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1.  Highly Prevalent Colorectal Cancer-Infiltrating LAP+Foxp3− T cells Exhibit More Potent Immunosuppressive Activity than Foxp3+ Regulatory T Cells 
Mucosal immunology  2013;7(2):428-439.
Although elevated CD4+Foxp3+ Treg frequencies within tumors are well-documented, functional and phenotypic characteristics of CD4+Foxp3+ and CD4+Foxp3− T cell subsets from matched blood, healthy colon and colorectal cancer require in-depth investigation. Flow cytometry revealed that the majority of intra-tumoral CD4+Foxp3+ T cells (Tregs) were Helios+ and expressed higher levels of CTLA-4 and CD39 than Tregs from colon and blood. Moreover, ~30% of intra-tumoral CD4+Foxp3− T cells expressed markers associated with regulatory functions, including LAP, LAG-3 and CD25. This unique population of cells produced IL-10 and TGF-β, and was ~50-fold more suppressive than Foxp3+ Tregs. Thus, intra-tumoral Tregs are diverse, posing multiple obstacles to immunotherapeutic intervention in colorectal malignancies.
PMCID: PMC3931584  PMID: 24064667
Colorectal Cancer; T cells; LAP; Foxp3; Suppressive
2.  Progression of carcinogen‐induced fibrosarcomas is associated with the accumulation of naïve CD4+ T cells via blood vessels and lymphatics 
The tumor microenvironment comprises newly formed blood and lymphatic vessels which shape the influx, retention and departure of lymphocytes within the tumor mass. Thus, by influencing the intratumoral composition of lymphocytes, these vessels affect the manner in which the adaptive immune system responds to the tumor, either promoting or impairing effective antitumor immunity. In our study, we utilized a mouse model of carcinogen‐induced fibrosarcoma to examine the composition of tumor‐infiltrating lymphocytes during tumor progression. In particular, we sought to determine whether CD4+Foxp3+ regulatory T cells (Tregs) became enriched during tumor progression thereby contributing to tumor‐driven immunosuppression. This was not the case as the proportion of Tregs and effector CD4+ T cells actually declined within the tumor owing to the unexpected accumulation of naïve T cells. However, we found no evidence for antigen‐driven migration of these T cells or for their participation in an antitumor immune response. Our data support the notion that lymphocytes can enter tumors via aberrantly formed blood and lymphatic vessels. Such findings suggest that targeting both the tumor vasculature and lymphatics will alter the balance of lymphocyte subpopulations that enter the tumor mass. A consideration of this aspect of tumor immunology may be critical to the success of solid cancer immunotherapies.
What's new?
It is well known that a tumors' microenvironment can impair the anti‐tumor immune response. The culprits are usually assumed to be various suppressor cells and cytokines. In this study, however, the authors found that seemingly innocuous, naïve T cells may also play a significant role—simply by accumulating and possibly out‐competing activated effector cells within the tumor. A better understanding of the signals produced by the tumor microenvironment may allow researchers to alter this T‐cell pool, thus enhancing the immune response.
PMCID: PMC4114538  PMID: 24142504
T cells; carcinogen; lymphatics
3.  Escalating regulation of 5T4-specific IFN-γ+ CD4+ T cells distinguishes colorectal cancer patients from healthy controls and provides a target for in vivo therapy 
Cancer immunology research  2013;1(6):10.1158/2326-6066.CIR-13-0035.
The relationship between the adaptive CD4+ T cell response and human cancer is unclear. The oncofetal antigen 5T4 is expressed on many human carcinomas, including colorectal cancer (CRC) cells, but has limited expression on normal tissues. We previously identified anti-5T4 CD4+ T cells in a proportion of CRC patients, and we extended this study to examine whether the quality or quantity of the T cell response reflects tumor stage. An overlapping peptide library spanning 5T4 was used as a target to enumerate cognate IFN-γ+CD4+ T-cells (measured as spot forming cells [SFC]/105 cultured T cells) in peripheral blood-derived lymphocytes following a 12-day in vitro culture period comparing patients pre-operatively (n = 27) to healthy controls (n = 17). Robust 5T4-specific T cell responses were present in 100% of healthy donors. There was a steady loss of T cell responses with advancing tumors with a significant negative correlation from stage I to III (P = 0.008). The predictability of the decline meant < 200 SFC/105 was only found in subjects with stage III CRC. The mechanism of loss of T cell response is independent of HLA-DR type or patient age, but does correspond to increases in Foxp3+ regulatory T cells (Tregs). Using low-dose cyclophosphamide to reduce the proportion of Tregs in vivo resulted in increased anti-5T4 T cell responses in CRC patients. The selective loss of 5T4-specific IFN-γ+CD4+ T cell responses implies a link between tumor stage and antitumor Th1 effector function; depleting Tregs can enhance such responses.
