Malcolm MacConmara

Impaired Regulatory T-Cell Response and Enhanced Atherosclerosis in the Absence of Inducible Costimulatory Molecule

Background— T-cell–mediated immunity contributes to the pathogenesis of atherosclerosis, but little is known about how these responses are regulated. We explored the influence of the inducible costimulatory molecule (ICOS) on atherosclerosis and associated immune responses. Methods and Results— Bone morrow chimeras were generated by transplanting ICOS-deficient or wild-type bone marrow into irradiated atherosclerosis-prone, LDR receptor–deficient mice, and the chimeric mice were fed a high-cholesterol diet for 8 weeks. Compared with controls, mice transplanted with ICOS-deficient marrow had a 43% increase in the atherosclerotic burden, and importantly, their lesions had a 3-fold increase in CD4+ T cells, as well as increased macrophage, smooth muscle cell, and collagen content. CD4+ T cells from ICOS-deficient chimeras proliferated more and secreted more interferon-γ and tumor necrosis factor-α than T cells from control mice, which suggests a lack of regulation. FoxP3+ regulatory T cells (Treg) were found to constitutively express high ICOS levels, which suggests a role for ICOS in Treg function. ICOS-deficient mice had decreased numbers of FoxP3+ Treg and impaired in vitro Treg suppressive function compared with control mice. Conclusions— ICOS has a key role in regulation of atherosclerosis, through its effect on regulatory T-cell responses.

Increased CD4+ CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity

Objectives:We recently reported increased CD4+ CD25+ T regulatory (Treg) activity after burn injury in mice. This study sought to determine if Tregs mediate the reduction in TH1-type immunity after serious injury in man and if Treg function is altered by injury. Methods:Peripheral blood was withdrawn from 19 consenting adult patients (35.1 ± 16.3 years of age) with Injury Severity Scores (ISS) 36.6 ± 13.9 on days 1 and 7 after trauma and from 5 healthy individuals. CD4+ T cells were purified and sorted into Treg (CD25high) and Treg-depleted populations. After activation of cells with anti-CD3/CD28 antibody, production of the TH1-type cytokine IFNγ, TH2-type cytokines (IL-4 and IL-5), and the inhibitory cytokine IL-10 was measured using cytometric bead arrays. Treg activity was measured by in vitro suppression of autologous CD4+ T cell proliferation. Results:All patients survived, 9 (47%) developed infection postinjury. IFNγ production by patient CD4+ T cells was decreased on day 1 and day 7, when compared with healthy controls. However, when Tregs were depleted from the CD4+ T cells, the IFNγ production increased to control levels. Tregs were the chief source of IL-4 and IL-5 as well as IL-10. Treg suppression of T cell proliferation increased significantly from day 1 to day 7 after injury. Conclusions:We demonstrate for the first time that human Tregs are increased in potency after severe injury. Most significantly, Tregs are important mediators of the suppression of T cell activation and the reduction in TH1 cytokine production found after injury.

Enhanced Regulatory T Cell Activity Is an Element of the Host Response to Injury

CD4+CD25+ regulatory T cells (Tregs) play a critical role in suppressing the development of autoimmune disease, in controlling potentially harmful inflammatory responses, and in maintaining immune homeostasis. Because severe injury triggers both excessive inflammation and suppressed adaptive immunity, we wished to test whether injury could influence Treg activity. Using a mouse burn injury model, we demonstrate that injury significantly enhances Treg function. This increase in Treg activity is apparent at 7 days after injury and is restricted to lymph node CD4+CD25+ T cells draining the injury site. Moreover, we show that this injury-induced increase in Treg activity is cell-contact dependent and is mediated in part by increased cell surface TGF-β1 expression. To test the in vivo significance of these findings, mice were depleted of CD4+CD25+ T cells before sham or burn injury and then were immunized to follow the development of T cell-dependent Ag-specific immune reactivity. We observed that injured mice, which normally demonstrate suppressed Th1-type immunity, showed normal Th1 responses when depleted of CD4+CD25+ T cells. Taken together, these observations suggest that injury can induce or amplify CD4+CD25+ Treg function and that CD4+CD25+ T cells contribute to the development of postinjury immune suppression.

Enhanced TLR4 reactivity following injury is mediated by increased p38 activation.

Severe injury primes the innate‐immune system for increased Toll‐like receptor 4 (TLR4)‐induced proinflammatory cytokine production by macrophages. In this study, we examined changes in TLR4 signaling pathways in splenic macrophages from burn‐injured or sham mice to determine the molecular mechanism(s) responsible for the increased TLR4 responsiveness. Using flow cytometry and specific antibodies, we first looked for injury‐induced changes in the expression levels of several TLR‐associated signaling molecules. We found similar levels of myeloid differentiation primary‐response protein 88 (MyD88) and interleukin‐1 receptor‐associated kinase‐M (IRAK‐M) and somewhat lower levels of total p38, extracellular signal‐regulated kinase (ERK), and stress‐activated protein kinase (SAPK)/c‐jun N‐terminal kinase (JNK) mitogen‐activated protein kinases (MAPKs) in burn compared with sham macrophages. However, with the use of antibodies specific for the phosphorylated (activated) forms of the three MAPKs, we found that macrophages from burn mice showed a twofold increase in purified lipopolysaccharide (LPS)‐stimulated p38 activation as compared with cells from sham mice on days 1 and 7 post‐injury, whereas ERK and SAPK/JNK activation was increased by burn injury only on day 1. Using the specific p38 inhibitor (SB203580), we confirmed that the increase in tumor necrosis factor α production by LPS‐stimulated burn macrophages requires p38 activation. Although we demonstrated that injury increases macrophage TLR4 mRNA expression and intracellular expression of TLR4‐myeloid differentiation protein‐2 (MD‐2) protein, macrophage cell‐surface expression of TLR4‐MD‐2 was not changed by burn injury. Our results suggest that the injury‐induced increase in TLR4 reactivity is mediated, at least in part, by enhanced activation of the p38 signaling pathway.

