Suppression of Activation and Costimulatory Signaling in Splenic CD4+ T Cells after Trauma-Hemorrhage Reduces T-Cell Function
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.081174
ISSN1525-2191
AutoresChi Hsun Hsieh, Jun Hsu, Ya-Ching Hsieh, Michael Frink, Raghavan Raju, William J. Hubbard, Kirby I. Bland, Irshad H. Chaudry,
Tópico(s)Immune Cell Function and Interaction
ResumoReduced immune function is frequently a consequence of serious injury such as trauma-hemorrhage (T-H). Injury may lead to reduced T-cell activation, resulting in decreased engagement of costimulatory molecules after antigen recognition and in subsequent immunological compromise and anergy. We hypothesized that inhibition of CD28 expression is one possible mechanism by which immune functions are suppressed after T-H. Male C3H/HeN mice (with or without ovalbumin immunization) were subjected to sham operation or T-H and sacrificed after 24 hours. Splenic T cells were then stimulated with concanavalin A or ovalbumin in vivo or in vitro, and CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD69, and phospho-Akt expression was determined. T-cell proliferation/cytokine production was measured in vitro. Stimulation-induced CD69, CD28, and phospho-Akt up-regulation were significantly impaired after T-H compared with sham-operated animals; however, CTLA-4 expression was significantly higher in the T-H group. Over a 3-day span, stimulated T cells from sham-operated animals showed significantly higher proliferation compared with the T-H group. IL-2 and IFN-γ were elevated in sham-operated animals, whereas IL-4 and IL-5 rose in the T-H group, revealing a shift from TH1 to TH2 type cytokine production after T-H. Dysregulation of the T-cell costimulatory pathway is therefore likely to be a significant contributor to post-traumatic immune suppression. Reduced immune function is frequently a consequence of serious injury such as trauma-hemorrhage (T-H). Injury may lead to reduced T-cell activation, resulting in decreased engagement of costimulatory molecules after antigen recognition and in subsequent immunological compromise and anergy. We hypothesized that inhibition of CD28 expression is one possible mechanism by which immune functions are suppressed after T-H. Male C3H/HeN mice (with or without ovalbumin immunization) were subjected to sham operation or T-H and sacrificed after 24 hours. Splenic T cells were then stimulated with concanavalin A or ovalbumin in vivo or in vitro, and CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD69, and phospho-Akt expression was determined. T-cell proliferation/cytokine production was measured in vitro. Stimulation-induced CD69, CD28, and phospho-Akt up-regulation were significantly impaired after T-H compared with sham-operated animals; however, CTLA-4 expression was significantly higher in the T-H group. Over a 3-day span, stimulated T cells from sham-operated animals showed significantly higher proliferation compared with the T-H group. IL-2 and IFN-γ were elevated in sham-operated animals, whereas IL-4 and IL-5 rose in the T-H group, revealing a shift from TH1 to TH2 type cytokine production after T-H. Dysregulation of the T-cell costimulatory pathway is therefore likely to be a significant contributor to post-traumatic immune suppression. It is now generally accepted that serious injury in humans or experimental animal models may be followed by systemic inflammatory response syndrome and may frequently lead to subsequent multiple organ dysfunction syndrome, which remains a major cause of death despite numerous advances in trauma research.1Noel G Guo X Wang Q Schwemberger S Byrum D Ogle C Postburn monocytes are the major producers of TNF-α in the heterogeneous splenic macrophage population.Shock. 2007; 27: 312-319Crossref PubMed Scopus (31) Google Scholar, 2Kher A Wang M Tsai BM Pitcher JM Greenbaum ES Nagy RD Patel KM Wairiuko GM Markel TA Meldrum DR Sex differences in the myocardial inflammatory response to acute injury.Shock. 2005; 23: 1-10Crossref PubMed Scopus (158) Google Scholar, 3Shelley O Murphy T Paterson H Mannick JA Lederer JA Interaction between the innate and adaptive immune systems is required to survive sepsis and control inflammation after injury.Shock. 2003; 20: 123-129Crossref PubMed Scopus (86) Google Scholar, 4Ayala A Herdon CD Lehman DL Ayala CA Chaudry IH Differential induction of apoptosis in lymphoid tissues during sepsis: variation in onset, frequency, and the nature of the mediators.Blood. 