PMCID: PMC3880929  PMID: 24409450
colorectal cancer; CD4+ T cells; 5T4; epitopes; regulatory T cells
4.  Neutrophils Recruited by IL-22 in Peripheral Tissues Function as TRAIL-Dependent Antiviral Effectors against MCMV 
Cell Host & Microbe  2014;15(4):471-483.
During primary infection, murine cytomegalovirus (MCMV) spreads systemically, resulting in virus replication and pathology in multiple organs. This disseminated infection is ultimately controlled, but the underlying immune defense mechanisms are unclear. Investigating the role of the cytokine IL-22 in MCMV infection, we discovered an unanticipated function for neutrophils as potent antiviral effector cells that restrict viral replication and associated pathogenesis in peripheral organs. NK-, NKT-, and T cell-secreted IL-22 orchestrated antiviral neutrophil-mediated responses via induction in stromal nonhematopoietic tissue of the neutrophil-recruiting chemokine CXCL1. The antiviral effector properties of infiltrating neutrophils were directly linked to the expression of TNF-related apoptosis-inducing ligand (TRAIL). Our data identify a role for neutrophils in antiviral defense, and establish a functional link between IL-22 and the control of antiviral neutrophil responses that prevents pathogenic herpesvirus infection in peripheral organs.
Graphical Abstract
•Neutrophils are critical antiviral effector cells during MCMV virus infection•Neutrophils directly inhibit virus replication in a TRAIL-dependent manner•IL-22 inhibits virus replication in peripheral but not secondary lymphoid tissues•IL-22 orchestrates CXCL1-dependent neutrophil recruitment
Murine cytomegalovirus (MCMV) targets multiple peripheral organs during infection. Stacey et al. report that in response to MCMV infection, NK, NKT, and T cells secrete the cytokine IL-22, which recruits antiviral neutrophils to infected peripheral organs in a CXCL1-dependent manner. Neutrophils exert antiviral effector functions via proapoptotic TRAIL expression.
PMCID: PMC3989063  PMID: 24721575
5.  The paradox of NKp46+ natural killer cells: drivers of severe hepatitis C virus-induced pathology but in-vivo resistance to interferon α treatment 
Gut  2013;63(3):515-524.
There is evidence that natural killer (NK) cells help control persistent viral infections including hepatitis C virus (HCV). The phenotype and function of blood and intrahepatic NK cells, in steady state and after interferon (IFN) α treatment has not been fully elucidated.
We performed a comparison of NK cells derived from blood and intrahepatic compartments in multiple paired samples from patients with a variety of chronic liver diseases. Furthermore, we obtained serial paired samples from an average of five time points in HCV patients treated with IFNα.
Liver NK cells demonstrate a distinct activated phenotype compared to blood manifested as downregulation of the NK cell activation receptors CD16, NKG2D, and NKp30; with increased spontaneous degranulation and IFN production. In contrast, NKp46 expression was not downregulated. Indeed, NKp46-rich NK populations were the most activated, correlating closely with the severity of liver inflammation. Following initiation of IFNα treatment there was a significant increase in the proportion of intrahepatic NK cells at days 1 and 3. NKp46-rich NK populations demonstrated no reserve activation capacity with IFNα treatment and were associated with poor viral control on treatment and treatment failure.
NKp46 marks out pathologically activated NK cells, which may result from a loss of homeostatic control of activating receptor expression in HCV. Paradoxically these pathological NK cells do not appear to be involved in viral control in IFNα-treated individuals and, indeed, predict slower rates of viral clearance.
PMCID: PMC3932740  PMID: 23665989
6.  Interleukin-6 limits influenza-induced inflammation and protects against fatal lung pathology 
European journal of immunology  2013;43(10):10.1002/eji.201243018.
