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Lymphopenia-Induced Homeostatic
Proliferation of CD8 + T Cells Is a
Mechanism for Effective Allogeneic Skin
Graft Rejection following Burn Injury
Robert Maile, Carie M. Barnes, Alma I. Nielsen, Anthony A.
Meyer, Jeffrey A. Frelinger and Bruce A. Cairns
J Immunol 2006; 176:6717-6726; ;
doi: 10.4049/jimmunol.176.11.6717
http://www.jimmunol.org/content/176/11/6717
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References
The Journal of Immunology
Lymphopenia-Induced Homeostatic Proliferation of CD8ⴙ T
Cells Is a Mechanism for Effective Allogeneic Skin Graft
Rejection following Burn Injury1
Robert Maile,*† Carie M. Barnes,* Alma I. Nielsen,* Anthony A. Meyer,* Jeffrey A. Frelinger,†
and Bruce A. Cairns2*†
M
odern organ transplantation began with the observation in burn patients during World War II that repeated
allogeneic skin grafts from a single donor to a single
recipient resulted in accelerated or second-set skin graft rejection
(1). This finding inspired Medawar (2) to perform his pioneering
skin grafting experiments in rabbits that led to the fundamental
understanding of the cellular basis for allogeneic rejection. Allogeneic organ transplantation has thrived since the mid-20th century resulting in improved patient survival and quality of life. A
seeming paradox after burn injury is that in the presence of profound immune dysfunction severely injured burn patients universally reject allogeneic skin grafts thus limiting their use to temporary wound coverage (3). The use of allogeneic skin grafts for burn
patients has therefore remained essentially unchanged since the
time of Medawar, relegated to use as a temporary wound cover that
is routinely rejected. Despite the development of recent biotechnologies such as cultured keratinocyte autografts (4) and dermal
substitutes (5), the problem of permanent wound coverage for the
patient with a massive burn wound remains unsolved. The failure
to close the burn wound continues to be a major contributor to the
development of infection, sepsis, multiple organ failure, and death
in severely injured burn patients (6). Now that composite tissue
allografts are being used for facial transplants (7, 8), it is imper*Department of Surgery and †Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599
Received for publication January 11, 2006. Accepted for publication March 14, 2006.
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.
ative that we better understand and control the cellular mechanism
of allogeneic skin graft rejection after injury.
In the most current model of immune response to serious burn
injury (9), an initial proinflammatory response is quickly followed
by a systemic inflammatory response syndrome (SIRS),3 which if
uncontrolled, results in early multiple organ dysfunction syndrome
(MODS) and death. In most patients however, after a period of
relative stability, a compensatory anti-inflammatory response syndrome (CARS) develops with associated immunosuppression and
increased risk of infection, that uncontrolled also can result in
MODS and death. The controlling mechanisms for initiating and
sustaining the development of SIRS and CARS have not been fully
elucidated and attempts to modulate either response with cytokine
therapy have largely been unsuccessful (10), except in very specific and controlled animal models of burn injury (11–16). The
failure of this intervention may be because the immune phenotype
after injury has not been fully defined, especially late after injury
when immune failure and subsequent infection, sepsis, and MODS
are most likely.
Although multiple cell populations are involved in the allogeneic skin graft rejection process, CD8⫹ T cells remain a major
obstacle in controlling chronic allograft rejection and inducing tolerance following solid organ transplantation (17, 18). Similarly in
sepsis, while increased interest in innate immunity (19), including
dendritic cells, macrophages, as well as other T cell populations,
including regulatory CD4⫹ T cells (20, 21), has resulted in significant progress in understanding the mechanism of burn-induced
adaptive and innate immune dysfunction, the CD8⫹ T cell population continues to be relevant in injury responses (22–24). TCR
transgenic and wild-type mouse models have been used to clarify
1
This work was supported by National Institutes of Health Grants KO8 GM067147
(to B.A.C.) and R01 GM067143 (to J.A.F.), and the North Carolina Jaycee Burn
Center.
2
Address correspondence and reprint requests to Dr. Bruce A. Cairns, Department of
Surgery and Department of Microbiology, University of North Carolina, CB No.
7290, Chapel Hill, NC 27599. E-mail address: [email protected]
Copyright © 2006 by The American Association of Immunologists, Inc.
3
Abbreviations used in this paper: SIRS, systemic inflammatory response syndrome;
MODS, multiple organ dysfunction syndrome; CARS, compensatory antiinflammatory response syndrome; TBSA, total body surface area.
0022-1767/06/$02.00
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Burn patients are immunocompromised yet paradoxically are able to effectively reject allogeneic skin grafts. Failure to close a
massive burn wound leads to sepsis and multiple system organ failure. Immune suppression early (3 days) after burn injury is
associated with glucocorticoid-mediated T cell apoptosis and anti-inflammatory cytokine responses. Using a mouse model of burn
injury, we show CD8ⴙ T cell hyperresponsiveness late (14 days) after burn injury. This is associated with a CD8ⴙ T cell pro- and
anti-inflammatory cytokine secretion profile, peripheral lymphopenia, and accumulation of a rapidly cycling, hyperresponsive
memory-like CD8ⴙCD44ⴙ IL-7Rⴚ T cells which do not require costimulation for effective Ag response. Adoptive transfer of
allospecific CD8ⴙ T cells purified 14 days postburn results in enhanced allogeneic skin graft rejection in unburned recipient mice.