1996; 87: 4261-4275Crossref PubMed Google Scholar, 5Hsieh YC Yu HP Frink M Suzuki T Choudhry MA Schwacha MG Chaudry IH G protein-coupled receptor 30-dependent protein kinase A pathway is critical in nongenomic effects of estrogen in attenuating liver injury after trauma-hemorrhage.Am J Pathol. 2007; 170: 1210-1218Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Perturbations of both the innate and adaptive immune systems after trauma-hemorrhage such as hyperactivity of macrophages and Kupffer cells, depressed dendritic cell functions, and impaired T-cell activities have been widely recognized as the cause of multiple organ dysfunction syndrome.6Hildebrand F Hubbard WJ Choudhry MA Frink M Pape HC Kunkel SL Chaudry IH Kupffer cells and their mediators: the culprits in producing distant organ damage after trauma-hemorrhage.Am J Pathol. 2006; 169: 784-794Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 7Alexander M Chaudry IH Schwacha MG Relationships between burn size, immunosuppression, and macrophage hyperactivity in a murine model of thermal injury.Cell Immunol. 2002; 220: 63-69Crossref PubMed Scopus (54) Google Scholar, 8Kawasaki T Fujimi S Lederer JA Hubbard WJ Choudhry MA Schwacha MG Bland KI Chaudry IH Trauma-hemorrhage induces depressed splenic dendritic cell functions in mice.J Immunol. 2006; 177: 4514-4520Crossref PubMed Scopus (77) Google Scholar, 9Schneider CP Schwacha MG Chaudry IH Influence of gender and age on T-cell responses in a murine model of trauma-hemorrhage: differences between circulating and tissue-fixed cells.J Appl Physiol. 2006; 100: 826-833Crossref PubMed Scopus (35) Google Scholar However, interactions between innate and adaptive immune systems and their relative contribution to multiple organ dysfunction syndrome after trauma-hemorrhage are not completely understood. The immune system has the remarkable ability to defend against various microbial pathogens and yet not to respond to self. This is due to the tightly regulated activation of T cells. T-cell activation requires two signals that are delivered by antigen-presenting cells (APCs).10Bour-Jordan H Blueston JA CD28 function: a balance of costimulatory and regulatory signals.J Clin Immunol. 2002; 22: 1-7Crossref PubMed Scopus (100) Google Scholar The first signal is the recognition by T-cell receptors of antigen presentation in the context of major histocompatability complex. The subsequent secondary or costimulatory signal is provided by molecules on APCs that engage cognate receptors on T cells. It is known that T cells will fail to recognize an antigen or enter a state of anergy in the absence of costimulation. The best-characterized T-cell costimulatory pathway involves the CD28 receptor, which binds to CD80 and CD86.11Carreno BM Collins M The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses.Annu Rev Immunol. 2002; 20: 29-53Crossref PubMed Scopus (746) Google Scholar The interaction of CD80 and CD86 with CD28 promotes the expansion of activated T cells and up-regulates cell survival protein expression. Our previous studies revealed that the antigen-presenting capacity of dendritic cells is suppressed after trauma-hemorrhage because of the down-regulation of major histocompatability complex II expression and the decreased ability to stimulate T-cell proliferation.8Kawasaki T Fujimi S Lederer JA Hubbard WJ Choudhry MA Schwacha MG Bland KI Chaudry IH Trauma-hemorrhage induces depressed splenic dendritic cell functions in mice.J Immunol. 2006; 177: 4514-4520Crossref PubMed Scopus (77) Google Scholar However, in that study, we also found that the expression of CD80 and CD86 on dendritic cells was not influenced by trauma-hemorrhage. However, in keeping with an overall pattern of immune unresponsiveness, we showed that T-cell activities were suppressed after trauma-hemorrhage.9Schneider CP Schwacha MG Chaudry IH Influence of gender and age on T-cell responses in a murine model of trauma-hemorrhage: differences between circulating and tissue-fixed cells.J Appl Physiol. 2006; 100: 826-833Crossref PubMed Scopus (35) Google Scholar, 12Suzuki T Shimizu T Yu HP Hsieh YC Choudhry MA Chaudry IH Salutary effects of 17β-estradiol on T-cell signaling and cytokine production after trauma-hemorrhage are mediated primarily via estrogen receptor-α.Am J Physiol Cell Physiol. 