Balancing the generation of immune responses capable of controlling virus replication with those causing immunopathology is critical for the survival of the host and resolution of influenza-induced inflammation. Based on the capacity of interleukin-6 (IL-6) to govern both optimal T-cell responses and inflammatory resolution, we hypothesised that IL-6 plays an important role in maintaining this balance. Comparison of innate and adaptive immune responses in influenza-infected wild-type control and IL-6 deficient mice revealed striking differences in virus clearance, lung immunopathology and generation of heterosubtypic immunity. Mice lacking IL-6 displayed a profound defect in their ability to mount an anti-viral T-cell response. Failure to adequately control virus was further associated with an enhanced infiltration of inflammatory monocytes into the lung and an elevated production of the pro-inflammatory cytokines IFN-α and TNF-α. These events were associated with severe lung damage, characterised by profound vascular leakage, and death. Our data highlight an essential role for IL-6 in orchestrating anti-viral immunity through an ability to limit inflammation, promote protective adaptive immune responses and prevent fatal immunopathology.
PMCID: PMC3886386  PMID: 23857287
Adaptive Immunity; Heterosubtypic Immunity; IL-6; Innate Immunity; Pulmonary Damage
7.  Rapid early innate control of hepatitis C virus during IFN-α treatment compromises adaptive CD4+ T cell immunity 
European journal of immunology  2012;42(9):2383-2394.
The ability to control and eradicate HCV with IFN-α-based treatments provides an opportunity in humans to study how the rate of viral clearance in vivo impinges on the development of anti-viral responses. Ex vivo (IFN-γ-producing) and cultured anti-viral CD4+ T cells, serum cytokines and viral loads were measured repeatedly in a cohort of chronically HCV-infected subjects (n = 33) receiving IFN-α. Rapid control of virus indicated by an increased calculated rate of viral clearance, occurred in those subjects (n=17) demonstrating absent/minimal T cell responses (p<0.0006). Surprisingly lower rates of virus, and presumably antigen, clearance were associated with an increase in T cell responses, and reduced serum IL-10 in treatment success in failure. A subsequent analysis of NK cell function in available subjects (n = 8) revealed an inverse correlation between pre-treatment NK cell expression of NKp46 and the potential to up-regulate cytotoxic function on exposure to IFN-α (p < 0.004), as well as the subsequent measured rate of viral clearance (p=0.045). Thus the CD4+ T cell response during IFN-α appears to be shaped by the rate of innate virus suppression. These data suggest that individuals who respond most effectively to immune intervention may be most in need of subsequent vaccination to prevent re-infection.
PMCID: PMC3781703  PMID: 22653709
Hepatitis C virus; CD4+ T cells; NK cells
8.  Rapid early innate control of hepatitis C virus during IFN-α treatment compromises adaptive CD4+ T-cell immunity 
European Journal of Immunology  2012;42(9):2383-2394.
The ability to control HCV with IFN-α-based treatments provides an opportunity in humans to study how the rate of viral clearance in vivo impinges on the development of antiviral responses. Ex vivo (IFN-γ-producing) and cultured antiviral CD4+ T cells, serum cytokines, and viral loads were measured repeatedly in a cohort of chronically HCV-infected subjects (n = 33) receiving IFN-α. Rapid control of virus indicated by an increased calculated rate of virus clearance, occurred in those subjects demonstrating absent/minimal T-cell responses (p < 0.0006). Surprisingly, in subjects who demonstrated the most robust T-cell responses (and reduced serum IL-10), there was actually a reduced rate of early virus clearance. A subsequent analysis of NK-cell function in available subjects (n = 8) revealed an inverse correlation between pretreatment NK-cell expression of NKp46 and the potential to upregulate cytotoxic function on exposure to IFN-α (p < 0.004), as well as the subsequent measured rate of viral clearance (p = 0.045). Thus, the CD4+ T-cell response during IFN-α treatment appears to be shaped by the rate of innate virus suppression. These data suggest that individuals who respond most effectively to immune intervention may be most in need of subsequent vaccination to prevent reinfection.
PMCID: PMC3781703  PMID: 22653709
CD4+ T cells; Hepatitis C virus; IFN-α; NK cells
9.  T Cell Trafficking Facilitated by High Endothelial Venules is Required for Tumor Control after Regulatory T Cell Depletion 
Cancer research  2012;72(21):5473-5482.