Chemical blockade of glucocorticoid-induced lymphocyte apoptosis early after burn injury abolishes both the late homeostatic
accumulation of CD8ⴙ memory-like T cells and the associated enhanced proinflammatory CD8ⴙ T cell response, but not the late
enhanced CD8ⴙ anti-inflammatory response. These data suggest a mechanism for the dynamic CD8ⴙ T cell response following
injury involving an interaction between activation, apoptosis, and cellular regeneration with broad clinical implications for
allogeneic skin grafting and sepsis. The Journal of Immunology, 2006, 176: 6717– 6726.
6718
LYMPHOPENIA-INDUCED MEMORY CD8⫹ T CELLS AFTER BURN INJURY
Materials and Methods
Animals
Transgenic mice (C57BL/6-TgN (TcrHY), HYTCR) that carry the transgenic TCR specific for male HY Ag (32) were obtained from the National
Institute of Allergy and Infectious Diseases via Taconic Farms. Normal
C57BL/6 (B6) mice were purchased from Charles River Laboratories. All
mice used in the study were maintained under specific pathogen-free conditions in the American Association of Laboratory Animal Care-accredited
University of North Carolina Department of Laboratory Animal Medicine
Facilities.
Mouse burn injury and RU486 treatment
Fifteen to 20 g, 6- to 8-wk-old female HYTCR or B6 mice were used as
subjects in all experiments. All protocols were performed in accordance
with the National Institutes of Health guidelines and approved by the University of North Carolina Institutional Animal Care and Use Committee.
Animals were anesthetized with inhalation of methoxyflurane vapor. Flank
and back hair was clipped. A full-thickness burn of ⬃20% total body
surface area (TBSA) was produced by applying a copper rod, heated in
boiling water, four times to the animal’s dorsum/flank for 10 s. Mice were
resuscitated with i.p. lactated Ringer’s solution (0.1 ml/g body weight) and
given s.c. buprenorphine (2 mg/kg body weight) for pain control immediately after injury and as needed after burn. There is a negligible mortality
(⬍1%) after burn injury with this protocol. Where noted, mice were given
20 ␮g/g mifepristone (RU486; Sigma-Aldrich) s.c. as recently described
(33) 30 min before burn, and two follow up injections performed at 24-h
periods after burn. Sham controls underwent all the described interventions
except for the application of the copper rod. They received buprenorphine
administration at the same dose as burn mice.
Peptide and tetramer preparation
HY (minor male histocompatibility Ag, KCSRNRQYL) and gp33 (glycoprotein peptide, KAVYNFATC) peptides were synthesized by the University of North Carolina microchemical facility, purified by HPLC, and tested
FIGURE 1. Functional immune response of splenocytes after burn injury is time dependent. A, splenocytes were harvested from wild-type B6 mice at
either 3 days (dashed line) or 14 days (solid line) after 20% TBSA full-thickness burn (F) or sham (E) injury. Proliferation was measured after 48 h of
culture with various concentrations of anti-CD3/anti-CD28 Ab. Data expressed as mean (of triplicate assays) ⫾ SEM, ⴱ, p ⱕ 0.05; ⴱⴱ, p ⱕ 0.005 compared
with matched sham controls by Student’s t test. Results are representative plots of four independent experiments. B, Normalized data from four independent
experiments (n ⫽ 24 mice/experimental group) showing percentage of proliferative response 3 or 14 days after burn injury, where sham response is defined
as 100%. Data expressed as mean ⫾ SEM; ⴱⴱ, p ⱕ 0.005 compared with sham controls by Student’s t test. C, Splenocytes were harvested from female
HYTCR mice 14 days after 20% TBSA full-thickness burn (F) or sham (E) injury. Splenocytes were cultured for 24 h with 10 ␮M HY peptide and then
cell proliferation was measured. Data are expressed as mean (of triplicate assays) ⫾ SEM, ⴱ, p ⱕ 0.05; ⴱⴱ, p ⱕ 0.005 compared with sham controls by
Student’s t test. Results are representative plots of three independent experiments. D, Cells were harvested from parallel proliferation plates and CD8⫹ T
cells magnetically purified; equivalent numbers used in an in vitro cytolytic assay to kill HY peptide-pulsed target cells. Data expressed as mean (of
triplicate assays) ⫾ SEM, ⴱ, p ⱕ 0.05; ⴱⴱ, p ⱕ 0.005 compared with controls by Student’s t test. Results are representative plots of three independent
experiments.
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the role of CD4⫹ and CD8⫹ T cells in the altered immune response after burn injury. Initially, it was demonstrated in an OVA
CD4⫹ TCR transgenic DO11.10 mouse model, that burn injury
induced a proinflammatory phenotype in transgenic CD4⫹ T cells
(25). We have previously used the minor histocompatibility Ag
HY TCR transgenic model (26) to demonstrate that while Agspecific proliferation is significantly impaired in TCR transgenic
CD8⫹ T cells 3 days after burn injury, activated CD8⫹ T cells also
express a proinflammatory phenotype with increased intracellular
IFN-␥ and IL-2. In addition, there are data that this proinflammatory profile can be exaggerated to lethal levels in CD8⫹ T cells and
not in CD4⫹ T cells when superantigen is given to wild-type mice
at the time of burn injury (27). We have also previously demonstrated that burn injury acutely impairs primary and secondary
CD8⫹ T cell responses (28 –31). An unexplained finding in these
studies was that 14 days after burn injury, the CD8⫹ T cell alloresponse recovered to greater than control.
