Homeostatic Expansion Occurs
Independently of Costimulatory Signals
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J Immunol 2001; 167:5664-5668; ;
doi: 10.4049/jimmunol.167.10.5664
http://www.jimmunol.org/content/167/10/5664
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References
Martin Prlic, Bruce R. Blazar, Alexander Khoruts, Traci Zell
and Stephen C. Jameson
Homeostatic Expansion Occurs Independently of
Costimulatory Signals1
Martin Prlic,* Bruce R. Blazar,† Alexander Khoruts,‡ Traci Zell,§ and Stephen C. Jameson2*
Naive T cells undergo homeostatic proliferation in lymphopenic mice, a process that involves TCR recognition of specific self
peptide/MHC complexes. Since costimulation signals regulate the T cell response to foreign Ags, we asked whether they also
regulate homeostatic expansion. We report in this study that homeostatic expansion of CD4 and CD8 T cells occurs independently
of costimulation signals mediated through CD28/B7, CD40L/CD40, or 4-1BB/4-1BBL interactions. Using DO11.10 TCR transgenic
T cells, we confirmed that CD28 expression was dispensable for homeostatic expansion, and showed that the presence of endogenous CD4ⴙCD25ⴙ regulatory cells did not detectably influence homeostatic expansion. The implications of these findings with
respect to regulation of T cell homeostasis and autoimmunity are discussed. The Journal of Immunology, 2001, 167: 5664 –5668.
Departments of *Laboratory Medicine and Pathology, †Pediatrics, ‡Medicine, and
§
Microbiology, Center for Immunology, University of Minnesota, Minneapolis, MN
55455
Received for publication August 22, 2001. Accepted for publication September
19, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by American Cancer Society Grant RPG 99-264 (to S.C.J.)
and National Institutes of Health Grants R01-AI38903 (to S.C.J.), R01-AI34495,
R37-HL56067, and P01-AI-35225 (to B.R.B.).
2
Address correspondence and reprint requests to Dr. Stephen C. Jameson, Department of Laboratory Medicine and Pathology, Center for Immunology, MMC
334, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455.
E-mail address: [email protected]
Copyright © 2001 by The American Association of Immunologists
true independence of homeostatic expansion from costimulation,
or could instead point to functional redundancy among costimulatory pathways. In this study, we show that homeostatic expansion does not require CD28 on the T cell, nor does it require CD40
or 4-1BBL expression on host APCs. This was true even when
combinations of costimulator deficiencies were studied (i.e.,
CD28⫺/⫺ T cells transferred into CD40⫺/⫺ or 4-1BBL⫺/⫺ hosts).
Indeed, in contrast to the critical role of CD28 in responses to
foreign Ags, we show that CD28-deficient cells proliferate as well
(or even slightly better) than wild-type cells during homeostatic
expansion of DO11.10 TCR transgenic T cells. This unexpected
result did not appear to correlate with the presence or absence of
the CD4⫹CD25⫹ regulatory T cell subset, as shown by depletion
experiments. The implications of these data on the discrimination
between conventional and homeostatic proliferation pathways are
discussed.
Materials and Methods
Mice
C57BL/6 (B6) background CD40⫺/⫺ mice were initially obtained from D.
Parker (Portland, OR), and additional CD40⫺/⫺ and B6 background
CD28⫺/⫺ mice were obtained from The Jackson Laboratory (Bar Harbor,
ME). The 4-1BBL⫺/⫺ mice (19) were obtained in collaboration with J.
Peschon (Immunex, Seattle, WA). BALB/c, BALB/c Rag⫺/⫺, B6, and
CD45.1 congenic B6 mice (carried under the strain name B6.Ly-5.2) were
obtained from the National Cancer Institute, The Jackson Laboratory, or
Taconic Farms. Normal and CD28⫺/⫺ DO11.10 TCR transgenic mice have
been described previously (20, 21) and were maintained at the University
of Minnesota. All mice were maintained under specific pathogen-free conditions and used at 6 –12 wk of age. Recipient mice were maintained on
antibiotic water (polymixin B sulfate and neomycin sulfate) throughout the
course of the experiment.
