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Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104
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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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Therefore, it is not surprising that the thymus will not only continue
to export naive T cells during lymphopenia, but peripheral T cells will
proliferate in the absence of exogenous stimulation (homeostatic
proliferation) to fill the "empty" lymphoid space
(10). This may occur physiologically following lymphoid
ablation of normal animals (as part of tolerance-inducing protocols or
cancer therapy). It is also observed in experimental systems where,
upon transfer of CD4+ and
CD8+ T lymphocytes into lymphopenic hosts, the T
cells proliferate. Many of the same factors are necessary for
homeostatic proliferation as for survival and maintenance of T
lymphocytes when peripheral lymphocyte counts are normal. These include
interaction with self-peptide-MHC complexes (11, 12, 13), as
well as cytokines, particularly those that signal through the common
cytokine receptor 
-chain (14, 15).
When naive peripheral CD4+ T lymphocytes encounter foreign peptides in a complex with self-MHC as well as a costimulatory signal, such as through CD28, a robust immune response is generated. This results in proliferation and differentiation into short-lived cytokine-secreting effector cells. However, some cells survive to become long-lived memory cells. The precise requirements for memory formation are unclear. Although somewhat controversial, most researchers agree that CD8+ T lymphocytes that have proliferated in a lymphopenic host in the absence of exogenous Ag have increased ability to produce cytokines and lyse targets compared with naive CD8+ lymphocytes (16, 17). Presently it is unclear whether the same holds true for CD4+ T lymphocytes. The requirement for costimulation during homeostatic proliferation of T lymphocytes is equally unclear. It has been shown that B7-deficient mice have decreased numbers of peripheral T cells, particularly CD4+ T cells, whereas the reverse is true for mice overexpressing B7 (18). One group (19) using CD28-knockout mice suggested that costimulation was not required for homeostatic proliferation of polyclonal populations of peripheral CD8+ T lymphocytes. In any event, it is unclear whether costimulation is required for homeostatic proliferation of Ag-specific peripheral CD4+ T cells.
Recently, we have used the dye CFSE to label Ag-specific CD4+ T lymphocytes before adoptive transfer into syngeneic wild-type mice (20). This has enabled us to quantify proliferation of T lymphocytes during a response to foreign Ag in vivo, and better determine the role of CD28 costimulation in promoting proliferation and differentiation of responding T cells. The studies presented here use this approach to determine whether B7-mediated costimulation is required for homeostatic proliferation of Ag-specific CD4+ T cells, as well as to determine whether CD4+ T cells acquire phenotypic and functional changes consistent with memory cells as a result of homeostatic proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombination-deficient DO11.10 (Thy1.2+) mice (21) on BALB/c background (>20 generations) were either obtained as DO11.10/recombination-activating gene (RAG)3-/- mice (W. Lee, State University of New York, Albany) or DO11.10 mice bred with SCID mice (The Jackson Laboratory, Bar Harbor, ME), and subsequently maintained as breeding colonies in our animal facility. PBL from all mice were screened by flow cytometry for the absence of B and nontransgenic T lymphocytes to ascertain that they were homozygotes for the SCID mutation or RAG deletion. RAG-/- mice were purchased from Taconic Farms (Germantown, NY). BALB/c (Thy 1.1+) mice were bred in our animal facility. All mice were used at 6–12 wk of age.
Peptide, Abs, and CFSE labeling
CTLA4Ig (22) was provided by R. Peach
(Bristol-Myers Squibb Pharmaceuticals, Princeton, NJ). The hybridoma
producing anti-CD28 mAb (37.51) was provided by J. Allison
(University of California, Berkley, CA). Control hamster IgG and
control human IgG were obtained from Jackson ImmunoResearch
Laboratories (West Grove, PA). OVA peptide (323–339)
(OVAp) was synthesized
by the Protein Chemistry Laboratory, University of Pennsylvania
(Philadelphia PA). Fluorochrome-labeled mAbs against CD4, CD44,
CD45RB, CD69, CD62 ligand (CD62L), IL-2, IFN-, CD16/CD32 (Fc
block), and isotype controls were purchased from BD PharMingen (San
Diego, CA). Fluorochrome-labeled anti-DO11.10 Ab, KJ1–26, was
purchased from Caltag Laboratories (Burlingame, CA). CFSE labeling of
lymphocytes was achieved as described (23), with some
modifications (24).
In vitro proliferation cultures
Labeled DO11.10/RAG-/- lymphocytes were plated at 10–20 x 103 transgenic cells per well in a round-bottom 96-well plate in RPMI 1640 medium containing 10% FCS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 5 µM 2-ME. They were stimulated with varying concentrations of OVAp for 4 days, with an excess of irradiated splenocytes as a source of APCs (APC/clonotypic T cell ratio >10) as well as anti-CD28 mAb and control human IgG or CTLA4Ig and control hamster IgG.