In this report, we confirm in two mouse models of burn injury
that there is a burn-dependent enhancement of CD8⫹ T cell responses 14 days after burn injury with dramatically elevated proand anti-inflammatory cytokine responses. We demonstrate that
apoptosis early after burn injury results in a persistent peripheral
CD8⫹ lymphopenia which, in turn, increases homeostatic proliferation of residual peripheral CD8⫹ T cells which form a highly
responsive cycling memory-like CD8⫹ T cell population which
not dependent on costimulatory signals for immune activation.
Chemically blocking acute lymphocyte apoptosis prevents formation of increased CD8⫹ memory-like T cells and enhanced proinflammatory cytokine secretion, yet preserves enhanced antiinflammatory cytokine secretion.
The Journal of Immunology
6719
for purity by mass spectroscopy. Recombinant HY or gp33-Db MHC class
I tetramer was prepared as we previously described (34). Samples was
routinely tested for endotoxin contamination (Pyrochrome kit; Cape Cod),
and was found to be within normal limits.
Paramagnetic CD8⫹ T cell purification from spleen
Splenic CD8⫹ T cells were negatively selected by depletion of CD4⫹,
MHC class II⫹, and CD11b⫹ cells using the MACS magnetic separation
system (Miltenyi Biotec) as we previously described (34).
Proliferation assay and multiplex cytokine analysis
Splenocytes or purified CD8⫹ T cells from burn and sham mice (1 ⫻ 106)
cells were stimulated with anti-CD3 and anti-CD28 Abs, peptide or MHC
class I tetramers as indicated (endotoxin and azide-free) in 200 ␮l of complete RPMI 1640 at the concentrations indicated. The cells were incubated
for 48 h with 1 ␮Ci [3H]thymidine for the last 12 h. 3H incorporation was
measured using a Beckman LS5000 scintillation counter. All data represent
average cpm of triplicate determinations and each experiment repeated at
least three times. Multiplex cytometric bead assay (BD Biosciences) allowed simultaneous measurement of IFN-␥, IL-2, IL-4, IL-5, TNF-␣, IL10, MCP-1, IL-6, and IL-12p70 in culture supernatants.
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Cytolytic assay
HYTCR splenocytes from burnt or sham-treated HYTCR mice were harvested 14 days postburn and 1 ⫻ 105 cells/well stimulated with 10 ␮M HY
peptide. After 24 h, the cells from each treatment were pooled, washed and
used as effector cells. EL4 cells were labeled with 51Cr, then pulsed with
10 ␮M HY or irrelevant (gp33) peptide and used as targets in a standard
CTL assay (34).
Flow cytometric analysis
The panel of mAbs used for flow cytometric analyses were of anti-CD8␣
(53-6.7), anti-CD3 (145-2C11), anti-CD4 (L3T4), anti-CD44 (Pgp-1,
IM7), anti-CD62L (MEL-14), anti-CD25 (PC61), anti-CD43 (1B11), antiIL4 (BVD4-1D11), anti-IFN-␥ (XMG-1.2) and anti-CD127 (A7R34) and
were purchased from BD Pharmingen. Intracellular staining for cytokines
was performed using standard methods (26). Examination for apoptosis
was determined by annexin V binding and 7-aminoactinomycin D viability
staining (Apoptosis Detection kit; BD Pharmingen). Cell cycle analysis
was performed using standard propidium iodide DNA staining. List mode
data were collected on a FACSCalibur (BD Biosciences) and analyzed
using Summit software (DakoCytomation).
Tail skin grafting
Tail skin grafting was performed as previously described (35, 36). Each
female recipient mouse received a male allograft and a female isograft as
a control. Glass tubes were placed over the grafted area for 3 days to
prevent removal of the graft by the mouse. Grafts that had failed to vascularize properly with apparent rejection at 3 days were classed as “technical failures” and removed from the analysis. Remaining grafts were
scored daily. Fully intact grafts were scored as 100% and when ⬍30% of
the graft remained, it was considered rejected.
BrdU incorporation assay
Twenty milligrams of BrdU was administered i.p. into mice 24 h before
harvest. Intracellular BrdU incorporation was assayed using an APC BrdU
Flow kit (BD Pharmingen) in conjunction with surface staining.
Statistical analysis
Data were analyzed using Student’s t test for differences in cell staining,
CTL activity and proliferation assays; log-rank analysis was used to test
differences in graft survival. GraphPad Prism version 4.03 was used for the
analyses. Statistical significance was defined as p ⱕ 0.05 unless indicated
otherwise.
Results
CD8⫹ T cell response to burn injury is dynamic and time
dependent
To characterize T cell function following burn injury, we used a
well-described scald burn injury in mice (26, 28, 31). In support of
our previous findings (31), we found that there is consistent
suppression in T cell proliferative response to anti-CD3 and
FIGURE 2. Splenocytes from burn mice demonstrate increased pro- and
anti-inflammatory cytokine secretion after activation in vitro. A, Splenocytes were harvested from wild-type B6 mice 3 or 14 days after 20% TBSA
full-thickness burn (f, n ⫽ 6) or sham (䡺, n ⫽ 5) injury and cultured for
48 h with 5 ␮g/ml anti-CD3/anti-CD28 Ab. Cytokine secretion into supernatant was assayed using flow cytometric bead array. Each data set represents mean (of triplicate assays) ⫾ SEM; ⴱ, p ⱕ 0.05, ⴱⴱ, p ⱕ 0.005 by
Student’s t test. Results are representative plots of three independent experiments. B, Splenocytes from the 14-day postburn cultures were then
harvested after the 48-h stimulation and surface staining for CD8 and CD4
and intracellular cytokine staining for IFN-␥ and IL-4 was performed.
