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Medline Plus Health Information
*Burns
Hazardous Substances DB
*RU-486
The Journal of Immunology, 2006, 176: 6717-6726.
Copyright © 2006 by The American Association of Immunologists

Lymphopenia-Induced Homeostatic Proliferation of CD8+ T Cells Is a Mechanism for Effective Allogeneic Skin Graft Rejection following Burn Injury1

Robert Maile*,{dagger}, Carie M. Barnes*, Alma I. Nielsen*, Anthony A. Meyer*, Jeffrey A. Frelinger{dagger} and Bruce A. Cairns2,*,{dagger}

* Department of Surgery and {dagger} Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 CD8+ T cell hyperresponsiveness late (14 days) after burn injury. This is associated with a CD8+ T cell pro- and anti-inflammatory cytokine secretion profile, peripheral lymphopenia, and accumulation of a rapidly cycling, hyperresponsive memory-like CD8+CD44+ IL-7R– T cells which do not require costimulation for effective Ag response. Adoptive transfer of allospecific CD8+ 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 CD8+ memory-like T cells and the associated enhanced proinflammatory CD8+ T cell response, but not the late enhanced CD8+ anti-inflammatory response. These data suggest a mechanism for the dynamic CD8+ T cell response following injury involving an interaction between activation, apoptosis, and cellular regeneration with broad clinical implications for allogeneic skin grafting and sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Modern 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 imperative 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, 12, 13, 14, 15, 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, 23, 24). TCR transgenic and wild-type mouse models have been used to clarify 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 Ag-specific 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-{gamma} 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, 29, 30, 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 pro- and 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 anti-inflammatory cytokine secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
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 µg/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 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 x 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 µl of complete RPMI 1640 at the concentrations indicated. The cells were incubated for 48 h with 1 µCi [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-{gamma}, IL-2, IL-4, IL-5, TNF-{alpha}, IL-10, MCP-1, IL-6, and IL-12p70 in culture supernatants.

Cytolytic assay

HYTCR splenocytes from burnt or sham-treated HYTCR mice were harvested 14 days postburn and 1 x 105 cells/well stimulated with 10 µM 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 µM 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{alpha} (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), anti-IL4 (BVD4-1D11), anti-IFN-{gamma} (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 ≤ 0.05 unless indicated otherwise.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
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 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.


Figure 1
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  		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 (•) or sham ({circ}) 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, *, p ≤ 0.05; **, p ≤ 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; **, p ≤ 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 (•) or sham ({circ}) injury. Splenocytes were cultured for 24 h with 10 µM HY peptide and then cell proliferation was measured. Data are expressed as mean (of triplicate assays) ± SEM, *, p ≤ 0.05; **, p ≤ 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, *, p ≤ 0.05; **, p ≤ 0.005 compared with controls by Student’s t test. Results are representative plots of three independent experiments.

 
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, 12, 13, 14, 15, 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 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-{gamma}, TNF-{alpha}, 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-{gamma} 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.


Figure 2
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  		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 ({blacksquare}, n = 6) or sham ({square}, n = 5) injury and cultured for 48 h with 5 µg/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; *, p ≤ 0.05, **, p ≤ 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-{gamma} 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.

 
Peripheral T cell lymphopenia and increased peripheral CD8+ T cell cycling 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 wild-type 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 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).


Figure 3
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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 ({blacksquare}) or sham ({square}) 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 (*, p ≤ 0.05; **, p ≤ 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 ({blacksquare}) or sham ({square}) 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 (*, p ≤ 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 (**, p ≤ 0.05, by Student’s t test) and is representative of three independent experiments.

 
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.

Cycling splenic CD3+CD8+CD44highCD62L+CD127– memory-like T cells increase in frequency and number late 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, 41, 42, 43, 44, 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 B6 mice compared with sham. Furthermore, we found that the increased CD8+ memory-like population in burn mice was CD127low (IL-7R{alpha}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.


Figure 4
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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; * p ≤ 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.

 
CD8+ T cells from burn mice exhibit costimulation-independent Ag activation

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.