The evolution of immune blockades in tumors limits successful anti-tumor immunity, but the mechanisms underlying this process are not fully understood. Depletion of regulatory T cells (Tregs), a T cell subset that dampens excessive inflammatory and autoreactive responses, can allow activation of tumor-specific T cells. However, cancer immunotherapy studies have demonstrated that a persistent failure of activated lymphocytes to infiltrate tumors remains a fundamental problem. In evaluating this issue, we found that despite an increase in T cell activation and proliferation following Treg depletion there was no significant association with tumor growth rate. In contrast, there was a highly significant association between low tumor growth rate and the extent of T cell infiltration. Further analyses revealed a total concordance between low tumor growth rate, high T cell infiltration and the presence of high endothelial venules (HEV). HEV are blood vessels normally found in secondary lymphoid tissue where they are specialized for lymphocyte recruitment. Thus, our findings suggest that Treg depletion may promote HEV neogenesis, facilitating increased lymphocyte infiltration and destruction of the tumor tissue. These findings are important as they point to a hitherto unidentified role of Tregs, the manipulation of which may refine strategies for more effective cancer immunotherapy.
PMCID: PMC3491872  PMID: 22962270
regulatory T cells; high endothelial venules; fibrosarcoma
10.  High endothelial venules 
Oncoimmunology  2013;2(5):e24272.
Recently, a number of studies have documented the presence of high endothelial venules in both mouse and human tumors. The significance of these highly specialized blood vessels within neoplastic lesions, and notably their capacity to influence anticancer immune responses and tumor progression, remains to be fully understood.
PMCID: PMC3667912  PMID: 23762806
HEV; blood vessels; carcinogenesis; fibrosarcoma; regulatory T cell
11.  Home Sweet Home: The Tumor Microenvironment as a Haven for Regulatory T Cells 
CD4+Foxp3+ regulatory T cells (Tregs) have a fundamental role in maintaining immune balance by preventing autoreactivity and immune-mediated pathology. However this role of Tregs extends to suppression of anti-tumor immune responses and remains a major obstacle in the development of anti-cancer vaccines and immunotherapies. This feature of Treg activity is exacerbated by the discovery that Treg frequencies are not only elevated in the blood of cancer patients, but are also significantly enriched within tumors in comparison to other sites. These observations have sparked off the quest to understand the processes through which Tregs become elevated in cancer-bearing hosts and to identify the specific mechanisms leading to their accumulation within the tumor microenvironment. This manuscript reviews the evidence for specific mechanisms of intra-tumoral Treg enrichment and will discuss how this information may be utilized for the purpose of manipulating the balance of tumor-infiltrating T cells in favor of anti-tumor effector cells.
PMCID: PMC3712544  PMID: 23874342
tumor immunology; regulatory T cells; intra-tumoral proliferation; chemokines; immunotherapy of cancer
12.  Hunting for clues 
Oncoimmunology  2012;1(7):1163-1164.
Colorectal cancer is a common disease which appears to be influenced by anti-tumor immune responses. Here we discuss our recent study1 which tracked anti-tumor CD4+ T cell responses before and after surgical resection, highlighting the importance of regulatory T cells (Tregs) in controlling these responses, and the influence of the tumor on these specific T‑cell populations.
PMCID: PMC3494631  PMID: 23170265
colorectal cancer; regulatory T cell; T cell
13.  The death receptor 3/TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity 
Death receptor 3 (DR3, TNFRSF25), the closest family relative to tumor necrosis factor receptor 1, promotes CD4+ T-cell-driven inflammatory disease. We investigated the in vivo role of DR3 and its ligand TL1A in viral infection, by challenging DR3-deficient (DR3KO) mice and their DR3WT littermates with the β-herpesvirus murine cytomegalovirus or the poxvirus vaccinia virus. The phenotype and function of splenic T-cells were analyzed using flow cytometry and molecular biological techniques. We report surface expression of DR3 by naive CD8+ T cells, with TCR activation increasing its levels 4-fold and altering the ratio of DR3 splice variants. T-cell responses were reduced up to 90% in DR3KO mice during acute infection. Adoptive transfer experiments indicated this was dependent on T-cell-restricted expression of DR3. DR3-dependent CD8+ T-cell expansion was NK and CD4 independent and due to proliferation, not decreased cell death. Notably, impaired immunity in DR3KO hosts on a C57BL/6 background was associated with 4- to 7-fold increases in viral loads during the acute phase of infection, and in mice with suboptimal NK responses was essential for survival (37.5%). This is the first description of DR3 regulating virus-specific T-cell function in vivo and uncovers a critical role for DR3 in mediating antiviral immunity.