Numbers shown are percentages of CD8⫹ or CD4⫹ T cells that are cytokine(s) positive. “Iso” designates use of appropriately conjugated isotype
control Abs to assess nonspecific staining. Results are representative plots
of three independent experiments.
6720
LYMPHOPENIA-INDUCED MEMORY CD8⫹ T CELLS AFTER BURN INJURY
anti-CD28 Ab stimulation in wild-type B6 whole splenocytes 3
days after burn injury compared with whole splenocytes from
sham mice (Fig. 1A). At 14 days after burn injury, the anti-CD3/
anti-CD28 T cell proliferative response from burn injured mice
was greater than sham (Fig. 1A). Fig. 1B compiles normalized data
from four independent experiments (24 mice per experimental
group) showing the percentage of T cell proliferative response after burn injury, with sham T cell response defined as 100%. This
illustrates the robustness and significance of the difference in T cell
responses to polyclonal stimulation between 3 (suppression) and
14 (enhancement) days after burn injury.
We then confirmed that enhanced T cell function 14 days after
burn injury resided in the CD8⫹ T cell compartment and was not
an artifact of nonspecific T cell stimulation or due to release or
exposure of self-Ag causing T cell priming or tolerance during
burn injury. We used MHC class-I restricted peptide epitope of the
minor histocompatibility Ag HY (a well-characterized minor
transplantation Ag (37)) to stimulate equivalent numbers of whole
splenocytes from burn or sham female HY-reactive CD8⫹ TCR
transgenic mice (36) which do not bear the male HY Ag. We have
demonstrated a suppressed Ag-specific CD8⫹ T cell proliferative
response to HY peptide 72 h after scald burn injury using this
model (26). Similar to wild-type B6 mice, naive female HYTCR
splenocytes proliferated more vigorously to HY peptide 14 days
after burn injury than sham controls (Fig. 1C). We then highly
purified effector HYTCR CD8⫹ CTL cells from these HY peptide
stimulated splenocyte cultures derived from burn or sham HYTCR
mice. Using an equivalent number of CD8⫹ T cell effectors we
demonstrated a significantly enhanced cytolytic activity against
HY peptide-pulsed target cells compared with sham controls (Fig.
1D). These data demonstrate that the CD8⫹ T cell response after
burn injury is associated with a late (14 days) enhanced proliferative and CTL activity after burn injury, in response to both Ag
specific and non-Ag-specific stimulation.
Increased responsiveness of CD8⫹ T cells late after burn injury
is not explained simply by altered cytokine profiles
We then examined cytokine secretion as a possible mechanism for
the late enhanced CD8⫹ T cell response to burn injury, as shown
in models of early immune dysfunction after burn injury (11–16).
We evaluated cytokine secretion from female wild-type B6 splenocytes after in vitro stimulation with anti-CD3 and anti-CD28 Abs
3 and 14 days post-burn injury. Splenocytes stimulated 3 days after
burn injury secreted significantly more IL-10 and IL-6 than sham
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FIGURE 3. Burn injury induces a loss of splenic CD8⫹ T cells and increased cell cycling of residual CD8⫹CD44⫹ cells. A, Splenocytes were harvested
from wild-type B6 mice as indicated 3, 7, 14, and 21 days after burn (f) or sham (䡺) injury and stained with anti-CD3 and anti-CD8 or anti-CD4 Ab.
The percentage of cells that were CD3⫹CD8⫹ or CD3⫹CD4⫹ T cells and absolute numbers of CD3⫹CD8⫹ or CD3⫹CD4⫹ per spleen was defined as the
total number of viable splenocytes (determined by trypan blue staining) multiplied by the percentage of total CD3⫹CD8⫹ or CD3⫹CD4⫹ T cells
(determined by flow cytometric staining). Each data set represents mean (five mice per group) ⫾ SEM (ⴱ, p ⱕ 0.05; ⴱⴱ, p ⱕ 0.005 by Student’s t test)
and is representative of three independent experiments. B, The absolute number of wild-type B6 splenocytes after burn (f) or sham (䡺) injury which was
apoptotic as defined by annexin V⫹ binding and 7-aminoactinomycin D⫺ (7-AAD⫺) exclusion. Each data set represents mean (of five mice) ⫾ SEM (ⴱ,
p ⱕ 0.05 by Student’s t test) and is representative of three independent experiments. C, Wild-type B6 mice were also subjected to burn (black line) or sham
(gray line) injury and BrdU injection 24 h before sacrifice 14 days after injury. Splenocytes were stained with anti-BrdU, anti-CD3, and anti-CD8 Abs.
Representative BrdU staining is shown after gating on CD3⫹CD8⫹ T cells. The profile shown is representative of three independent experiments. D,
Splenocytes were harvested from female HYTCR mice 14 days after 20% TBSA full-thickness burn or sham injury as indicated. The percentage of
splenocytes that were CD8⫹CD3⫹ T cells were quantified using flow cytometry, as detailed in Fig. 2. Each data set represents mean (⬎4 mice/group) ⫾
SEM (ⴱⴱ, p ⱕ 0.05, by Student’s t test) and is representative of three independent experiments.