Adoptive transfer and immunization
A total of 1.5–15 x 106 CFSE-labeled transgenic cells (obtained from the lymph nodes and spleens of DO11.10/RAG-/- or DO11.10/SCID mice) were injected i.v. into RAG-/- mice in a total volume of 0.2 ml. One to four days later mice were either left unimmunized or immunized with 50 µg of OVAp mixed in IFA (OVAp/IFA) into two sites in the lower back, at the base of the tail, and three sites on the upper back (total of 300 µg of OVAp/mouse). In some cases the mice were treated at the time of adoptive transfer, before immunization, and twice per week thereafter with 200 µg i.p. of either CTLA4Ig or human IgG.
Flow cytometry
Cells were washed in PBS containing 2% FCS and 0.02% azide at
4°C. Unlabeled anti-CD16/CD32
(anti-FcRIII/anti-FcRII) were used to block FcR binding.
Between 0.2 and 3.0 x 106 cells were
stained with either 1) PE-conjugated anti-CD4 and
CyChrome-conjugated anti-CD44 or 2) PE-conjugated Abs against
CD45RB or L-selectin, and PerCP-conjugated anti-CD4. In both cases,
cells were also stained with APC-conjugated Thy1.2 and/or
FITC-conjugated KJ1–26 to identify the transgenic cells. Four-color
flow cytometry was performed on a FACSCalibur (BD Immunocytometry
Systems, San Jose, CA), and cells were acquired and analyzed using
CellQuest and FlowJo software (BD Immunocytometry Systems).
Between 20 x 103 and 1.5 x
106 events were collected.
Intracellular cytokine staining
Cells were stimulated in vitro with 2.5 µg/ml OVAp in the
presence of 2 µg/ml anti-CD28 and 2 µM monensin (Boehringer
Mannheim, Indianapolis, IN) (25). After staining for
surface receptors, the cells were fixed in a final concentration of 1%
formaldehyde at 4°C overnight. Subsequently they were washed in PBS,
then in PBS containing 2% FCS, 0.02% azide, and 0.1% saponin (Sigma,
St. Louis, MO). PE-conjugated anti-IL-2 or anti-IFN- mAb (BD
PharMingen) mixed in 30 µl of PBS/saponin buffer was used to stain
each sample for 60 min (30 min at 4°C followed by 30 min at room
temperature).
Quantitation of T cell-proliferative dynamics from CFSE profiles
The quantitative analysis of proliferation using CFSE has been previously described (20, 24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To better understand the requirements for, and consequences of, homeostatic proliferation of CD4+ T cells, we adoptively transferred CFSE-labeled OVA-specific T lymphocytes into RAG-/- mice. As a source of naive Ag-specific CD4+ T cells, we used DO11.10/RAG-/- or DO11.10/SCID mice.
Three weeks after adoptive transfer we identified two populations of
OVA-specific T lymphocytes based on CFSE brightness (Fig. 1
). When 1.5 x
106 cells were adoptively transferred, some of
the input cells divided minimally (0–2 rounds of division), and they
are visualized as distinct peaks of proliferation, whereas others
divided multiple times (>6–8 rounds of division, Fig. 1A).
In the latter case, the CFSE stain became very diluted, and individual
peaks of proliferation were no longer identified. The loss of discrete
peaks in the multiply divided population precludes us from
extrapolating the ratio of input cells that generated these two
populations (20). However, the input cells that divided
multiple times generated 75.5 ± 11.4% of the OVA-specific T
lymphocytes present in the spleen (Fig. 1, A and
G). The rest, or 24.3 ± 11.4%, were generated by
input cells that proliferated minimally or not at all. It is unclear
why two distinct homeostatic proliferative potentials exist. As the
OVA-specific T cells that we adoptively transferred were derived from
TCR-transgenic mice on a RAG-/- background,
this eliminates the possibility that the population generated by
multiple rounds of division was the progeny of memory cells generated
by exposure to an environmental Ag through stimulation of a second TCR
(26). However, it is possible that the different
proliferative behavior reflects distinct stimuli that the cells
encountered in the recipient mouse, e.g., endogenous Ag presentation by
professional vs nonprofessional APCs. Alternatively, the input cells
may have an intrinsic variability in proliferative potential.
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, A and B, as well as
C and D). However, despite this increase in
proliferation, there was no difference in the total number of cells
collected 3 and 5 wk after adoptive transfer (data not shown). This
suggests that in the recipient RAG-/- mice
homeostatic proliferation continues but is balanced by ongoing cell
death. Homeostatic proliferation–requirements
Two parameters that are known to regulate Ag-driven T cell proliferation in vivo are the availability of costimulation through molecules such as CD28, and the relative vacancy of the lymphoid compartment (13, 19, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). To understand how these factors regulate homeostatic proliferation, we altered the number of input cells that were adoptively transferred and/or blocked B7-mediated costimulation.