Injury Enhances Resistance to Escherichia coli Infection by Boosting Innate Immune System Function

Major injury is widely thought to predispose the injured host to opportunistic infections. This idea is supported by animal studies showing that major injury causes reduced resistance to polymicrobial sepsis induced by cecal ligation and puncture. Although cecal ligation and puncture represents a clinically relevant sepsis model, we wanted to test whether injury might also lead to greater susceptibility to peritoneal infection caused by a single common pathogen, Escherichia coli. Contrary to our expectation, we show herein that the LD50 for sham-injured mice was 103 CFU of E. coli, whereas the LD50 for burn-injured mice was 50 × 103 CFU at 7 days postinjury. This injury-associated enhanced resistance was apparent as early as 1 day after injury, and maximal resistance was observed at days 7 and 14. We found that burn-injured mice had higher numbers of circulating neutrophils and monocytes than did sham mice before infection and that injured mice were able to recruit greater numbers of neutrophils to the site of infection. Moreover, the peritoneal neutrophils in burn-injured mice were more highly activated than neutrophils from sham mice as determined by Mac-1 expression, superoxide generation, and bactericidal activity. Our findings suggest that the enhanced innate immune response that develops following injury, although it is commonly accepted as the mediator of the detrimental systemic inflammatory response syndrome, may also, in some cases, benefit the injured host by boosting innate immune antimicrobial defenses.

Regulatory T cells suppress antigen-driven CD4 T cell reactivity following injury

Injury initiates local and systemic host responses and is known to increase CD4 Treg activity in mice and humans. This study uses a TCR transgenic T cell adoptive transfer approach and in vivo Treg depletion to determine specifically the in vivo influence of Tregs on antigen‐driven CD4 T cell reactivity following burn injury in mice. We report here that injury in the absence of recipient and donor Tregs promotes high antigen‐driven CD4 T cell expansion and increases the level of CD4 T cell reactivity. In contrast, CD4 T cell expansion and reactivity were suppressed significantly in injured Treg‐replete mice. In additional experiments, we found that APCs prepared from burn‐ or sham‐injured, Treg‐depleted mice displayed significantly higher antigen‐presenting activity than APCs prepared from normal mice, suggesting that Tregs may suppress injury responses by controlling the intensity of APC activity. Taken together, these findings demonstrate that Tregs can actively control the in vivo expansion and reactivity of antigen‐stimulated, naïve CD4 T cells following severe injury.

Effective immunotherapy of weakly immunogenic solid tumours using a combined immunogene therapy and regulatory T-cell inactivation

Obstacles to effective immunotherapeutic anti-cancer approaches include poor immunogenicity of the tumour cells and the presence of tolerogenic mechanisms in the tumour microenvironment. We report an effective immune-based treatment of weakly immunogenic, growing solid tumours using a locally delivered immunogene therapy to promote development of immune effector responses in the tumour microenvironment and a systemic based T regulatory cell (Treg) inactivation strategy to potentiate these responses by elimination of tolerogenic or immune suppressor influences. As the JBS fibrosarcoma is weakly immunogenic and accumulates Treg in its microenvironment with progressive growth, we used this tumour model to test our combined immunotherapies. Plasmids encoding GM-CSF and B7-1 were electrically delivered into 100 mm3 tumours; Treg inactivation was accomplished by systemic administration of anti-CD25 antibody (Ab). Using this approach, we found that complete elimination of tumours was achieved at a level of 60% by immunogene therapy, 25% for Treg inactivation and 90% for combined therapies. Moreover, we found that these responses were immune transferable, systemic, tumour specific and durable. Combined gene-based immune effector therapy and Treg inactivation represents an effective treatment for weakly antigenic solid growing tumours and that could be considered for clinical development.

Murine dendritic cell antigen-presenting cell function is not altered by burn injury

Severe injury disrupts normal immune regulation causing a transient hyperinflammatory reaction and suppressed adaptive immune function. This report addresses the potential contribution of dendritic cells (DC) to changes in adaptive immune function after injury by specifically measuring injury‐induced changes in splenic DC numbers and subsets, cell‐surface markers, TLR responses, and APC function. Using a mouse burn injury model, we found that injury did not markedly alter the relative percentage of lymphoid, myeloid, or plasmacytoid DC in the spleens of burn‐injured mice. Moreover, we did not observe a significant reduction in cell‐surface expression of several major costimulatory molecules, CD40, CD80, CD86, programmed death 1 ligand, ICOS ligand, and B7‐H3, on DC. Instead, we observed increased cell‐surface expression of CD86 at 1 day after injury with no significant changes in costimulatory molecule expression at 7 days after injury, suggesting that burn injury causes an early activation of DC. In addition, injury did not suppress DC reactivity to TLR2, TLR4, or TLR9 agonists. Most important, DC prepared from injured mice were able to present peptide antigen to naïve OTII TCR transgenic CD4+ T cells as efficiently and effectively as DC from sham‐injured mice. We also found that CD4 T cells stimulated with antigen presented by DC from sham or burn mice showed similar levels of IL‐2, IFN‐γ, IL‐10, and IL‐13 production. Taken together, these findings support the conclusion that DC do not acquire a suppressive phenotype following severe injury in mice.