Figure 5
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  		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 ({blacksquare}, n = 6) or sham ({circ}, n = 4) injury. A total of 1 x 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 (•, n = 5) or sham ({circ}, n = 3) injury. 1 x 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 µg/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.

 
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 µg/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 demonstrate 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.


Figure 6
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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; *, p ≤ 0.05, **, p ≤ 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{alpha}-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; *, p ≤ 0.05 by Student’s t test. D, CD8+ T cells were also magnetically purified and stimulated with 0.025 to 0.1 µg/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, *, p < 0.05; **, p ≤ 0.005 by Student’s t test. A representative plot is shown from three independent experiments.

 
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-{gamma} 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 anti-CD28 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). 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 anti-inflammatory responses unaffected.


Figure 7
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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 µg/ml anti-CD3 and anti-CD28 Abs for 24 h. The supernatant was assayed for IFN-{gamma} (A) and IL-4 and IL-10 (B) by flow cytometric bead array. Each data set represents mean ± SEM, * p ≤ 0.05, ** p ≤ 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. 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.

 
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 x 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 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
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
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 anti-inflammatory 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 memory-like 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 population that triggers CD8+ T cell activation or cross-priming. We can exclude release of self-Ag in the experiments in which we use burn female HYTCR transgenic mice which do not contain any HY (self) Ag, but cannot exclude this as a mechanism in the experiments which use burn wild-type mice. Similarly, we cannot exclude release of cross-reactive Ags or production of nonspecific immune mediators in any experiment. These mechanisms likely play roles in this enhancement of the CD8+ T cell response late after burn injury.

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 this study as these cells are recalcitrant to homeostatic division due to low affinity for MHC-peptide molecules (50, 51) and a higher requirement for IL-7 (52). We are currently investigating whether burn injury induces a state of IL-7 sufficiency or more effective (self) MHC-peptide presentation allowing female HY CD8+ T cells to divide homeostatically.

These findings are clinically relevant for several reasons. First, attempts to improve the T cell response to burn injury by manipulating cytokines have been largely been unsuccessful and therefore not generally used in the clinic. However, some studies indicate that treating burn mice with IL-12 (14, 53, 54), IL-4 antagonist (16), or IL-10 antagonist Abs (55, 56) helps protect mice from polymicrobial sepsis or viral infection. Our data suggest why cytokine manipulation may be unsuccessful—i.e., cytokine CD8+ T cell phenotype after burn injury may already be proinflammatory when proinflammatory cytokines (such as IFN-{gamma} or IL-2) are being administered. Indeed, it appears that CD8+ T cells late after burn injury have the capacity to generate both pro- and anti-inflammatory cytokines (57, 58). Second, apoptosis has been suggested as a major mechanism responsible for the impaired T cell response to injury and sepsis. In fact, several investigators have suggested that blocking apoptosis may be the key to improving this response (59). In our model, cellular apoptosis creates 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 number of cells undergoing apoptosis is very low in comparison to the much higher extent of apoptosis observed after sepsis (60). 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 HYTCR CD8+ T cells transferred from RU486-treated burnt mice did not impart any significant tolerogenic effect to male graft when compared with cells from non-RU486-treated burnt mice. We predict that the "sensitivity" of the HY allograft model is unsuitable to demonstrate any differences in the ablation of a late CD8+ T cell proinflammatory response by early apoptosis blockade as there are multiple HY epitopes driving both CD8+ and CD4+ T cell alloresponses (61, 62). Thus it is not entirely clear that blocking apoptosis after burn injury results in the desired clinical result with regard to T cell function and further study is required. This question is one of great clinical importance, particularly with regard to injury and sepsis. We are pursuing the cellular mechanism for this, for instance selective apoptosis of proinflammatory T cells after burn injury, or activation of APCs by uptake of apoptotic cells. Third, the majority of lymphocyte studies focuses solely on the early response to burn injury (<10 days) or involves another insult (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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 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. Back

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: bac{at}unc.edu Back

3 Abbreviations used in this paper: SIRS, systemic inflammatory response syndrome; MODS, multiple organ dysfunction syndrome; CARS, compensatory anti-inflammatory response syndrome; TBSA, total body surface area. Back

Received for publication January 11, 2006. Accepted for publication March 14, 2006.


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