Adoptive transfer
Cells were purified by negative selection utilizing magnetic cell sorting
using MACS microbeads (Miltenyi Biotec, Auburn, CA). To purify
CD44low T cell, lymph node cells were depleted from adherent cells (90min incubation on tissue culture-treated flasks at 37°C) and labeled with
FITC-coupled Abs to B220 (clone RA3-6B2), I-Ab (AF6-120.1), and CD44
(IM-7) (0.0125 ␮g anti-B220 and anti-I-Ab per 1 ⫻ 106 cells; 0.004 ␮g
anti-CD44 per 1 ⫻ 106 cells) (all from BD PharMingen, San Diego, CA).
In some experiments, CD4 cells were also labeled with FITC anti-CD4
(GK1.5; BD PharMingen) (0.0125 ␮g per 1 ⫻ 106 cells). Following staining, cells were subject to depletion using anti-FITC MACS microbeads.
Flow through cells were ⬎90% pure (established by flow cytometric
analysis).
0022-1767/01/$02.00
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L
ymphocyte numbers in the periphery are tightly controlled through various homeostatic mechanisms. The size
of the T and B cell subsets is regulated independently, as
are naive and memory T cell pools (1, 2). Recent data from a
number of groups indicated that a reduction in the number of circulating T cells is compensated by proliferation of remaining lymphocytes, a process called homeostatic expansion (3– 8). Since T
cell numbers may be reduced during life as a result of both infections and diminished thymic output, homeostatic expansion may
play an important role in maintaining peripheral T cell numbers.
T cell recognition of specific self peptide/MHC ligands is important for optimal homeostatic expansion, and cells undergoing
this process become phenotypically and functionally indistinguishable from memory T cells (5, 6, 9). Together, these data suggest
that homeostatic expansion involves a TCR signal that induces
functional differentiation. As is well established, T proliferative
responses toward conventional foreign peptide/MHC ligands require a second signal, referred to as costimulation, to avoid T cell
anergy or death. Costimulation is mediated by a variety of cell
surface molecules of which CD28/B7 and CD40L/CD40 interactions are the best characterized (reviewed in Refs. 10 and 11),
although several other molecules may synergize with or even substitute for these classic costimulators (reviewed in Refs. 12–14).
Prominent among these is the 4-1BB/4-1BBL interaction that can
both augment and substitute for CD28/B7 interactions in both CD4
and CD8 T cell responses (12, 15–18). However, the role of costimulation in homeostatic expansion has not been well defined.
Unpublished data from two groups suggest that CD28 is not essential for homeostatic expansion (6, 8), a result that might reflect
The Journal of Immunology
5665
CD4⫹ cells from DO11.10 animals were purified utilizing a similar
protocol using biotinylated (bio)3 anti-CD8 (53.6-7) plus bio-anti-B220,
followed by streptavidin (SA) microbeads. In some cases, CD25⫹ cells
were depleted at the same time using bio-anti-CD25 (7D4) (if applicable)
(PharMingen). The percentage of CD4⫹CD25⫹ T cells before and after
depletion was assessed by staining an aliquot of the cells with both
CD25-PE Ab and SA-PE (to reveal cells bound by the depleting bio-CD25
Ab) and counterstaining for CD4 with CD4-APC or CD4-PerCP.
After purification, cells were stained with CFSE (Molecular Probes,
Eugene, OR), as described previously (7, 22). Labeled donor cells were
suspended in PBS, and 1–3 million cells were injected via the tail vein of
recipient mice. In some cases, host mice were sublethally irradiated (700
cGy) 1 day before cell transfer. In some experiments, recipient mice were
primed i.v. with 2 mg OVA (whole protein) plus 25 ␮g LPS (Sigma, St.