The Journal of Immunology
6721
controls (Fig. 2A), indicative of the suppressed phenotype seen at
this time point after burn injury (38, 39). In contrast, splenocytes
harvested 14 days after burn injury secreted significantly more
IFN-␥, TNF-␣, IL-10, IL-4, IL-6, and MCP-1 but not IL-2, IL-5,
and IL-12p40 compared with sham counterparts (Fig. 2A). At day
14 post-burn injury, intracellular cytokine staining of IFN-␥ and
IL-4 revealed that the CD8⫹ T cell population, but not the CD4⫹
T cell population, had increased cytokine expression (Fig. 2B).
These data demonstrate that enhanced T cell activity 14 days after
burn injury is associated with increased secretion of both pro- and
anti-inflammatory cytokines and thus is not fully explained by altered cytokine profiles.
were cycling more than sham CD8⫹ T cells (Fig. 3C). We observed similar results in the female HYTCR mouse at 14 days after
burn injury; namely reduced numbers of CD8⫹ T cells (Fig. 3D)
which were undergoing increased cell cycling (data not shown).
We did not detect any significant difference in CD8⫹ T cell trafficking to lymphoid organs after adoptive transfer of purified
GFP⫹ expressing CD8⫹ T cells 14 days after burn injury into
unburned recipients (data not shown).
These data indicate that peripheral CD8⫹ T cell numbers late
after burn injury are decreased as a consequence of burn-induced
apoptosis and that residual splenic CD8⫹ T cells undergo increased cell cycling.
Peripheral T cell lymphopenia and increased peripheral CD8⫹
T cell cycling after burn injury
Cycling splenic CD3⫹CD8⫹CD44highCD62L⫹CD127⫺
memory-like T cells increase in frequency and number late
after burn injury
It is known that lymphocyte apoptosis develops within 2 days after
burn injury (33). We find there is also a significant decrease in both
percentage and absolute number of splenic CD8⫹ T cells in wildtype B6 mice 3–14 days after burn injury compared with sham
(Fig. 3A). There was a significant but less dramatic loss of CD4⫹
T cells at 14 days but not at earlier time points. The loss of peripheral CD8⫹ T cells 14 days postburn was associated with apoptosis defined by annexin-V binding (Fig. 3B). BrdU uptake demonstrated that the residual CD8⫹ T cells 14 days after burn injury
Homeostatic proliferation has been implicated in situations
where lymphopenia results in residual T cells acquiring memory-like hyperresponsiveness leads to T cell autoimmunity and
chronic transplant rejection (40 – 45). We hypothesized that
CD8⫹ T cell lymphopenia after burn injury results in the generation of CD8⫹ memory T cells. We found an increased frequency and number of a population of memory-like
CD3⫹CD8⫹CD44highCD62LhighCD25⫺CD69⫺ T cells in burn
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FIGURE 4. Splenocytes from burn mice exhibit enrichment of memory-like CD8⫹ T cells. Splenocytes were harvested from wild-type B6 mice at 3,
7, or 14 days after 20% TBSA full-thickness burn or sham injury. CD8⫹ T cells were purified and cells were then surface stained for CD3, CD44, CD62L,
and CD127. A, Representative staining profiles of CD8⫹ T cells from sham or burn mice after gating on CD3⫹ staining. The numbers in each quadrant
represent percentage of CD3⫹CD8⫹ T cells in each quadrant. The smaller gate designates the CD44highCD62L⫹ T cell population; the associated number
is percentage of CD3⫹CD8⫹ T cells that are of this surface phenotype. Representative anti-CD127 staining of this population is shown in the single-color
histogram. The number refers to the percentage of CD3⫹CD8⫹ CD44highCD62L⫹ T cells that were defined as CD127high. Representative staining is shown
from four independent experiments (n ⫽ 3–5 mice/experimental group). B, The percentage of CD3⫹CD8⫹ T cells that were CD44highCD62L⫹ plotted
against time postinjury and absolute number of CD3⫹CD8⫹CD44highCD62L⫹ T cells from burn- or sham-treated mice. For each data set, horizontal bar
represents the mean percentage; ⴱ p ⱕ 0.05 by Student’s t test. A representative plot is shown from four independent experiments. C, Wild-type B6 mice
were also subjected to burn or sham injury and BrdU injection 24 h before sacrifice 14 days after injury. CD8⫹ T cells were purified and stained with
anti-BrdU, anti-CD3, and anti-CD44 Abs. Representative BrdU and CD44 staining is shown after gating on CD3⫹ T cells from three independent
experiments. The number refers to the percentage of CD3⫹CD8⫹ T cells that were defined as BrdU⫹. The difference in fluorescence intensity of CD44
staining between a and c is due to differing anti-CD44 Ab-conjugated fluorophores.
6722
LYMPHOPENIA-INDUCED MEMORY CD8⫹ T CELLS AFTER BURN INJURY
B6 mice compared with sham. Furthermore, we found that the increased CD8⫹ memory-like population in burn mice was CD127low
(IL-7R␣low), therefore neither effector (CD62L⫺CD127high) nor central (CD62LhighCD127high) memory CD8⫹ T cells (Fig. 4A). The
percentage of CD3⫹CD8⫹ T cells of the memory-like phenotype significantly increased 7 days after burn injury compared with sham,
mirroring the onset of CD8⫹ T cell peripheral apoptosis at day 7 (Fig.
3A), and absolute numbers of CD8⫹CD44highCD62L⫹ T cells were
significantly increased 14 days postburn compared with sham (Fig.