The proliferative capacity of the input cells was clearly affected by
the number of cells adoptively transferred into the
RAG-/- mice. When 10 times more cells (15
x 106) were adoptively transferred, the input
cells again proliferated either minimally or multiple times and
generated two discrete populations of cells. However, the relative size
of these two populations was very different compared with when fewer
cells were adoptively transferred (Fig. 1, A, E, and
G). Thus, only 30.0 ± 1.5% of the population was
generated by input cells that had proliferated multiple times compared
with 75.5 ± 11.4% when 10 times fewer cells were adoptively
transferred (p = 0.05). CD28 costimulation
signals also regulated the size of the highly proliferative pool. When
1.5 x 106 input cells were adoptively
transferred and mice were treated with CTLA4Ig, only 9.0 ± 6.0%
of the OVA-specific T lymphocyte population was generated by cells that
had proliferated maximally (p < 0.05) (Fig. 1F). The effect of CTLA4Ig was less evident when 15 x
106 cells were adoptively transferred, although
the difference seen in the spleen did reach statistical difference
(p < 0.05) (Fig. 1G). Taken
together, these data suggest that two known checkpoints of Ag-specific
proliferation, costimulation and pool size, also regulate homeostatic
proliferation.
Surface phenotype of Ag-specific CD4+ T lymphocytes after homeostatic proliferation and after priming by the specific Ag
When naive T lymphocytes encounter their specific Ag they increase in size and up-regulate "early activation markers," such as CD69. Subsequently, they proliferate and differentiate into effector and memory cells. To determine whether the same holds true for naive cells that have undergone homeostatic proliferation we examined expression of early activation markers as well as expression of classical markers of memory.
OVA-specific T lymphocytes were adoptively transferred into syngeneic
RAG-/- mice. One to two days after adoptive
transfer, the mice were either left untreated (adoptively
transferred–untreated) or immunized with OVAp mixed in IFA s.c.
(adoptively transferred–immunized) (Fig. 2). The OVA-specific T cells exposed to
the OVAp became activated as noted by their increase in size and
up-regulation of CD69 as early as 24 h after immunization. In
contrast, the OVA-specific T lymphocytes that were adoptively
transferred and not exposed to OVAp remained small and did not
up-regulate CD69 at 48 h after adoptive transfer. Thus, consistent
with previous reports (16, 17, 38, 39), we find that
OVA-specific T lymphocytes adoptively transferred into lymphopenic
hosts do not show hallmark signs of early activation after adoptive
transfer. However, at later time points (4–5 wk), the OVA-specific T
cells that had undergone maximal levels of homeostatic proliferation
were enlarged, consistent with blastogenesis (data not shown).
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). As
expected, OVA-specific T lymphocytes exposed to OVA/IFA in vivo changed
their surface phenotype within 4 wk after adoptive transfer from naive
(CD45RBhigh and CD62Lhigh)
to that of memory/effector cells (CD45RBlow and
CD62Llow) (n = 7, Fig. 3).
Interestingly, the surface phenotype of OVA-specific T lymphocytes that
had undergone homeostatic proliferation also converted to that of
memory cells, but this conversion required a minimum of 6 wk
(n = 3). It should be noted that although the entire
population of OVA-specific T cells undergoing homeostatic proliferation
down-regulated CD45, only a proportion down-regulated CD62L. This also
holds true for the memory/effector population, although in this
instance a larger proportion down-regulated CD62L. Partial
down-regulation of CD62L may in part be due to the triphasic expression
of CD62L (40). In addition, down-regulation of both CD45
and CD62L is associated with cell cycle number, as both were most
markedly reduced in the cells that had divided multiple times (Fig. 3B). These data suggest that specific foreign Ag is not
required for conversion from a naive to a memory surface phenotype.
However, the rate of conversion is slower than that of T lymphocytes
primed with the specific peptide, possibly as a result of much slower
progression through the cell cycle (our unpublished observations and
Ref. 39).
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Memory markers are expressed on both memory and effector T lymphocytes and do not inform us of the ability of expressing T cells to produce cytokines or proliferate. Having found that OVA-specific T cells that had undergone homeostatic proliferation acquired phenotypic characteristics of memory cells, we next wished to determine their functional characteristics as well. To study this, we adoptively transferred OVA-specific T lymphocytes into RAG-/- mice as described above, and either left the mice untreated (homeostatic prolif.-naive) or immunized the mice with OVAp/IFA s.c. to generate the memory/effector population. The function of these cells was then compared with that of freshly isolated OVA-specific T lymphocytes (fresh naive).
Proliferation of OVA-specific T lymphocytes
In contrast to effector cells, which undergo activation-induced
cell death upon Ag encounter (41, 42, 43, 44), the proliferative
response of memory cells to specific foreign Ags is faster and
quantitatively greater than that of naive cells (45, 46, 47).