PMCID: PMC3460213  PMID: 22593543
murine cytomegalovirus; vaccinia virus
14.  Anti-CD8 antibodies can trigger CD8+ T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining 
CD8+ T-cells recognize immunogenic peptides presented at the cell surface bound to major histocompatibility complex class I (MHCI) molecules. Antigen recognition involves the binding of both T-cell receptor (TCR) and CD8 co-receptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates effects on antigen sensitivity. Anti-CD8 antibodies have been used extensively to examine the role of CD8 in CD8+ T-cell activation. However, as previous studies have yielded conflicting results, it is unclear from the literature whether anti-CD8 antibodies per se are capable of inducing effector function. Here, we report on the ability of seven monoclonal anti-human CD8 antibodies to activate six human CD8+ T-cell clones with a total of five different specificities. Six out of seven anti-human CD8 antibodies tested did not activate CD8+ T-cells. In contrast, one anti-human CD8 antibody, OKT8, induced effector function in all CD8+ T-cells examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining and the visualization of antigen-specific CD8+ T-cells. The anti-mouse CD8 antibodies, CT-CD8a and CT-CD8b, also activated CD8+ T-cells despite opposing effects on pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 antibodies to trigger T-cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of antibody-mediated CD8-engagement to deliver an activation signal underscores the importance of CD8 in CD8+ T-cell signalling.
PMCID: PMC3145095  PMID: 21677135
anti-CD8 antibody; CD8+ T-cell activation; pMHCI tetramer; T-cells; surface plasmon resonance
15.  Suppression of tumour-specific CD4+ T cells by regulatory T cells is associated with progression of human colorectal cancer 
Gut  2011;61(8):1163-1171.
There is indirect evidence that T cell responses can control the metastatic spread of colorectal cancer (CRC). However, an enrichment of CD4+Foxp3+ regulatory T cells (Tregs) has also been documented.
To evaluate whether CRC promotes Treg activity and how this influences anti-tumour immune responses and disease progression.
A longitudinal study of Treg activity on a cohort of patients was performed before and after tumour resection. Specific CD4+ T cell responses were also measured to the tumour associated antigens carcinoembryonic antigen (CEA) and 5T4.
Tregs from 62 preoperative CRC patients expressed a highly significant increase in levels of Foxp3 compared to healthy age-matched controls (p=0.007), which returned to normal after surgery (p=0.0075). CD4+ T cell responses to one or both of the tumour associated antigens, CEA and 5T4, were observed in approximately two-thirds of patients and one third of these responses were suppressed by Tregs. Strikingly, in all patients with tumour recurrence at 12 months, significant preoperative suppression was observed of tumour-specific (p=0.003) but not control CD4+ T cell responses.
These findings demonstrate that the presence of CRC drives the activity of Tregs and accompanying suppression of CD4+ T cell responses to tumour-associated antigens. Suppression is associated with recurrence of tumour at 12 months, implying that Tregs contribute to disease progression. These findings offer a rationale for the manipulation of Tregs for therapeutic intervention.
PMCID: PMC3388728  PMID: 22207629
Regulatory T cells; colorectal cancer; imaging; T lymphocytes; colorectal cancer; colorectal diseases; histopathology; cancer immunobiology; hepatitis; immunoregulation; hepatitis C; α β T cells; hepatitis B
16.  Analysis of the T cell receptor repertoires of tumor-infiltrating conventional and regulatory T cells reveals no evidence for conversion in carcinogen-induced tumors 
Cancer research  2010;71(3):736-746.