4B). We did not find any significant changes in the number or frequency of CD4⫹CD44high memory-like T cells. We also found that it
was specifically the memory-like CD8⫹CD44high T cells that were
actively cycling by BrdU uptake in both B6 (Fig. 4C) and HYTCR
(data not shown) mice 14 days after burn injury.
CD8⫹ T cells from burn mice exhibit costimulation-independent
Ag activation
Blocking apoptosis abolishes T cell hyperresponsiveness 14 days
after burn injury
To pursue lymphopenia-induced homeostatic proliferation as a
mechanism for enhanced CD8⫹ T cell activity, we reasoned that
blocking apoptosis early after burn injury should decrease or prevent homeostatic proliferation and the subsequent development of
enhanced, memory-like CD8⫹ T cells. To test this hypothesis, we
used the glucocorticoid receptor inhibitor mifepristone (RU486)
which has been used previously to block acute lymphocyte apoptosis in the first hours after burn injury (33, 47).
We treated four groups of wild-type female B6 mice as follows:
1) 20% TBSA burn; 2) 20% TBSA burn and three daily injections
of 20 ␮g/g RU486 (first injection administered 30 min before burn
injury); 3) sham; and 4) sham ⫹ RU486. We confirmed by flow
cytometry that RU486 prevented the burn-induced apoptosis of
thymic double positive (DP) T cells, highly sensitive to burn-induced apoptosis (33) (Fig. 6A), and prevented the decrease of absolute numbers and percentage of splenic CD8⫹ T cells at 14 days
(Fig. 6B). The number and frequency of CD8⫹ T cells in sham
mice was not affected by RU486. RU486 significantly prevented
the accumulation of CD8⫹CD44highCD62L⫹ T cells in the spleen
14 days after burn (Fig. 6C) in contrast to non-RU486-treated burn
mice. Additionally, highly purified CD8⫹ T cells from burn ⫹
RU486 mice were not hyperresponsive and responded to limiting
amounts of anti-CD28 and anti-CD3 Ab similarly to CD8⫹ T cells
from sham and sham ⫹ RU486 mice (Fig. 6D). These data dem-
FIGURE 5. CD8⫹ T cells from HYTCR burn mice are less sensitive to
a lack of costimulation. A, Splenocytes were harvested from female
HYTCR mice 14 days after 20% TBSA full-thickness burn (f, n ⫽ 6) or
sham (E, n ⫽ 4) injury. A total of 1 ⫻ 106/ml purified CD8⫹ T cells were
stimulated in vitro with either relevant HY-Db (solid line) or irrelevant
gp33-Db (dashed line) MHC class I tetramer for 48 h, as indicated. Proliferation was measured by [3H]thymidine incorporation. Data are expressed as mean cpm ⫾ SEM. A representative plot is shown from three
independent experiments. B, Splenocytes were harvested from B6 mice 14
days after 20% TBSA full-thickness burn (F, n ⫽ 5) or sham (E, n ⫽ 3)
injury. 1 ⫻ 106/ml purified CD8⫹ T cells were stimulated in vitro with
increasing concentrations of unbound anti-CD3 Ab with (solid line) or
without (dashed line) 1 ␮g/ml unbound anti-CD28 Ab for 48 h. Proliferation was measured by [3H]thymidine incorporation. Data are expressed as
mean (of triplicate samples) ⫾ SEM. A representative plot is shown from
five independent experiments.
onstrate that blocking early apoptosis ablates subsequent lymphopenia-induced homeostatic proliferation and expansion of
memory-like costimulation-independent CD8⫹CD44highCD62L⫹
T cells 14 days after burn injury.
Blocking apoptosis reduces ability of CD8⫹ T cells to mount
proinflammatory responses 14 days after burn injury
We then investigated the impact of blocking apoptosis on the cytokine profile secreted by activated purified CD8⫹ T cells 14 days
after burn injury. We observed that less IFN-␥ was secreted by
splenocytes (Fig. 7A, and compare with Fig. 2A) and CD8⫹ T cells
(data not shown) from burn ⫹ RU486 mice in response to antiCD28 and anti-CD3 Ab stimulation compared with untreated burn
mice. Furthermore, CD8⫹ T cells from burn ⫹ RU486 mice secreted significantly more anti-inflammatory IL-10 and IL-4 compared with burn, sham, and sham ⫹ RU486 controls (Fig. 7B).
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Given that the requirement for costimulation is much less in memory T cells than in naive T cells (41, 46), we hypothesized that
enhanced CD8⫹ T cell activity 14 days after burn injury is costimulation independent. We tested this in two different assays of
costimulation dependency using purified CD8⫹ T cells. First, we
took advantage of our findings that soluble MHC class I tetramers
bearing HY peptide can effectively stimulate purified CD8⫹ T
cells in vitro in the absence of costimulatory molecules provided
by APCs (34). We found that equivalent numbers of purified
splenic CD8⫹ T cells from female HYTCR mice 14 days after
burn injury generated a much greater proliferative response after
simulation with equivalent HY-Db tetramer compared with T cells
from sham mice (Fig. 5A). Second, CD8⫹ T cells from sham mice
were responsive to limiting amounts of anti-CD3 plus anti-CD28
Ab but not to anti-CD3 alone; however, under the same conditions,
equivalent numbers of CD8⫹ T cells from burn mice responded
more vigorously in both conditions (Fig. 5B). These data suggest
that enhanced CD8⫹ T cell activity 14 days after burn injury is
costimulation independent, characteristic of memory CD8⫹ T cell
responses.