Memory cells also proliferate in response to a lower dose of Ag and are
less dependent on costimulatory signals compared with naive cells
(47). To determine whether cells that had undergone
homeostatic proliferation had a similar proliferative potential as
memory cells, we labeled OVA-specific T lymphocytes that had previously
undergone homeostatic proliferation or been primed in vivo with
specific peptide (memory/effector) or freshly isolated OVA-specific T
lymphocytes (fresh naive) with CFSE and stimulated them in vitro at
varying doses of OVAp for 4 days (Fig. 4). An excess of irradiated splenocytes
served as a source of APCs, and cultures were stimulated either in the
presence of anti-CD28 to provide maximal costimulation, or CTLA4Ig
to block costimulation.
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A). In fact, quantifying proliferation based on CFSE
brightness showed that only 36% of the input OVA-specific T
lymphocytes initiated proliferation. However, when maximal
costimulation was provided, this response increased to 78%. In
contrast, the memory/effector population proliferated efficiently,
independent of costimulatory signals. In the absence of costimulation,
69% of the input OVA-specific memory/effector T lymphocytes initiated
proliferation. When maximal costimulation was provided there was a
minor increment to 76%. Therefore, the proliferative potential of the
effector/memory population was clearly greater than that of freshly
isolated naive OVA-specific T lymphocytes. Proliferation of
OVA-specific T lymphocytes that had previously undergone homeostatic
proliferation was similar to that of the memory/effector population. In
the absence of costimulation, 66% of the input OVA-specific T
lymphocytes initiated proliferation. When maximal costimulation was
provided, this response increased to 82%. Taken together, these data
suggest that the proliferative response of Ag-specific
CD4+ T lymphocytes that have undergone
homeostatic proliferation is similar to that of memory/effector
CD4+ T lymphocytes.
We also examined proliferative responses as a function of Ag
concentration under conditions of CD28 blockade. This showed that in
the absence of costimulation, freshly isolated OVA-specific T
lymphocytes required 10-fold more peptide to obtain the same amount of
proliferation compared with the memory/effector population (Fig. 4B). This is consistent with published data comparing
proliferation of memory and naive T lymphocytes (45, 46, 47).
The proliferative response of OVA-specific T lymphocytes that had
previously undergone homeostatic proliferation was again comparable to
the memory/effector cell population. In the absence of CD28-mediated
costimulation they required a 10-fold lower Ag concentration to achieve
a similar proliferative response as freshly isolated naive
CD4+ T cells. Taken together, these data show
that CD4+ T lymphocytes that have previously
undergone homeostatic proliferation show a similar proliferative
response as our memory/effector cell population.
Cytokine production by OVA-specific T lymphocytes
To determine whether the OVA-specific T lymphocytes that had undergone homeostatic proliferation had comparable ability to make cytokines as memory cells, we restimulated these cell populations for a short period (4–16 h) in vitro with OVAp and anti-CD28 in the presence of irradiated splenocytes.
When naive, OVA-specific T lymphocytes freshly isolated from the spleen
were stimulated in vitro, only 3.4 ± 5.8 and 0.43 ± 0.2%
made IL-2 or IFN-, respectively (Fig. 5 and Table I). This
is consistent with published data
indicating that during a brief activation period only a few naive T
cells produce cytokines (48, 49). In contrast, 33.8
± 5.9 and 20.5 ± 13.1% of effector/memory cells isolated from
the spleen made these cytokines (p < 0.05).
The percentage of OVA-specific T lymphocytes that had undergone
homeostatic proliferation and made these cytokines was intermediate
between the freshly isolated naive and memory/effector population.
Thus, 18.1 ± 12.6 and 9.1 ± 6.5%, respectively, made IL-2
and IFN-. Therefore, the data suggested that Ag-specific
CD4+ T lymphocytes that had undergone homeostatic
proliferation acquired the ability to make the cytokines IL-2 and
IFN-. However, they were not quite as likely to make cytokines as
the memory/effector T cells.
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), we next wished to
determine whether these two populations differed in their functional
capacities. As we (20) and others (50, 51)
have previously shown a link between cell division history and the
ability of cells to produce cytokines such as IFN- and IL-4, but not
IL-2, we chose cytokine production as a functional readout. For that
purpose we adoptively transferred CFSE-labeled OVA-specific T cells
into RAG-/- mice. Four weeks later we
sacrificed the mice and briefly restimulated the T cells in vitro
before intracellular staining for IL-2 and IFN-. As seen in Fig. 6, there was a strong relationship
between cell division and the ability of the T cells to produce
IFN-. Only the OVA-specific T cells that divided more than four to
five times were able to produce IFN-. This was in sharp contrast to
the capacity for IL-2 production,
which was not associated with cell cycle production.
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, C and E),
unlike freshly isolated naive OVA-specific T cells (Fig. 5, lower
left panel), which do not produce IL-2 under the conditions
studied. This suggests that a maturational/differentiative process has
occurred uncoupled from cell division.