Louis, MO) 1 day cell posttransfer. Cells were analyzed on day 3
postpriming.
Flow cytometry
Results
Experimental system
To test the role of CD28/B7 interactions in regulating homeostatic
expansion, we studied proliferation of CFSE-labeled normal and
CD28⫺/⫺ donor T cells after transfer into normal or lymphopenic
B6 hosts. To minimize mouse to mouse variation, we employed a
cotransfer system in which we premixed CD45.1⫹ wild-type and
CD45.2⫹ CD28⫺/⫺ T cells before transfer and distinguished the
two donor populations during analysis with CD45 allele-specific
Abs. Similar transfers were performed using CD40⫺/⫺ or
4-1BBL⫺/⫺ hosts to determine the effect of disrupting CD40L/
CD40 and 4-1BB/4-1BBL interactions (alone or in combination
with CD28 deficiency) on homeostatic expansion.
No requirement for CD28, CD40, or 4-1BBL in T cell
homeostatic expansion
Using the transfer scheme outlined above, we tested whether CD28
expression on T cells and CD40 or 4-1BBL expression on host
APCs was required for T cell homeostatic expansion. As expected,
neither wild-type nor CD28⫺/⫺ T cells proliferate after transfer
into unmanipulated syngeneic B6 hosts (Fig. 1, a– d). Also as expected, normal CD8 and CD4 T cells undergo one to four rounds
of proliferation 14 days after transfer into irradiated syngeneic
hosts (Fig. 1, e and g, respectively). Interestingly, CD28⫺/⫺ T cells
proliferated to the same extent as wild-type cells when transferred
into irradiated wild-type hosts (Fig. 1, f and h). This suggests that
CD28 is not required for homeostatic expansion of either CD8 or
CD4 subsets.
It was possible that CD40L/CD40 or 4-1BB/4-1BBL costimulation could partially or completely compensate for CD28 deficiency. Indeed, 4-1BB and CD40L interactions can synergize with
CD28 or even replace it in some T cell responses (17, 19, 23, 24).
To test this, we transferred both wild-type and CD28⫺/⫺ T cells
into CD40-deficient hosts (Fig. 1, i–l) or into 4-1BBL-deficient
hosts (Fig. 1, m–p). Once again, homeostatic proliferation of both
CD4 and CD8 T cell subsets was similar regardless of CD28 expression on the donor cells and CD40 or 4-1BBL expression in
the host.
3
Abbreviations used in this paper: bio, biotinylated; SA, streptavidin.
FIGURE 1. Role of CD28 and CD40 interactions in homeostatic expansion of cotransferred CD4 and CD8 T cells. One million CD44low T
cells from CD28⫺/⫺ and from CD45.1 congenic B6 animals were mixed at
a 1:1 ratio, CFSE labeled, and injected i.v. into the indicated host mice. In
some cases (irrad.), recipient mice were sublethally irradiated (700 cGy) 1
day before transfer. Recipient mice were sacrificed 14 days posttransfer,
and lymph node cells were analyzed by flow cytometry using Abs to CD4,
CD8, and CD45.1 to distinguish the different subsets of cells. The CFSE
profile of CD4 and CD8 subsets and of CD28⫺/⫺ and wild-type populations is shown separately, but comes from the same mice in each row.
Because the recipient mice are all CD45.1⫺, host cells appear in the analysis of the CD28⫺/⫺ subsets as a CFSE-negative population. Data are
representative of two to three mice per group, and comparable results were
obtained in at least three experiments.
In all of these experiments, we observed similar donor T cell
recoveries for both the normal and CD28⫺/⫺ donors after expansion in the various irradiated host strains (data not shown), which
argues against a survival role for these costimulator molecules in
homeostatic expansion, at least in the short term.