The Journal of Immunology
6723
These data demonstrate that while blocking apoptosis results in a
substantial impairment of CD8⫹ T cells from burn mice to generate a proinflammatory response, it appears to leave antiinflammatory responses unaffected.
CD8⫹ T cells adoptively transferred from lymphopenic mice 14
days after burn injury accelerate allogeneic skin graft rejection
in unburned mice
We hypothesized that hyperresponsive CD8⫹ T cells purified late
after burn injury and adoptively transferred to unburned recipients
would confer enhanced activity in vivo and be dependent on early
burn injury associated apoptosis. To test this, we used a mouse
model of allogeneic tail skin graft rejection. Female HYTCR mice
were anesthetized and treated as follows: 1) 20% TBSA burn; 2)
20% TBSA burn and three daily injections of RU486; 3) sham and
4) sham with RU486. Mice were sacrificed 14 days after burn
injury, splenic CD8⫹ T cells were purified and adoptively transferred i.v. into female wild-type B6 mice (1 ⫻ 106 cells/mouse).
Transferred cells were allowed to redistribute for 48 h, then each
mouse received allogeneic male (HY⫹) skin and control isogeneic
female (HY⫺) tail skin grafts. Mice that received CD8⫹ HYTCR
T cells isolated after 14 days after burn injury rejected male skin
graft significantly faster than mice that receive CD8⫹ T cells from
sham mice (11 days vs 17 days, p ⫽ 0.019), confirming that transferred CD8⫹ T cells from burn mice had enhanced activity in vivo
(Fig. 7C). The effect was blocked when RU486 was administered
to burn mice, CD8⫹ T cells were adoptively transferred and skin
graft rejection is compared between burn without RU486 (18 vs 11
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FIGURE 6. RU486 prevents burn-induced apoptosis in thymus and spleen and subsequent evidence of homeostatic proliferation 14 days after burn
injury. A, Thymocytes were harvested 36 h after burn injury from wild-type B6 after burn or sham in the presence or absence of RU486 treatment. Cells
were stained, directly ex vivo, for surface CD3, CD4 and CD8 and analyzed by flow cytometry. A representative profile after gating on CD3⫹ staining is
shown. The numbers refer to the percentage of CD3⫹ thymocytes that were CD4⫹CD8⫹ double positive. B, Splenocytes were harvested from wild-type
B6 mice after burn or sham in the presence or absence of RU486 treatment. Cells were stained, directly ex vivo, for surface CD3 and CD8 and analyzed
by flow cytometry. Percentage and absolute numbers of peripheral splenic CD3⫹CD8⫹ T cells 14 days after burn were plotted. For each data set the mean
percentage ⫾ SEM of total cells that were CD3⫹CD8⫹ or absolute number was plotted; ⴱ, p ⱕ 0.05, ⴱⴱ, p ⱕ 0.005 by Student’s t test. Numbers under
bars represent number of mice in each group. A representative plot is shown from three independent experiments. C, Splenocytes were harvested from
wild-type B6 14 days after burn, or sham treatment in the presence or absence of RU486 treatment (“⫹RU”). Cells were stained directly ex vivo with
anti-CD8␣-PerCP, anti-CD3-FITC, anti-CD25-PE and anti-CD44-allophycocyanin and analyzed by flow cytometry. The percentage of CD3⫹CD8⫹ T cells
that were CD44highCD62L⫹ plotted against treatment is shown. For each data set, horizontal bar represents the mean percentage; ⴱ, p ⱕ 0.05 by Student’s
t test. D, CD8⫹ T cells were also magnetically purified and stimulated with 0.025 to 0.1 ␮g/ml anti-CD3 and anti-CD28 Abs for 24 h and then proliferation
assessed using standard [3H]thymidine uptake. Each data set represents mean (of at least three mice) ⫾ SEM, ⴱ, p ⬍ 0.05; ⴱⴱ, p ⱕ 0.005 by Student’s t
test. A representative plot is shown from three independent experiments.
6724
LYMPHOPENIA-INDUCED MEMORY CD8⫹ T CELLS AFTER BURN INJURY
days, p ⫽ 0.0013; Fig. 6B), sham (18 vs 17 days, p ⫽ 0.12), sham
with RU486 (18 vs 15 days, p ⫽ 0.09) and no cells transferred (18
vs 17 days, p ⫽ 0.10, Fig. 7D).
These data demonstrate that CD8⫹ T cells transferred from mice
14 days after burn injury mediate accelerated allogeneic skin graft
rejection and that blocking apoptosis early after burn injury prevents enhanced graft rejection by these cells.
Discussion
C and D, CD8⫹ T cells were magnetically purified from naive female
HYTCR mice after burn or sham in the presence (“⫹RU”) or absence of
RU486 treatment and adoptively transferred in female B6 mice (n ⬎6) as
indicated. These were tail grafted with allogeneic male and isogeneic female skin. Survival of grafts was monitored over time. Numbers in parentheses represent median survival time of male skin graft. A representative
survival plot is shown from two independent experiments.
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FIGURE 7. Blocking apoptosis reduces burn-induced proinflammatory
cytokine secretion, but not anti-inflammatory cytokine secretion. CD8⫹ T
cells transferred from lymphopenic mice 14 days after burn injury mediate
enhanced graft rejection in unburned mice. A and B, Splenocytes were
harvested from wild-type B6 14 days after burn or sham injury in the
presence (“⫹RU”) or absence of RU486 treatment. Cells were stimulated
with 1.5 ␮g/ml anti-CD3 and anti-CD28 Abs for 24 h. The supernatant was
assayed for IFN-␥ (A) and IL-4 and IL-10 (B) by flow cytometric bead
array. Each data set represents mean ⫾ SEM, ⴱ p ⱕ 0.05, ⴱⴱ p ⱕ 0.005 by
Student’s t test. Numbers in parentheses represent number of mice in each
group. A representative plot is shown from three independent experiments.