As noted above, although cells that had undergone homeostatic
proliferation developed functional capabilities similar to cells that
had been stimulated by Ag, on a cell-by-cell basis they had a lower
frequency of IL-2 and IFN- production than Ag-activated
memory/effector cells. One possibility is that this difference was due
to the presence of effector cells in the latter population, which among
CD4+ T lymphocytes, are the most efficient
cytokine producers (52, 53). This might occur if the OVAp
was not cleared adequately from the immunized mouse during this time,
as it could continuously stimulate proliferation of OVA-specific T
lymphocytes and differentiation into effector cells. To test that, we
adoptively transferred 10 x 106
CFSE-labeled OVA-specific T lymphocytes into
RAG-/- mice that 4 wk earlier had received
unlabeled OVA-specific T lymphocytes and were either immunized with
OVA/IFA s.c. or left untreated (as control). The CFSE-labeled
OVA-specific T lymphocytes that were adoptively transferred into the
unimmunized RAG-/- mice did not
proliferate, whereas the OVA-specific T lymphocytes that were
adoptively transferred into the previously immunized
RAG-/- mice increased in size and
proliferated (data not shown). These proliferating T cells became 10%
of the CFSE-labeled population. This clearly suggests that residual
OVAp is present 4 wk after immunization of the
RAG-/- mice, in quantities large enough
to stimulate blast formation and proliferation of a proportion of
OVA-specific T lymphocytes, and thus suggests the presence of effector
cells in the memory/effector population.
To determine whether OVA-specific T lymphocytes that had undergone
homeostatic proliferation produced cytokine comparable to "true"
memory cells, it was important to eliminate the effector cells from our
primed memory/effector population. To do so, we adoptively transferred
OVA-specific T lymphocytes from the RAG-/- mice
(previously adoptively transferred with OVA-specific T lymphocytes and
either immunized with OVAp/IFA or left unimmunized) into wild-type Thy
1.1+ BALB/c mice (Fig. 7). This allows the
OVA-specific T cells to rest in a physiologic environment, away from
the specific peptide. By labeling the cells before adoptive transfer
with CFSE, we confirmed that the cells were resting (as measured by
lack of division) 4 and 7 days after adoptive transfer into BALB/c mice
(data not shown). Due to down-regulation of the TCR, after in vitro
culture we used Thy 1.1+ BALB/c mice to better
identify our cell population, which is Thy 1.2+.
IL-2 production by OVA-specific T lymphocytes that had previously
undergone homeostatic proliferation was now equal to the IL-2
production of the memory cell population (12.4 ± 7.7% and
15.9 ± 5.6% in the lymph nodes and 26.68 ± 18.63% and
30.15 ± 21.57% in the spleen, respectively). This was
significantly higher than that of freshly isolated OVA-specific T
lymphocytes adoptively transferred into BALB/c mice (1.4 ± 0.8%)
(Fig. 7). Collectively, these data show that Ag-specific
CD4+ T lymphocytes that have undergone
homeostatic proliferation are equally efficient cytokine producers as
resting memory CD4+ T lymphocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several groups have studied homeostatic proliferation both in
CD8+ and CD4+ T
lymphocytes. Most of the studies on CD8+ T cells
indicate that they change their surface phenotype to that of memory
cells (11, 17, 54). Some studies report no functional
alteration of CD8+ T cells during homeostatic
proliferation compared with naive cells (54), whereas
others disagree (16, 17, 19). The discrepancy in the
reported studies is probably due to differences in the experimental
system. Thus, it was recently shown that adoptive transfer of
transgenic CD8+ T lymphocytes into irradiated
wild-type mice allowed cells to proliferate and reach the "optimum"
number of peripheral cells (17). During this
proliferation, the CD8+ T cells changed their
surface phenotype to that of memory cells and acquired increased CTL
activity and the ability to make IFN-. When the peripheral pool was
"full", the T cells reverted back to quiescence, converted back to
a naive surface phenotype, and were less efficient in lysing targets
and making IFN-. However, when the same CD8+ T
cells were adoptively transferred into RAG-/-
mice, the behavior of the cells was quite different. They never reached
the same "desired" peripheral number of cells and never ceased
proliferating. Accordingly, the T cells never reverted back to a naive
phenotype and never lost the ability to lyse targets or make IFN-.
Others have also reported that the amount of proliferation of
transgenic CD8+ T cells is dependent on the
number of cells adoptively transferred as well as the time
(19). Therefore, the discrepancy in the published studies
can be explained by differences in factors such as the length of time
since adoptive transfer, numbers of cells that were adoptively
transferred, as well as the type of recipient mouse used.
Somewhat to our surprise, we identified two populations of Ag-specific
T lymphocytes that were generated during homeostatic proliferation.