CD28⫺/⫺ DO11.10 TCR transgenic T cells undergo greater
homeostatic expansion, but less Ag-driven proliferation than
their CD28⫹ counterparts
The experiments above utilized polyclonal T cells and, although
the overall degree of homeostatic expansion was similar with or
without CD28, it was possible that different populations of T cells
were responding, perhaps based on TCR specificity dictating
CD28 dependence. Furthermore, we had to date looked exclusively in the C57BL/6 system, whereas there is some evidence that
the impact of CD28/B7 deficiency is more extreme in certain
BALB/c responses (25). Accordingly, we also studied homeostatic
expansion of T cells from DO11.10 TCR transgenic mice, which
are maintained on the BALB/c background. Data from several
groups have shown that CD28 deficiency impairs the peptide/
MHC (OVA/I-Ad) response of these T cells, both in vitro and in
vivo (21, 26).
Thus, we transferred DO11.10 and DO11.10/CD28⫺/⫺ cells
into irradiated BALB/c hosts and analyzed their homeostatic expansion. As shown in Fig. 2, not only did we fail to see a requirement for CD28 in DO11.10 homeostatic expansion, there was evidence that CD28⫺/⫺ DO11.10 cells proliferate moderately better
than their wild-type counterparts. The degree of this improved response was somewhat variable between individual animals, as exemplified in Fig. 2. Thus, at a minimum we find that more CD28deficient DO11 cells commit to homeostatic expansion than normal
DO11 cells (see proportions of cells undergoing zero vs three divisions, Fig. 2a), and that in some cases this was seen as an extra
round of proliferation by the CD28⫺/⫺ DO11.10 cells (Fig. 2b).
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Recipient mice were sacrificed at the time points indicated, and single cell
suspensions were prepared separately from spleen and a pool of major
lymph nodes. Lymph node and spleen cells were then stained with the
fluorescently conjugated Abs to CD4, CD8, CD44, and CD45RB. Biotinylated Abs to CD25, CD45.1, CD45.2, CD28, and KJ1-26 (specific for the
DO11.10 TCR) were used, staining being revealed using SA-TriColor,
SA-PerCP, or SA-PE (all reagents from BD PharMingen). Cells were analyzed by using a FACSCalibur (BD Biosciences, San Jose, CA) and analyzed using FLOWJO (TreeStar, San Carlos, CA) software.
5666
HOMEOSTATIC EXPANSION OCCURS IN THE ABSENCE OF COSTIMULATION
tion was slightly greater than that of wild-type transgenic cells
(Fig. 3, g vs f).
CD4⫹CD25⫹ cells do not contribute to the difference seen
between normal and CD28⫺/⫺ DO11.10 T cells
FIGURE 2. Homeostatic expansion of normal vs CD28⫺/⫺ DO11.10
cells. One million CFSE-labeled DO11.10 or CD28⫺/⫺ DO11.10 T cells
were transferred into sublethally irradiated BALB/c hosts and analyzed 21
days after transfer. The CFSE profiles shown were gated on CD4⫹ KJ126⫹ cells. The number of rounds of proliferation (derived from comparison
with CFSE levels in cells transferred into unirradiated BALB/c hosts) are
indicated over the traces. Normal DO11.10 cells are represented by the
shaded histogram; CD28⫺/⫺ DO11.10 cells are shown as the unfilled solid
line. The data for a and b come from separate host mice in the same
experiment and represent the range seen among three separate experiments
involving three mice per group.
FIGURE 3. Impact of CD28 expression and CD4⫹CD25⫹ T cell depletion (Depl.) on foreign Ag reactivity and homeostatic expansion of
DO11.10 cells in irradiated hosts. Normal, CD28⫺/⫺, and CD25 T celldepleted DO11.10 T cells were transferred into normal (a– e) or irradiated
(f– h) BALB/c hosts. The percentages of CD4⫹CD25⫹ cells in the normal,
CD28⫺/⫺, and CD25-depleted populations were 4.2%, 1.2%, and 0.5%,
respectively, in the experiment shown. The unirradiated hosts were challenged with OVA/LPS 1 day after transfer and sacrificed 3 days later.