Burn injury results in substantial immune dysfunction leading to
an increased risk of infection yet the immunobiology of the response remains poorly understood. The most current hypothesis is
that severe burn injury results in a complex interaction of both
innate and adaptive immunity that initially generates a proinflammatory response followed by a counterregulatory antiinflammatory response that frequently leads to immune dysfunction, infection and sepsis. Yet this paradigm does not explain the
essential clinical finding that allogeneic skin graft rejection occurs,
even in the face of profound immune suppression following extensive burn injury. This was clearly demonstrated by Medawar in
1943 (1), and the relevance of allogeneic skin graft rejection is
now longer limited to the management of the acute burn wound.
Refinement in surgical techniques has made it possible for composite tissue allografts, including hand or face, to be allotransplanted. The skin elements remain the most immunologically susceptible component of these transplants and a clinical rejection
episode in such a transplant could have devastating cosmetic and
psychological effects. This report clarifies the CD8⫹ T cell response and thus provides a mechanism for allogeneic skin graft
rejection following burn injury.
The peripheral T lymphocyte pool is composed of naive T cells
exported from the thymus and those generated by homeostatic proliferation as well as Ag-experienced memory T cells. Burn injury
leads to lymphocyte apoptosis in the thymus early (hours to 3
days) after injury, which is mediated in part by the acute phase
response via glucocorticoids (33) and results in a significant decrease in thymic output of naive T cells in to the periphery. A
persistent CD8⫹ T cell apoptosis also occurs in the periphery later
after injury via an unknown mechanism (48), though it can be
ablated by the glucocorticoid inhibitor RU486. Between the time
of initial burn injury and ⬃14 days, peripheral lymphocyte pools
are maintained by expansion of memory pools and lymphopenia
driven homeostatic proliferation, similar to other models including
induction of T cell autoimmunity and chronic transplant rejection
(40, 41, 44, 45). The resultant CD8⫹ T cell population is low in
absolute cell numbers, but enriched with actively cycling memorylike CD8⫹ T cells which are costimulation independent and hyperresponsive to antigenic stimuli, including alloantigens. It is
likely that homeostatic proliferation is only one of many mechanisms leading to CD8⫹ T cell dysfunction at this later time point.
It is possible that Ags (e.g., self or cross-reactive Ags, gut-derived
Ags) or proinflammatory or counterinflammatory mediators are
released or exposed to the immune system after injury and these
are involved in this change in CD8 expansion and phenotype after
injury. Apoptotic cells could prime a particular Ag-presenting cell
The Journal of Immunology
(such as cecal ligation and puncture) within a week of burn injury
and thus may have missed an important characteristic of the T cell
response to injury. Finally, of particular relevance to burn injury is
the effect of enhanced memory-like CD8⫹ T cell activity on allograft rejection (29, 31). We have demonstrated that the cycling
memory-like CD8⫹ T cells which arise late after burn injury are
costimulation independent and hyperresponsive to antigenic stimuli, including alloantigens. It will be important to further characterize the ability of these cells to prevent induction of allograft
tolerance, for example by a reduced requirement of CD4⫹ T cell
help or reduced suppression by regulatory T cell populations, as
we have demonstrated with memory CD8⫹ T cells (63). Indeed,
failure to definitively close the burn wound is a major contributor
to prolonged length of stay, complications, and death (64). Little
progress has been made in using allogeneic skin for permanent
wound coverage (65, 66). Homeostatic proliferation of T cells is
recognized as an important mechanism preventing the induction of
allogeneic tolerance (45, 67) and may provide a mechanism for
why severely immunocompromised burn patients are able to reject
allogeneic skin grafts (29, 65).
It appears that the cellular mechanism of immune dysfunction
following burn injury is due to a dynamic interplay between injury,
apoptosis, activation, and cellular regeneration. This study provides a new paradigm on the CD8⫹ T cell response and the highly
controversial role of apoptosis in burn injury that both have important implications on therapeutic strategies that address the immune response to injury.
Acknowledgments
We thank Katherine Midkiff, Michael Johnson, and Maria A. Roldan for
technical assistance and members of the Frelinger and Collins laboratories
for helpful discussion.
Disclosures
The authors have no financial conflict of interest.
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The phenotype of the CD8⫹ T cell population arising later after
burn injury is intriguing as it does not fit into existing defined
effector or central memory T cell phenotypes. A recent study revealed a similar rapidly dividing CD8⫹CD44highCD127(IL-7R)low
novel population of T cells in a model of CD8⫹ T cell homeostatic
proliferation (49). Surprisingly, we were able to identify homeostatic proliferation in female HYTCR transgenic CD8⫹ T cells in
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requirement for IL-7 (52). We are currently investigating whether
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These findings are clinically relevant for several reasons. First,
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lymphopenia that drives homeostatic proliferation resulting in a
memory-like T cell phenotype and hyperresponsiveness. It is important to note that our model is not one of sepsis and that the total
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Blocking apoptosis not only prevents lymphopenia and homeostatic proliferation, it also impairs the capacity of T cells to generate a proinflammatory, but not an anti-inflammatory, response to
challenge by an unknown mechanism. However, we found that
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6725
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