A, the
CD62LloET and CD62LhiET subsets
exhibit disparate activation profiles to antigenic and
anti-CD3-mediated stimuli. The CD62LloET subset
proliferates vigorously in response to Ag/APC or anti-CD3 in the
presence of II+ or II-
APC. By contrast, the CD62LhiET subset is
hyporesponsive to anti-CD3/II+ APC, while it
proliferates well in response to Ag/APC and
anti-CD3/II- APC, similar to the activation
profile observed with ex vivo isolated resting memory CD4 T cells (see
Fig. 2C).
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B, CD62LloET
proliferation in response to either
anti-CD3/II+APC or
anti-CD3/II-APC increases 2-fold between
48 and 72 h. By contrast, CD62LhiET proliferation
in response to anti-CD3/II+APC is negligible
at 48 h and remains low after 72 h in culture, while
proliferation in response to anti-CD3/II-APC
at 48 h is comparable to that in the CD62LloET
subset and doubles at 72 h (Fig. 5B, right).
Taken together, the results in Fig. 5 demonstrate that the sorted
CD62LloET and CD62LhiET subsets
exhibit distinct activation profiles. The CD62LloET
subset has a high proliferative capacity and is hyper-responsive to
stimulation by Ag/APC, anti-CD3/II+ APC, and
anti-CD3/II- APC similar to effector CD4 T
cells, whereas the CD62LhiET subset has a lower
proliferative capacity overall and is hyporesponsive to stimulation by
anti-CD3/II+ APC, similar to resting memory
CD4 T cells.
During the course of functional analyses of the sorted CD62L subsets,
we also found that spontaneous proliferation in the presence of
II+APC differs between
CD62LET subsets. As shown in Fig. 6, only the CD62LloET
population exhibited significant background proliferation in response
to II+APC alone, whereas the
CD62LhiET subset, and both
CD62LN subsets sorted from fresh naive HA-TCR CD4
T cells exhibited negligible background proliferation to
II+APC. This CD62LloET-specific
proliferation to II+APC was consistently present
in all the sorting experiments performed. Interestingly, neither the
CD62LET nor the CD62LN
subset exhibited background proliferation to
II-APC, indicating that the proliferation in
response to II+APC most likely derives from
interaction with MHC class II+ and endogenous
peptide. We also found that in vitro-generated effectors, but not ex
vivo-derived memory CD4 T cells defined by
CD45RBlow expression, proliferate in response to
MHC class II+ but not MHC class
II- APC (data not shown), suggesting that MHC
class II-dependent turnover may be an effector-specific property.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been difficult to follow the differentiative fate of effector cells in vivo and characterize memory T cell heterogeneity due to the lack of phenotypic markers and functional assays that reliably distinguish effector and memory T cells. Cell surface phenotypes have been shown to delineate effector and memory CD8 T cells in mice and humans (14, 28); however, clear-cut phenotypic or functional differences between effector and memory CD4 T cells have not yet been reported. To characterize the cells that comprise the memory CD4 T cell pool, we first established novel functional and activation differences between effector and memory CD4 T cells. As our standard for resting memory CD4 T cells generated in vivo, we used the CD45RBlow subset of splenic CD4 T cells, because this subset bears the phenotypic and functional properties of memory CD4 T cells (29). As presented here, ex vivo-derived memory CD4 T cells can be clearly distinguished from effector CD4 T cells based on activation profile to antigenic and anti-CD3-mediated stimuli. Specifically, effector CD4 T cells exhibit a greater proliferative capacity than memory CD4 T cells and are hyper-responsive to anti-CD3/II+APC and Ag, whereas memory CD4 T cells are hyporesponsive to anti-CD3/II+APC relative to Ag.
The differences in activation parameters identified here suggest that effector and memory CD4 T cells signal differently through the TCR/CD3 complex. We have previously demonstrated striking differences in the tyrosine phosphorylation profile of naive, effector, and memory CD4 T cells (10) and in proximal kinase phosphorylation in ex vivo derived naive and memory subsets (30). Differences in the expression of CD8-associated p56lck kinase have also been identified in naive vs effector/memory CD8 T cells (31). These functional and biochemical disparities suggest that alterations in the TCR-coupled signaling machinery control the distinct functional properties of naive, effector, and memory T cells. We are currently examining both proximal and distal TCR-mediated signal intermediates in these three subsets to address this hypothesis.
The identification of a functional assay to distinguish between effector and memory CD4 T cells enabled analysis of the differentiative fate of effector CD4 T cells in vivo and the cellular composition of the memory T cell pool. The memory T cell pool is defined here as the Ag-specific HA-TCR CD4 T cells that persist 8–13 wk following transfer of HA-TCR effectors into RAG2-/- adoptive hosts, analogous to other CD4 and CD8 adoptive transfer systems (25, 26, 32). By applying our activation profile assay, we found that the persisting memory population (designated CD4ET) exhibited an effector-specific activation profile in both its high proliferative capacity and its hyper-responsiveness to anti-CD3/II+APC. This finding was consistent with our previous results demonstrating that CD4ET exhibited an effector-specific biochemical profile (10). When taken together, our data suggest that effector CD4 T cells may persist for long periods of time in vivo, longer than previous estimates of effector T cell life span (33), yet consistent with recent findings that effector B cells or plasma cells are likewise long-lived in vivo (34).