There are several factors that might explain why this has not been a
consistent finding in published papers. Clearly, the ability to
identify these two populations is dependent on the ability to
differentiate the input cells from the cells of the recipient by a
specific marker, because the population generated by highly
proliferative cells becomes CFSE negative. Also, the appearance of this
population is dependent on the number of cells that are adoptively
transferred, as well as on time. Others studying polyclonal unsorted
CD4+ T lymphocytes have identified two
populations (39). Interestingly, in these studies the
population generated by multiple divisions (more than six to eight
times) expressed a surface phenotype consistent with that of memory
cells. In contrast, the population generated by slowly dividing cells
expressed a surface phenotype consistent with naive cells. Thus it was
proposed that the population of cells that divided maximally was
generated by homeostatic proliferation of memory cells within the
polyclonal CD4+ T cell population. Our data would
argue that the highly proliferative cells are not derived from
pre-existing memory cells because we used
DO11.10/RAG-/- or DO11.10/SCID cells. These
cells are unable to express a second TCR on their surface, which in the
presence of an unknown foreign Ag can lead to memory T cell formation
(26). Consistently, all the DO11.10 cells express a naive
phenotype. Rather, our data would suggest that either a subset of the
donor CD4+ T lymphocytes are already inherently
different or, alternatively, that the cells are exposed to different
signals in the lymphopenic mouse. This would include competition for
signals delivered by professional APCs as has been found to occur in
vivo (55, 56). Although not mutually exclusive, we do
favor the latter explanation because the highly proliferative donor
cells were dependent upon B7 signaling for their proliferation, whereas
the other population was not. It is controversial whether or not
CD4+ T cells that divide in lymphopenic hosts
acquire phenotypic and functional changes comparable to that of memory
cells. Most studies find that naive CD4+ T
lymphocytes do not convert to a surface memory phenotype during
homeostatic proliferation (14, 39, 57); however, some have
reported partial up-regulation of CD44 (11). Our data
would suggest that CD4+ T cells might need a long
period of time (>4 wk) to acquire the phenotypic changes of memory
cells. This is in contrast to CD8+ T lymphocytes,
which convert to a surface phenotype much earlier, possibly because of
a brisker proliferative response during lymphopenia (11).
Our data also suggest that CD4+ T cells that have
undergone homeostatic proliferation acquire function comparable to
memory T cells. First, their ability to proliferate in response to low
doses of Ag and relative independence of costimulation is similar to
the memory population. Second, their ability to make the cytokines IL-2
and IFN- in a short restimulation assay is greater than that of
naive CD4+ T cells, and their IL-2 production is
similar to that of memory cells. Finally, functional memory-like cells
are more likely to be found following homeostatic proliferation in
the population of cells that have divided multiple times and whose
generation requires CD28 signals. This conclusion is based on the
finding that the multiply divided cells have the greatest
down-regulation of CD45RB (Fig. 3B), and more importantly,
are the only population of cells that produced IFN- (Fig. 6A).
The question remains whether or how proliferation and differentiation are mechanistically linked. Signals from both the TCR and from cytokine receptors are critical in determining the cytokine profile of naive T cells. Multiple investigators have shown a correlation between cell cycle progression and cytokine production (20, 50, 58). This may be explained in part by the remodeling of chromatin during cell cycle progression (50, 51). Despite the chromatin changes that take place during proliferation, some have found that cell cycle progression is not required for cytokine production of naive T cells (59, 60). The controversy may, in part, be explained by different nonphysiologic situations used by the investigators to induce cytokine production in the absence of division. There are also multiple known factors, such as transcription factors and their repressors that might induce cytokine production independent of proliferation (61, 62, 63, 64, 65, 66, 67, 68). Taken together, cell division seems to be an important parameter determining differentiation of naive T cells into effector cells, by remodeling DNA. It remains to be determined whether these epigenetic modifications of DNA are stably expressed in resting memory cells and whether similar changes occur in T lymphocytes that have undergone homeostatic proliferation.
Collectively therefore, the data suggest that the specific foreign Ag is not required for formation of memory CD4+ T cells. Rather, we find that endogenous peptides and necessary costimulatory molecules during lymphopenia are sufficient to stimulate formation of memory cells. It remains to be determined whether their ability to survive and compete for niches is similar to that of memory cells generated after encounter with the specific exogenous Ag.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Laurence A. Turka, Department of Medicine, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104. E-mail address: turka{at}mail.med.upenn.edu
3 Abbreviations used in this paper: RAG, recombination-activating gene; OVAp, OVA peptide (323–339).
Received for publication April 2, 2001. Accepted for publication July 23, 2001.