2007; 292: C2103-C2111Crossref PubMed Scopus (36) Google Scholar Hence, it is not clear whether posttraumatic T-cell suppression is secondary to suppression of APC functions or alternatively whether costimulatory molecules on T cells also play an important role in producing T-cell dysfunction. We hypothesized that the costimulatory factor CD28 is down-regulated after trauma-hemorrhage even though the expression of CD80 and CD86 on APCs is not affected under those conditions, and the decreased expression of CD28 on T-cell surface is another key mechanism that explains the impaired T-cell function after trauma-hemorrhage. Male C3H/HeN mice 8 weeks old and weighing 22 to 25 g (Charles River Laboratories, Wilmington, MA) were fasted overnight before the experiment but were allowed water ad libitum. All experiments were performed in adherence with National Institutes of Health (Bethesda, MD) Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham (Birmingham, AL). Animals were subjected to either the trauma-hemorrhage procedure or a sham operation. To study the impact of trauma-hemorrhage on T-cell activation and CD28 expression, T cells were activated either in vivo or in vitro and were activated either by concanavalin A (ConA) or by ovalbumin (OVA) immunization followed by OVA challenge. Animals were anesthetized with isoflurane (Minrad, Bethlehem, PA) and restrained in a supine position.13Hsieh CH Frink M Hsieh YC Kan WH Hsu JT Schwacha MG Choudhry MA Chaudry IH The role of MIP-1α in the development of systemic inflammatory response and organ injury following trauma hemorrhage.J Immunol. 2008; 181: 2806-2812PubMed Google Scholar A midline laparotomy (2 cm) was performed, which was then closed in two layers with sutures (Ethilon 6/0, Ethicon, Somerville, NJ). Both femoral arteries and a femoral vein were cannulated with polyethylene-10 tubing (Becton Dickinson, Sparks, MD). Blood pressure was measured via one of the arterial catheters using a blood pressure analyzer (Micro-Med, Louisville, KY). The animals were then restrained in a supine position in a small plastic box covered with a dark cloth to prevent the mice from seeing the surrounding environment and thus keeping them calm. On awakening, the mice were bled rapidly through the other arterial catheter to a mean arterial blood pressure of 35 ± 5 mmHg within 10 minutes. The rapid bleeding puts the animals in a state of depressed sensibility. Blood was further withdrawn slowly to reach a blood loss of 60% of the total circulating blood volume while still maintaining mean arterial pressure at 35 ± 5 mmHg. The entire hemorrhagic shock time lasted for 90 minutes. At the end of that interval, the animals were resuscitated with four times the shed blood volume with Ringer’s lactate over 30 minutes via the venous line. After resuscitation, the restraint was removed, and the animals were lightly anesthetized with isoflurane. After the blood vessels were ligated, catheters were removed, and the incision sites were bathed with lidocaine and closed with sutures. The animals were then allowed to awaken and were returned to their cages. Sham-operated animals underwent the same surgical procedures but were neither subjected to hemorrhage nor resuscitated. The animals were sacrificed at 24 hours after resuscitation or sham operation. For in vivo ConA stimulation, a nonhepatotoxic dosage of Con A (3 mg/kg b.wt.14Wang J Sun R Wei H Dong Z Tian Z Pre-activation of T lymphocytes by low dose of concanavalin A aggravates toll-like receptor-3 ligand-induced NK cell-mediated liver injury.Int Immunopharmacol. 2006; 6: 800-807Crossref PubMed Scopus (16) Google Scholar) dissolved in 100 μl of Ringer’s lactate was injected via the venous line at the end of resuscitation. Sham-operated animals were also injected at the same time point. For OVA immunization, each mouse was injected s.c. with 0.1 ml of complete Freund’s adjuvant mixed with 0.1 mg of OVA 14 days before trauma-hemorrhage. After trauma-hemorrhage, mice were challenged with 0.1 mg of OVA again at the end of resuscitation or at the respective time point for sham-operated animals. ConA, OVA, and complete Freund’s adjuvant were all purchased from Sigma-Aldrich (St. Louis, MO). The animals were anesthetized with isoflurane 24 hours after resuscitation or sham operation. Spleens were removed aseptically and were isolated as described previously.15Hildebrand F Hubbard WJ Choudhry MA Thobe BM Pape HC Chaudry IH Are the protective effects of 17β-estradiol on splenic macrophages and splenocytes after trauma-hemorrhage mediated via estrogen-receptor (ER)-α or ER-β?.J Leukoc Biol. 2006; 79: 1173-1180Crossref PubMed Scopus (35) Google Scholar In brief, spleens were gently ground to produce a single cell suspension, which was centrifuged at 400 × g for 10 minutes at 4°C. The erythrocytes were lysed with lysis buffer (BD Biosciences, San Jose, CA), and the remaining cells were washed with PBS by centrifugation (400 × g, 10 minutes, 4°C). After centrifugation, cells were resuspended in PBS to get a final concentration of 1 × 107 cells/ml. Splenocyte suspensions were divided into two parts for either flow cytometric analysis or further purification of CD4+ T cells and macrophages by magnetic separation. Isolated splenocytes were resuspended in MACS buffer (Miltenyi Biotec, Auburn, CA) and incubated with CD4 or CD11b microbeads (Miltenyi Biotec) at 4°C for 15 minutes according to the manufacturer’s instructions. After washing steps, CD4+ T cells and CD11b+ macrophages were positively selected by applying splenocytes onto MS columns that were placed on a MACS separator (all from Miltenyi Biotec). The magnetically labeled CD4+ T cells and CD11b+ macrophages were eluted and collected. The CD11b+ macrophages were used as antigen-presenting cells for experiments involving in vitro stimulation of T cells (see below). Only macrophages of sham animals were isolated. A regulatory T-cell staining kit was used to stain CD4+ T cells and regulatory T cells (Tregs) (eBioscience, San Diego, CA). The fraction of Tregs in CD4+ T cells was analyzed for splenocytes in the sham and trauma-hemorrhage groups. Furthermore, Tregs and CD4+ T cells were purified from splenocytes by using a CD4+CD25+ regulatory T-cell isolation kit and anti-CD4 microbeads according to the manufacturer’s instruction (all from Miltenyi Biotec, Auburn, CA). Three anti-mouse antibodies against surface markers were used in this analysis: APC anti-CD4 antibody (Ab), phycoerythrin (PE) anti-CD28 Ab, and PE Cy7-anti-CD69 Ab (all from eBioscience). Splenocytes were washed and resuspended in staining buffer (PBS containing 0.5% bovine serum albumin and 0.09% sodium azide, Sigma-Aldrich) at a concentration of 1 × 107 cells/ml and were stained as described previously.8Kawasaki T Fujimi S Lederer JA Hubbard WJ Choudhry MA Schwacha MG Bland KI Chaudry IH Trauma-hemorrhage induces depressed splenic dendritic cell functions in mice.J Immunol. 2006; 177: 4514-4520Crossref PubMed Scopus (77) Google Scholar In brief, after blocking with anti-CD16/32 Ab (eBioscience) for 15 minutes on ice, cells were stained with the above indicated antibodies for 45 minutes at 4°C in the dark. Cells were then washed, resuspended in staining buffer, and analyzed using a BD LSRII flow cytometer (BD Biosciences, Mountain View, CA). A total of 50,000 events were collected for analysis. A PE-conjugated American hamster anti-mouse cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal antibody (eBioscience) and an Alexa Fluor 488-conjugated anti-phospho-Akt (p-Akt) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA) were used in this study. Cells were surface-stained with APC anti-CD4 Ab as described in the previous section and were then fixed in 2% formaldehyde for 20 minutes at room temperature. After washing, cells were resuspended in permeabilization buffer (eBioscience) and incubated with either anti-p-Akt Ab or anti-CTLA4 Ab for 30 minutes. After 2 additional washes, cells were resuspended in staining buffer and analyzed on a flow cytometer. A total of 50,000 events were collected for analysis. To determine any changes in proliferative capacity of T cells after trauma-hemorrhage or sham operation, T cells were cultured and challenged with either ConA or OVA. For ConA stimulation, purified CD4+ T cells from sham or trauma-hemorrhage animals were resuspended in complete RPMI 1640 medium (10% fetal bovine serum, 2 mmol/L glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, and 20 μg/ml gentamicin, all from Invitrogen, Carlsbad, CA) with or without 5 μg/ml of ConA and plated in a 96-well plate at a cell density of 1 × 105 cells/well. For OVA challenge, isolated splenic macrophages from a sham mouse were first incubated for 30 minutes (37°C, 5% CO2, in the dark) with 50 μg/ml mitomycin C (Sigma-Aldrich) to serve as APCs. After washing, these APCs were cultured with T cells (1 × 105 cells/well at a 1:1 ratio) purified from OVA-immunized sham or trauma-hemorrhage animals in culture medium with or without 0.1 mg/ml of OVA. The proliferation capacities of T cells were measured after 24, 48, and 72 hours of culture, respectively. Cell proliferation was determined using a Cell Proliferation Biotrak ELISA System (Amersham Biosciences Inc., Piscataway, NJ) as described previously.8Kawasaki T Fujimi S Lederer JA Hubbard WJ Choudhry MA Schwacha MG Bland KI Chaudry IH Trauma-hemorrhage induces depressed splenic dendritic cell functions in mice.J Immunol. 