We and others have found that the memory T cell population persisting after adoptive transfer of effectors was heterogeneous for the expression of CD62L (7, 10, 25). CD62L heterogeneity has likewise been observed in memory T cells that persist following viral (6, 35) or bacterial (5) infection. We found that the CD62Llo and CD62Lhi subsets differed strikingly in activation profile, proliferative capacity, and activation kinetics, similar to distinctions between effector and resting memory CD4 T cells, respectively. The association of effector-like functions with CD62Llo phenotype has been suggested in studies in which splenic LCMV-specific memory CD8 T cells heterogeneous for CD62L expression exhibited more rapid effector function than lymph node memory CD8 T cells that were primarily CD62Lhi (35). Similarly, the CD62Llo fraction of long-lived Sendai virus-specific memory CD8 T cells was found to exhibit a higher proliferative capacity in vitro than the CD62Lhi fraction, as assessed by CFSE staining (6). Although loss of CD62L expression is typically associated with effector/memory function, CD62Lhi memory CD4 T cells have been found in unmanipulated mice (36) and were found to predominate in aged mice (37). These results suggest that resting memory T cells may reacquire CD62L expression over time in vivo.
Subsets of human memory CD4 T cells have recently been isolated by Lanzavecchia and colleagues based on expression of the chemokine receptor CCR7 (4). The CCR7- memory subset was designated as effector-memory (TEM) due to rapid activation kinetics, whereas the CCR7+ subset was designated central memory (TCM) due to slower activation kinetics. Although the TCM subset is predominantly CD62Lhi (4) and may correspond to the mouse CD62Lhi resting memory subset identified here, the TEM subset is heterogeneous for CD62L expression (4). Taken together, our results and those reported by Lanzavecchia suggest that both the CD62L homing receptor and the CCR7 chemokine receptor may be differentially expressed on memory T cell subsets. Once the murine CCR7-specific reagents are available, we can assess the pattern of CCR7 expression in the mouse CD62L memory subsets and determine whether additional memory subsets can be defined by coordinate expression of these two markers.
Current models for the generation of memory T cells do not take into
account heterogeneity of the memory T cell pool (38). The
linear model predicts the generation of resting memory T cells directly
from effectors that have reverted to the resting state. By contrast, a
divergent model predicts distinct generation of effectors and memory T
cells directly from a naive T cell precursor. We present in Fig. 7 a modified model for the generation of
memory in which the two memory subsets diverge either from an effector
intermediate (scheme 1) or from a pre-effector intermediate (scheme 2).
Two other possibilities that cannot be ruled out are that resting
memory T cells derive from persisting effectors (Fig. 7, scheme 3) and,
conversely, persisting effectors may derive from a memory intermediate
as has been suggested by Lanzavecchia (4). Following the
development of each memory subset in vivo will enable identification of
the cellular precursor for each memory T cell subset.
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What is the purpose of these two subsets of memory T cells in anamnestic responses? It is feasible that each memory subset provides a unique role in memory responses in both the type of protection provided and the type of APC they encounter or activate. The persisting effector population with its rapid recall response and effector function may form the first line of protection in peripheral tissues. It has been shown that the CD62Llo effector subset rapidly migrates to inflammatory sites such as bronchial tissue to eradicate influenza infection (40) or to tumor sites to combat fibrosarcoma (41). In the tissues the CD62Llo subset may also recruit and prime tissue macrophages and immature dendritic cells. By contrast, the CD62Lhi resting memory subset migrates to secondary lymph nodes to be reprimed by resident APC (40), enabling them to provide help to B cells. We are currently examining the role of each CD62L memory subset in recall responses and their potential protective capacity in vivo.
In addition to playing distinct roles in recall responses, these two memory subsets may have different life spans and/or requirements for long term maintenance in vivo. Although it has recently been demonstrated that CD4 T cell memory can persist in the absence of MHC class II (42), it is unclear whether this phenomenon applies to both subsets of memory T cells. Our results that only the CD62Llo memory subset exhibited spontaneous proliferation to MHC class II in the absence of cognate Ag suggests that subsets of memory CD4 T cells may be differentially maintained via MHC class II-dependent turnover. These subsets may also be differentially maintained in the presence of Ag. Although many studies have focussed on the question of memory persistence in the absence of Ag (43, 44, 45), in vivo Ag may indeed persist for an extended period of time depending on the nature of the pathogen or Ag (46). Whether the longevity of persisting effectors is dependent on the presence or the absence of Ag remains to be determined.
In conclusion, the results presented here demonstrate that the memory CD4 T cell pool can be subdivided into two subsets that differ in phenotype and multiple functional parameters. Elucidation of the generation and maintenance of these subsets in vivo and their roles in protective immunity can lead to improved vaccine strategies to generate the appropriate type of anamnestic response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Donna L. Farber, Department of Surgery, University of Maryland, MSTF Building, Room 400, 685 West Baltimore Street, Baltimore, MD 21201.
3 Abbreviations used in this paper: CD62L, CD62 ligand; HA, hemagglutinin; RAG2, recombinase-activating gene 2.
Received for publication August 8, 2000. Accepted for publication October 24, 2000.
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define distinct epitopes, one of which may interact with CD4 during T cell activation. J. Immunol. 142:4169.[Abstract]
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