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  X. Wang, X. Chang, V. Facchinetti, Y. Zhuang, and B. Su MEKK3 Is Essential for Lymphopenia-Induced T Cell Proliferation and Survival J. Immunol., March 15, 2009; 182(6): 3597 - 3608. [Abstract] [Full Text] [PDF]
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C. J. Winstead, J. M. Fraser, and A. Khoruts Regulatory CD4+CD25+Foxp3+ T Cells Selectively Inhibit the Spontaneous Form of Lymphopenia-Induced Proliferation of Naive T Cells J. Immunol., June 1, 2008; 180(11): 7305 - 7317. [Abstract] [Full Text] [PDF]
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T. Suzuki, S. Ogawa, K. Tanabe, H. Tahara, R. Abe, and H. Kishimoto Induction of Antitumor Immune Response by Homeostatic Proliferation and CD28 Signaling J. Immunol., April 1, 2008; 180(7): 4596 - 4605. [Abstract] [Full Text] [PDF]
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V. F. Moxham, J. Karegli, R. E. Phillips, K. L. Brown, T. T. Tapmeier, R. Hangartner, S. H. Sacks, and W. Wong Homeostatic Proliferation of Lymphocytes Results in Augmented Memory-Like Function and Accelerated Allograft Rejection J. Immunol., March 15, 2008; 180(6): 3910 - 3918. [Abstract] [Full Text] [PDF]
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S. Xiao, D.-m. Su, and N. R. Manley Atypical Memory Phenotype T Cells with Low Homeostatic Potential and Impaired TCR Signaling and Regulatory T Cell Function in Foxn1{Delta}/{Delta} Mutant Mice J. Immunol., December 15, 2007; 179(12): 8153 - 8163. [Abstract] [Full Text] [PDF]
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E. Shklovskaya and B. Fazekas de St. Groth Severely Impaired Clonal Deletion of CD4+ T Cells in Low-Dose Irradiated Mice: Role of T Cell Antigen Receptor and IL-7 Receptor Signals J. Immunol., December 15, 2006; 177(12): 8320 - 8330. [Abstract] [Full Text] [PDF]
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C. Bourgeois and B. Stockinger CD25+CD4+ Regulatory T Cells and Memory T Cells Prevent Lymphopenia-Induced Proliferation of Naive T Cells in Transient States of Lymphopenia J. Immunol., October 1, 2006; 177(7): 4558 - 4566. [Abstract] [Full Text] [PDF]
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F. Song, Z. Guan, I. E. Gienapp, T. Shawler, J. Benson, and C. C. Whitacre The Thymus Plays a Role in Oral Tolerance in Experimental Autoimmune Encephalomyelitis J. Immunol., August 1, 2006; 177(3): 1500 - 1509. [Abstract] [Full Text] [PDF]
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T. L. Vanasek, S. L. Nandiwada, M. K. Jenkins, and D. L. Mueller CD25+Foxp3+ Regulatory T Cells Facilitate CD4+ T Cell Clonal Anergy Induction during the Recovery from Lymphopenia J. Immunol., May 15, 2006; 176(10): 5880 - 5889. [Abstract] [Full Text] [PDF]
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D. C. Neujahr, C. Chen, X. Huang, J. F. Markmann, S. Cobbold, H. Waldmann, M. H. Sayegh, W. W. Hancock, and L. A. Turka Accelerated Memory Cell Homeostasis during T Cell Depletion and Approaches to Overcome It. J. Immunol., April 15, 2006; 176(8): 4632 - 4639. [Abstract] [Full Text] [PDF]
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H. Su, D. S. Chang, S. S. Gambhir, and J. Braun Monitoring the Antitumor Response of Naive and Memory CD8 T Cells in RAG1-/- Mice by Positron-Emission Tomography J. Immunol., April 1, 2006; 176(7): 4459 - 4467. [Abstract] [Full Text] [PDF]
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 B. Knoechel, J. Lohr, E. Kahn, J. A. Bluestone, and A. K. Abbas Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen J. Exp. Med., November 21, 2005; 202(10): 1375 - 1386. [Abstract] [Full Text] [PDF]
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 H. Zhang and G. G. Meadows Chronic alcohol consumption in mice increases the proportion of peripheral memory T cells by homeostatic proliferation J. Leukoc. Biol., November 1, 2005; 78(5): 1070 - 1080. [Abstract] [Full Text] [PDF]
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 S. P Hickman and L. A Turka Homeostatic T cell proliferation as a barrier to T cell tolerance Phil Trans R Soc B, September 29, 2005; 360(1461): 1713 - 1721. [Abstract] [Full Text] [PDF]
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C. Bourgeois, G. Kassiotis, and B. Stockinger A Major Role for Memory CD4 T Cells in the Control of Lymphopenia-Induced Proliferation of Naive CD4 T Cells J. Immunol., May 1, 2005; 174(9): 5316 - 5323. [Abstract] [Full Text] [PDF]
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W. C. Kieper, A. Troy, J. T. Burghardt, C. Ramsey, J. Y. Lee, H.-Q. Jiang, W. Dummer, H. Shen, J. J. Cebra, and C. D. Surh Cutting Edge: Recent Immune Status Determines the Source of Antigens That Drive Homeostatic T Cell Expansion J. Immunol., March 15, 2005; 174(6): 3158 - 3163. [Abstract] [Full Text] [PDF]