2006; 177: 4514-4520Crossref PubMed Scopus (77) Google Scholar In brief, 5-bromo-2′-deoxyuridine was added to the cell suspension, which was then given 3-hour exposure to the substrate. The cells were fixed, and their DNA was denatured and then incubated with peroxidase-labeled anti-5-bromo-2′-deoxyuridine Ab according to the manufacturer’s instructions. The optical density of each well was determined by a spectrophotometer at 450 nm after the peroxidase substrate 3,3′,5,5′-tetramethylbenzide was added to the cells. CD28 and Akt activation were measured in cultured T cells by flow cytometry. Cells were cultured in the same way as those for measuring cellular proliferation capacity. After 3 days of culture, cells were harvested, stained for CD4, CD28, and p-Akt, and analyzed using flow cytometry as described in the previous section. The mean fluorescence intensity (MFI) of CD28 and p-Akt of those events gated as CD4+ were measured. The cytokine concentrations in culture supernatant were determined with a cytometric bead array using flow cytometry according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA) as described previously.16Frink M Hsieh YC Thobe BM Choudhry MA Schwacha MG Bland KI Chaudry IH TLR4 regulates Kupffer cell chemokine production, systemic inflammation and lung neutrophil infiltration following trauma-hemorrhage.Mol Immunol. 2007; 44: 2625-2630Crossref PubMed Scopus (53) Google Scholar In brief, 50 μl of mixed capture beads were incubated with 50 μl of sample for 1 hour at 25°C, and then 50 μl of mixed PE-conjugated detection antibody was added. After incubation for 1 hour at 25°C in the dark, the complexes were washed twice and analyzed using the LSRII flow cytometer. Data analysis was performed using the FACSDiva and FCAP array software (BD Biosciences). Statistical analysis was performed using Sigma-Stat computer software (SPSS, Chicago, IL). Statistical significance was assumed when probability values of less than 0.05 were obtained. Comparisons between groups were performed using one-way analysis of variance followed by Tukey’s test. Results are expressed as mean ± SE. The expression of CD69, CD28, and CTLA-4 on splenic CD4+ T cells was analyzed 24 hours after in vivo stimulation. As shown in Figure 1, A and B, CD69 expression was significantly increased after ConA stimulation compared with that for matched unstimulated controls. In addition, when one compares both stimulated groups, CD69 expression by sham-treated animals was significantly higher than that in the trauma-hemorrhage group. There were no differences between unstimulated sham and trauma-hemorrhage groups. The expression of CD28 with or without ConA stimulation showed a similar pattern as observed for CD69. As shown in Figure 1, C and D, although ConA stimulation induced a significant increase in CD28 expression, such an increase was suppressed by the trauma-hemorrhage insult. No differences in CD28 expression were noted between the unstimulated groups. In contrast, as shown in Figure 1, E and F, the expression of CTLA-4 was significantly higher in the ConA-stimulated trauma-hemorrhage group than in all other groups. The OVA challenge induced increased CD69 and CD28 expression on T cells similar to those stimulated by ConA; however, such increases were suppressed by the trauma-hemorrhage insult (Figure 2, A–D). On the other hand, although CTLA-4 expression was elevated after OVA challenge in sham and trauma-hemorrhage groups, trauma-hemorrhage was found to induce a further increase in CTLA-4 compared with that in the sham group (Figure 2, E and F).Figure 2Expression of CD69, CD28, and CTLA-4 on splenic CD4+ T cells after OVA immunization and in vivo OVA challenge. In this experiment, all of the animals were immunized with OVA 14 days before trauma-hemorrhage by injection of 0.1 ml of complete Freund’s adjuvant mixed with 0.1 mg of OVA. Another 0.1 mg of OVA or vehicle (PBS) was injected at the end of resuscitation in trauma-hemorrhage groups or at corresponding time points in sham groups. Twenty-four hours after the end of resuscitation, splenocytes with or without OVA challenge were isolated and stained with APC anti-CD4 Ab plus either PE Cy7-anti-CD69 Ab, PE anti-CD28 Ab, or PE anti-CTLA-4 Ab as described in Materials and Methods. Representative histograms were gated on CD4+ T cells and showed MFI of CD69 (A), CD28 (C), and CTLA-4 (E). MFI data for CD69 (B), CD28 (D), and CTLA-4 (F) are shown as mean ± SEM. *P < 0.05 versus all of the other groups. **P < 0.05 versus vehicle groups. n = 5/group. The vertical dotted line in each histogram is intended for better recognition of the shift of MFI among different treatment groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As shown in Figure 3, A and B, the expression of p-Akt was significantly increased in ConA-stimulated sham