A significant enrichment of CD4+Foxp3+ T cells (Tregs) is frequently observed in murine and human carcinomas. As Tregs can limit effective anti-tumor immune responses, thereby promoting tumor progression, it is important that the mechanisms underpinning intra-tumoral accumulation of Tregs are identified. Due to evidence gathered mostly in vitro, the conversion of conventional T cells (Tconv) into Tregs has been proposed as one such mechanism. We assessed the contribution of conversion in vivo by analyzing the TCR repertoires of Tconv and Treg cells in carcinogen-induced tumors in mice. Our results indicate that the TCR repertoires of Treg and Tconv cells within tumor-infiltrating lymphocytes (TILs) are largely distinct. Indeed, the cell population with the greatest degree of repertoire similarity with tumor-infiltrating Tregs was the Treg population from the tumor draining lymph node. These findings demonstrate that conversion of Tconv cells does not contribute significantly to the accumulation of tumor-infiltrating Tregs; rather, Tconv and Treg cells arise from different populations with unique TCR repertoires. Enrichment of Tregs within TILs most likely, therefore, reflects differences in the way that Treg and Tconv cells are influenced by the tumor microenvironment. Elucidating the nature of these influences may indicate how the balance between tumor-infiltrating Treg and Tconv cells can be manipulated for therapeutic purposes.
PMCID: PMC3128990  PMID: 21156649
Conversion; methylcholanthrene; regulatory T cell; TCR repertoire; tumor immunology
17.  Paracetamol reduces influenza-induced immunopathology in a mouse model of infection without compromising virus clearance or the generation of protective immunity 
Thorax  2011;66(5):368-374.
Seasonal influenza A infection affects a significant cohort of the global population annually, resulting in considerable morbidity and mortality. Therapeutic strategies are of limited efficacy, and during a pandemic outbreak would only be available to a minority of the global population. Over-the-counter medicines are routinely taken by individuals suffering from influenza, but few studies have been conducted to determine their effectiveness in reducing pulmonary immunopathology or the influence they exert upon the generation of protective immunity.
A mouse model of influenza infection was utilised to assess the efficacy of paracetamol (acetaminophen) in reducing influenza-induced pathology and to examine whether paracetamol affects generation of protective immunity.
Administration (intraperitoneal) of paracetamol significantly decreased the infiltration of inflammatory cells into the airway spaces, reduced pulmonary immunopathology associated with acute infection and improved the overall lung function of mice, without adversely affecting the induction of virus-specific adaptive responses. Mice treated with paracetamol exhibited an ability to resist a second infection with heterologous virus comparable with that of untreated mice.
Our results demonstrate that paracetamol dramatically reduces the morbidity associated with influenza but does not compromise the development of adaptive immune responses. Overall, these data support the utility of paracetamol for reducing the clinical symptoms associated with influenza virus infection.
PMCID: PMC3088439  PMID: 21310755
Influenza; pulmonary; inflammation; paracetamol; virus; not applicable; infection control; innate immunity; respiratory infection; viral infection
18.  CD59 Blockade Enhances Antigen Specific CD4+ T Cell Responses In Humans: A New Target For Cancer Immunotherapy?1 
CD59, a broadly expressed GPI anchored molecule, regulates formation of the membrane attack complex of the complement cascade. We previously demonstrated that mouse CD59 also down-modulates CD4+ T cell activity in vivo. In this study, we explored the role of CD59 on human CD4+ T cells. Our data demonstrate that CD59 is up regulated on activated CD4+ T cells and serves to down-modulate their activity in response to polyclonal and antigen- specific stimulation. The therapeutic potential of this finding was explored using T cells isolated from colorectal cancer patients. The findings were striking and indicated that blockade of CD59 significantly enhanced the CD4+ T cell response to two different tumor antigens. These data highlight the potential for manipulating CD59 expression on T cells for boosting weak immune responses, such as those found in individuals with cancer.
PMCID: PMC2704398  PMID: 19380765
Human; T cells; Tumour immunity
19.  Regulatory T cells inhibit Fas ligand-induced innate and adaptive tumour immunity 
European Journal of Immunology  2007;37(3):758-767.
CD4+CD25+ regulatory T cells (Treg) are known to influence T cell responses to tumours. Here we have explored the role of Treg in inhibiting not only adaptive, but also innate immune responses to tumours. To this end we used a Fas ligand (FasL)-expressing melanoma cell line in a mouse model. In this system, innate immunity is sufficient to reject the tumour. All mice depleted of Treg and challenged with FasL-expressing melanoma remained tumour-free. Investigation of the underlying cellular effector mechanisms revealed that depletion of Treg enhanced an NK cell response capable of tumour lysis. Furthermore, this initial innate immune response primed mice to make an effective adaptive immune response leading to complete rejection of challenge with the parental melanoma. Both antigen-specific antibody and CD4+ T cells were implicated in protection via adaptive immunity. We conclude that removal of Treg and vaccination with whole tumour cells expressing FasL activates multiple arms of the immune system, leading to efficient tumour rejection. These findings highlight a novel role for FasL in inducing innate immune responses that are normally inhibited by Treg and uncover an adjuvant effect of FasL that can be used to stimulate tumour immunity after depletion of Treg.