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 T. J. Fry, M. Sinha, M. Milliron, Y.-W. Chu, V. Kapoor, R. E. Gress, E. Thomas, and C. L. Mackall Flt3 ligand enhances thymic-dependent and thymic-independent immune reconstitution Blood, November 1, 2004; 104(9): 2794 - 2800. [Abstract] [Full Text] [PDF]
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K. A. Hagen, C. T. Moses, E. F. Drasler, K. M. Podetz-Pedersen, S. C. Jameson, and A. Khoruts A Role for CD28 in Lymphopenia-Induced Proliferation of CD4 T Cells J. Immunol., September 15, 2004; 173(6): 3909 - 3915. [Abstract] [Full Text] [PDF]
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M. P. Eggena, L. S.K. Walker, V. Nagabhushanam, L. Barron, A. Chodos, and A. K. Abbas Cooperative Roles of CTLA-4 and Regulatory T Cells in Tolerance to an Islet Cell Antigen J. Exp. Med., June 21, 2004; 199(12): 1725 - 1730. [Abstract] [Full Text] [PDF]
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J.-Y. Choi and J. Craft Activation of Naive CD4+ T Cells In Vivo by a Self-Peptide Mimic: Mechanism of Tolerance Maintenance and Preservation of Immunity J. Immunol., June 15, 2004; 172(12): 7399 - 7407. [Abstract] [Full Text] [PDF]
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 B. Min, G. Foucras, M. Meier-Schellersheim, and W. E. Paul Spontaneous proliferation, a response of naive CD4 T cells determined by the diversity of the memory cell repertoire PNAS, March 16, 2004; 101(11): 3874 - 3879. [Abstract] [Full Text] [PDF]
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J. H. Xie, N. Nomura, M. Lu, S.-L. Chen, G. E. Koch, Y. Weng, R. Rosa, J. Di Salvo, J. Mudgett, L. B. Peterson, et al. Antibody-mediated blockade of the CXCR3 chemokine receptor results in diminished recruitment of T helper 1 cells into sites of inflammation J. Leukoc. Biol., June 1, 2003; 73(6): 771 - 780. [Abstract] [Full Text] [PDF]
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N. J. Hill, K. Van Gunst, and N. Sarvetnick Th1 and Th2 Pancreatic Inflammation Differentially Affects Homing of Islet-Reactive CD4 Cells in Nonobese Diabetic Mice J. Immunol., February 15, 2003; 170(4): 1649 - 1658. [Abstract] [Full Text] [PDF]
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C. T. Moses, K. M. Thorstenson, S. C. Jameson, and A. Khoruts Competition for self ligands restrains homeostatic proliferation of naive CD4 T cells PNAS, February 4, 2003; 100(3): 1185 - 1190. [Abstract] [Full Text] [PDF]
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J.-I. Rodriguez-Barbosa, Y. Zhao, G. Zhao, A. Ezquerra, and M. Sykes Murine CD4 T Cells Selected in a Highly Disparate Xenogeneic Porcine Thymus Graft Do Not Show Rapid Decay in the Absence of Selecting MHC in the Periphery J. Immunol., December 15, 2002; 169(12): 6697 - 6710. [Abstract] [Full Text] [PDF]
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B. Adkins, Y. Bu, and P. Guevara Murine Neonatal CD4+ Lymph Node Cells Are Highly Deficient in the Development of Antigen-Specific Th1 Function in Adoptive Adult Hosts J. Immunol., November 1, 2002; 169(9): 4998 - 5004. [Abstract] [Full Text] [PDF]
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M. Eyrich, T. Croner, C. Leiler, P. Lang, P. Bader, T. Klingebiel, D. Niethammer, and P. G. Schlegel Distinct contributions of CD4+ and CD8+ naive and memory T-cell subsets to overall T-cell-receptor repertoire complexity following transplantation of T-cell-depleted CD34-selected hematopoietic progenitor cells from unrelated donors Blood, August 13, 2002; 100(5): 1915 - 1918. [Abstract] [Full Text] [PDF]
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 H.-M. Hu, C. H. Poehlein, W. J. Urba, and B. A. Fox Development of Antitumor Immune Responses in Reconstituted Lymphopenic Hosts Cancer Res., July 15, 2002; 62(14): 3914 - 3919. [Abstract] [Full Text] [PDF]
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M. H. Yen, N. Lepak, and S. L. Swain Induction of CD4 T Cell Changes in Murine AIDS Is Dependent on Costimulation and Involves a Dysregulation of Homeostasis J. Immunol., July 15, 2002; 169(2): 722 - 731. [Abstract] [Full Text] [PDF]
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M. Prlic, B. R. Blazar, A. Khoruts, T. Zell, and S. C. Jameson Homeostatic Expansion Occurs Independently of Costimulatory Signals J. Immunol., November 15, 2001; 167(10): 5664 - 5668. [Abstract] [Full Text] [PDF]
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6 times) ± SD obtained from the spleen between 3 and 5 wk after
adoptive transfer as a function of the number of clonotypic cells that
were adoptively transferred in three separate experiments.