animals compared with that in other groups. However, in the ConA-stimulated trauma-hemorrhage animals, it was significantly lower than in sham-operated animals and was similar to that in those without stimulation. In the experiments in which animals were challenged with OVA in vivo, the p-Akt levels increased significantly after OVA challenge compared with those without OVA challenge. Moreover, sham animals challenged with OVA had significantly higher p-Akt levels compared with trauma-hemorrhage animals challenged with OVA (Figure 3, C and D). Although we demonstrated that trauma-hemorrhage suppressed the responses of T cells to in vivo activation, we further examined whether this suppression also occurred in vitro. As shown in Figure 4, A and B, ConA significantly induced an increase in CD28 expression in both sham and trauma-hemorrhage groups compared with unstimulated groups. We also found that cells of the ConA-stimulated sham group had higher CD28 expression compared with that of the ConA-stimulated trauma-hemorrhage group. Moreover, OVA stimulation induced a marked increase in CD28 expression in cells of the sham group, an effect not seen in the OVA-stimulated trauma-hemorrhage group (Figure 4, C and D). As shown in Figure 5, A and B, comparison between the stimulated and unstimulated subsets, ConA treatment induced a marked increase in p-Akt in both sham and trauma-hemorrhage groups. Moreover, the increase was more pronounced in the sham group compared with that in the trauma-hemorrhage group. There was no difference in p-Akt expression in unstimulated groups. In the experiments in which the cells were stimulated by OVA, a result similar to that in the ConA-stimulated group was observed. Whereas p-Akt levels of T cells were increased after OVA stimulation, they were significantly elevated in the sham versus the trauma-hemorrhage group (Figure 5, C and D). As shown in Figure 6A, T cells proliferated minimally throughout 3 days of culture in nonstimulated groups. Beginning 24 hours after culture, the proliferation of ConA-stimulated T cells was markedly increased compared with that in those without stimulation. Moreover, although the extent of T-cell proliferation in the stimulated sham and trauma-hemorrhage groups was similar after 24 hours of culture, the ConA-stimulated sham T cells showed increased proliferative capacity after 48 and 72 hours of culture compared with ConA-stimulated trauma-hemorrhage T cells. For those T cells stimulated by APC-processed OVA (Figure 6B) after 48 hours in culture, T cells of the OVA-stimulated sham group proliferated more compared with all of the other groups. However, by 72 hours after culture the proliferation of T cells in OVA-stimulated trauma-hemorrhage groups was significantly increased compared with that in the nonstimulated groups. As shown in Figure 7A, unstimulated T cells produced minimal amounts of interleukin (IL)-2, interferon (IFN)-γ, IL-4, and IL-5. The cytokine response of T cells to OVA stimulation was different between sham and trauma-hemorrhage groups. T cells in the sham group produced significantly more IL-2 compared with those in the trauma-hemorrhage group. In contrast, cells in the trauma-hemorrhage group produced more IL-4 and IL-5 compared with those in the sham group. Furthermore, TH1 (IL-2 and IFN-γ)/TH2 (IL-4 and IL-5) cytokine production ratios in the trauma-hemorrhage group were significantly lower than those in the sham group, indicating that T-cell cytokine production shifted from TH1 type to TH2 type after trauma-hemorrhage (Figure 7B). In addition, to exclude the possibility that the increased IL-2 and IFN-γ production in the sham group was due to increased numbers of T cells being generated during culture, intracellular cytokine staining was performed to determine cytokine production on a per cell basis. These measurements revealed that intracellular cytokine production of IL-2 and IFN-γ was significantly suppressed after trauma-hemorrhage compared with those of the sham group (supplementary Figure 1, A and B, see http://ajp.amjpathol.org). The immune system is well designed to defend against diverse pathogens, while avoiding a response to self. In their role as the key mediators of the immune system, T-cell activation is tightly regulated to maintain immune homeostasis.17Lenschow DJ Walunas TL Bluestone JA CD28/B7 system of T cell costimulation.Annu Rev Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2358) Google Scholar As an example of a fail-safe mechanism, T-cell activation requires two signals that are delivered by APCs. 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