PMCID: PMC2435420  PMID: 17294404
Fas ligand; Innate tumour immunity; Regulatory T cells
20.  CD59a deficiency exacerbates influenza-induced lung inflammation through complement-dependent and-independent mechanisms 
European Journal of Immunology  2007;37(5):1266-1274.
Influenza-specific immune activity not only promotes virus clearance but also causes immunopathology, thereby underlining the importance of mounting a measured anti-viral immune response. Since complement bridges both the innate and adaptive immune systems and has been implicated in defence against influenza, the role of the complement regulator CD59a in modulating the response to influenza was explored. For this purpose, immune responses to influenza virus, strain E61-13-H17, in mice deficient in the complement regulator protein CD59a (Cd59a–/– mice) were compared to those in wild-type mice. The severity of lung inflammation was significantly enhanced in the lungs of Cd59a–/– mice with increased numbers of infiltrating neutrophils and CD4+ T cells. When complement was inhibited using soluble complement receptor1, the frequency of lung-infiltrating neutrophils in influenza-infected Cd59a–/– mice was much reduced whilst numbers of CD4+ T cells remained unchanged. These results demonstrate that CD59a, previously defined as a complement regulator, modulates both the innate and adaptive immune response to influenza virus by both complement-dependent and-independent mechanisms.
PMCID: PMC2435422  PMID: 17429844
Inflammation; Lung; T cells; Virus
21.  Potent T cell agonism mediated by a very rapid TCR/pMHC interaction 
European Journal of Immunology  2007;37(3):798-806.
The interaction between T cell receptors (TCR) and peptide-major histocompatibility complex (pMHC) antigens can lead to varying degrees of agonism (T cell activation), or antagonism. The P14 TCR recognises the lymphocytic choriomeningitis virus (LCMV)-derived peptide, gp33 residues 33–41 (KAVYNFATC), presented in the context of H-2Db. The cellular responses to various related H-2Db peptide ligands are very well characterised, and P14 TCR-transgenic mice have been used extensively in models of virus infection, autoimmunity and tumour rejection. Here, we analyse the binding of the P14 soluble TCR to a broad panel of related H-2Db-peptide complexes by surface plasmon resonance, and compare this with their diverse cellular responses. P14 TCR binds H-2Db-gp33 with a KD of 3 µM (±0.5 µM), typical of an immunodominant antiviral TCR, but with unusually fast kinetics (koff=1 s−1), corresponding to a half-life of 0.7 s at 25°C, outside the range previously observed for murine agonist TCR/pMHC interactions. The most striking feature of these data is that a very short half-life does not preclude the ability of a TCR/pMHC interaction to induce antiviral immunity, autoimmune disease and tumour rejection.
PMCID: PMC2435421  PMID: 17295390
Biophysics; Protein-protein interactions; TCR
22.  Circulating neutrophils maintain physiological blood pressure by suppressing bacteria and IFNγ-dependent iNOS expression in the vasculature of healthy mice 
Blood  2008;111(10):5187-5194.
Whether leukocytes exert an influence on vascular function in vivo is not known. Here, genetic and pharmacologic approaches show that the absence of neutrophils leads to acute blood pressure dysregulation. Following neutrophil depletion, systolic blood pressure falls significantly over 3 days (88.0 ± 3.5 vs 104.0 ± 2.8 mm Hg, day 3 vs day 0, mean ± SEM, P < .001), and aortic rings from neutropenic mice do not constrict properly. The constriction defect is corrected using l-nitroarginine-methyl ester (L-NAME) or the specific inducible nitric oxide synthase (iNOS) inhibitor 1400W, while acetylcholine relaxation is normal. iNOS- or IFNγ-deficient mice are protected from neutropenia-induced hypotension, indicating that iNOS-derived nitric oxide (NO) is responsible and that its induction involves IFNγ. Oral enrofloxacin partially inhibited hypotension, implicating bacterial products. Roles for cyclooxygenase, complement C5, or endotoxin were excluded, although urinary prostacyclin metabolites were elevated. Neutrophil depletion required complement opsinization, with no evidence for intravascular degranulation. In summary, circulating neutrophils contribute to maintaining physiological tone in the vasculature, at least in part through suppressing early proinflammatory effects of infection. The speed with which hypotension developed provides insight into early changes that occur in the absence of neutrophils and illustrates the importance of constant surveillance of mucosal sites by granulocytes in healthy mice.