Irradiated (f– h) and control unirradiated hosts (e) were not immunized and
were analyzed 21 days after transfer. CFSE profiles shown are gated on
CD4⫹ KJ1-26⫹ cells, except in a, which was gated on CD4⫹ KJ1-26⫺
cells as a control for Ag nonspecific proliferation. Data are representative
of three mice in each group.
FIGURE 4. Impact of CD28 expression and CD4⫹CD25⫹ T cell depletion (Depl.) on homeostatic expansion in Rag⫺/⫺ hosts. Three million
CFSE-labeled normal, CD28⫺/⫺, or CD25 T cell-depleted DO11.10 cells
were transferred into BALB/c Rag-2⫺/⫺ hosts. The percentages of
CD4⫹CD25⫹ cells in the normal, CD28⫺/⫺, and CD25-depleted populations were 4.1, 2.8, and 0.6%, respectively. Recipient mice were harvested
22 days after transfer, and the data shown are representative of three mice
in each group.
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Given this surprising result, it was important to test that, in our
hands, CD28 deficiency had the predicted effect of blunting the
DO11.10 Ag-specific response, as reported by others (21, 26).
Thus, we transferred CFSE-labeled wild-type or CD28⫺/⫺
DO11.10 T cells into irradiated or unirradiated BALB/c hosts. The
unirradiated hosts were then immunized with OVA/LPS, and the
proliferative response of the DO11.10 population was monitored.
As expected, CD28 deficiency severely impaired the Ag-specific
DO11.10 response (Fig. 3, c vs b). However, as in Fig. 2, homeostatic proliferation of the same CD28⫺/⫺ DO11.10 donor popula-
Looking for an explanation for the slightly enhanced proliferation
of DO11.10 CD28⫺/⫺ cells, we considered the contribution of
CD4⫹CD25⫹ regulatory cells (27, 28). This subset, which can
suppress autoimmune T cell responses, is underrepresented in
CD28⫺/⫺ animals (29). This is also true in mice expressing the
DO11.10 TCR transgene, in which CD28⫺/⫺ DO11.10 mice have
fewer CD4⫹CD25⫹ cells compared with wild-type DO11.10 animals (3–5% vs 5–10%, respectively; data not shown). Thus, we
tested the impact of depleting the donor DO11.10 cells of CD25⫹
cells. If the CD25⫹ subset were restraining normal DO11.10 homeostatic expansion, then removal of these cells should lead to
even greater homeostatic expansion than seen for the CD28⫺/⫺
DO11.10 group. CD25⫹ regulatory cell depletion did not improve
the robust Ag-specific T cell response in vivo, perhaps reflecting
the fact that this response was already optimal (Fig. 3d). Interestingly, however, CD25⫹ subset depletion failed to impact DO11.10
homeostatic expansion (Fig. 3, compare h with f).
Since sublethal irradiation does not deplete all T cells in the
host, it was possible that endogenous CD4⫹CD25⫹ cells could
influence homeostatic expansion of the transferred cells. Hence,
we also transferred these same DO11.10 populations into BALB/c
Rag⫺/⫺ hosts. Similar to the results in the irradiated hosts, proliferation of CD25-depleted DO11.10 cells did not differ significantly
from that of normal DO11.10 cells, whereas there was once again
a subtle, but consistent increase in proliferation by the CD28⫺/⫺
DO11.10 population (Fig. 4). Intriguingly, after expansion of
DO11.10 cells transferred into Rag-deficient hosts, a sizeable proportion of the cells had proliferated sufficiently to dilute out the
CFSE dye, which was not observed in irradiated hosts (compare
Fig. 4 with Figs. 2 and 3). This may relate to the fact that sublethally irradiated hosts recover endogenous T cell numbers during
The Journal of Immunology
the course of the experiment (therefore limiting T cell space),
whereas this will not occur in Rag⫺/⫺ hosts. We also noticed a
slight increase in the total number of donor T cells recovered after
transfer of CD25-depleted DO11.10 cells compared with wild-type
or CD28⫺/⫺ cells, which was also not observed in irradiated hosts
(data not shown).
Thus, these results do not support a role for the DO11.10
CD25⫹ subset in restraining homeostatic expansion of DO11.10 T
cells.
Discussion
In fact, our data suggested that lack of CD28 expression mildly
enhanced DO11.10 T cell homeostatic expansion in both sublethally irradiated and Rag⫺/⫺ hosts. It is important to note that this
effect was subtle and somewhat variable in its magnitude (as exemplified in Fig. 2). Furthermore, we could not see consistent evidence for enhanced proliferation of CD28⫺/⫺ polyclonal T cells
(Fig. 1 and data not shown): subtle differences in the CD28⫺/⫺
population may be obscured if there is variability in the degree of
homeostatic proliferation among polyclonal T cells (as is most
likely based on analysis of numerous TCR transgenic systems) (9,
33) or may imply that the enhancing effect of CD28 deficiency is
unique to DO11.10 T cells. Further investigation will be needed to
explore the significance of this finding.
While our data rule out a requirement for CD28 in homeostatic
expansion, it is still possible that such responses are regulated by
CTLA-4, which negatively regulates T cell proliferation in response to foreign Ag. Indeed, since CD28/B7 interactions are involved (but not essential) (34, 35) for CTLA-4 expression itself,
cells proliferating during homeostatic expansion may not express
CTLA-4 at sufficient levels to inhibit the expansion process. This
in turn may allow these cells to evade normal regulatory control.
One potential explanation for the mild positive effect of CD28
deficiency on homeostatic expansion could be the decreased representation of CD4⫹CD25⫹ regulatory cells in CD28⫺/⫺ animals
(29). However, elimination of this population in the DO11.10 system did not result in enhanced homeostatic expansion in either
irradiated or Rag⫺/⫺ recipients. These data do not rule out the
possibility that CD4⫹CD25⫹ cells may be able to inhibit homeostatic expansion if present in sufficient numbers or if activated
appropriately (an apparent requirement for their suppressive effect)
(36, 37). Rather, our data argue that the endogenous CD4⫹CD25⫹
population does not inhibit homeostatic expansion, in contrast to
the proposed role of such cells in preventing autoimmune or alloreactive responses (27, 28, 37–39). Interestingly, this might be
expected if inhibition by CD4⫹CD25⫹ regulatory cells was mediated through disruption of IL-2 production and/or IL-2R␣ upregulation (as has been proposed; Ref. 28), since IL-2 appears not
to be important in homeostatic expansion and IL-2R␣ is not expressed during this response (7, 8, 40, 41). Thus, these data further
emphasize the differences in regulation that control homeostatic
expansion compared with other T cell responses.
Note added in proof. While this manuscript was in press, Gudmundsdottir et al. (42) reported that CTLA4Ig blocked homeostatic expansion of a subset of DO11.10 Rag⫺/⫺ T cells in Rag⫺/⫺
recipients. Specifically, CTLA4Ig administration caused a selective loss of the rapidly proliferating subset and this inhibition was
influenced by the number of donor T cells transferred. These data
suggest B7 interactions might influence homeostatic proliferation
of certain subsets under specific conditions, while our data argue
that CD28 interactions are not obligatory for this response. The
basis for these discordant results presumably lies in the different
experimental approaches used.
Acknowledgments
We thank David Parker and Jacques Peschon for knockout mice, Marc
Jenkins for providing other mouse strains and suggestions, Kristen Thorstenson and Brent Koehn for excellent technical assistance, and Kris
Hogquist and members of the Hogquist and Jameson labs for critical input.
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