PMCID: PMC2602588  PMID: 18281503
23.  Interleukin-6 Is Crucial for Recall of Influenza-Specific Memory CD4+ T Cells 
PLoS Pathogens  2008;4(2):e1000006.
Currently, our understanding of mechanisms underlying cell-mediated immunity and particularly of mechanisms that promote robust T cell memory to respiratory viruses is incomplete. Interleukin (IL)-6 has recently re-emerged as an important regulator of T cell proliferation and survival. Since IL-6 is abundant following infection with influenza virus, we analyzed virus-specific T cell activity in both wild type and IL-6 deficient mice. Studies outlined herein highlight a novel role for IL-6 in the development of T cell memory to influenza virus. Specifically, we find that CD4+ but not CD8+ T cell memory is critically dependent upon IL-6. This effect of IL-6 includes its ability to suppress CD4+CD25+ regulatory T cells (Treg). We demonstrate that influenza-induced IL-6 limits the activity of virus-specific Tregs, thereby facilitating the activity of virus-specific memory CD4+ T cells. These experiments reveal a critical role for IL-6 in ensuring, within the timeframe of an acute infection with a cytopathic virus, that antigen-specific Tregs have no opportunity to down-modulate the immune response, thereby favoring pathogen clearance and survival of the host.
Author Summary
Influenza virus poses a serious global health threat, particularly in light of newly emerging strains such as the avian virus H5N1. The generation of cell-mediated vaccines against influenza virus requires an understanding of mechanisms underlying effective virus-specific T cell memory. This study explored the impact of a cytokine, interleukin-6 (IL-6), on generation of effective influenza-specific T cell memory. This cytokine was considered important based on previous studies revealing its role in promoting survival and activity of conventional T cells whilst inhibiting the activity of T cells involved in dampening down immunity (regulatory T cells). We found that the activity of a subset of influenza-specific memory T cells (CD4+ T cells) was diminished in the absence of IL-6 due to the inhibitory effects of regulatory T cells—an effect that compromised protective anti-viral immunity. Since a robust CD4+ T cell response is likely to be central to the success of a vaccine against influenza virus, these findings highlight the importance of IL-6 in promoting effective cell-mediated immune responses, thereby facilitating successful virus clearance.
PMCID: PMC2279258  PMID: 18389078
24.  CD4+CD25+FOXP3+ Regulatory T Cells Suppress Anti-Tumor Immune Responses in Patients with Colorectal Cancer 
PLoS ONE  2006;1(1):e129.
A wealth of evidence obtained using mouse models indicates that CD4+CD25+FOXP3+ regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4+ T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients.
Methodology and Principal Findings
Treg were identified and characterized as CD4+CD25+FOXP3+ using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4+ T cell responses, as observed by IFNγ release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group.
Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy.
PMCID: PMC1762416  PMID: 17205133
25.  Murine CD59a Modulates Anti-Viral CD4+ T Cell Activity in a Complement-Independent Manner 
CD59 blocks formation of the membrane attack complex of complement by inhibiting binding of C9 to the C5b-8 complex. To investigate a role for CD59 in promoting T cell responses, we compared T cell activation in CD59a-deficient (Cd59a−/−) and wild-type (WT) mice after in vitro stimulation and after infection with recombinant vaccinia virus. Virus-specific CD4+ T cell responses were significantly enhanced in Cd59a−/− mice compared to WT mice. Similarly, Cd59a−/− T cells responded more vigorously to in vitro stimulation with CD3-specific antibodies compared to WT mice. This effect of CD59a on T cell proliferation was found to be complement-independent. Collectively, these results demonstrate that CD59a down-modulates CD4+ T cell activity in vitro and in vivo, thereby revealing another link between complement regulators and T cell activation.
PMCID: PMC1455165  PMID: 16301611
viral; Cell proliferation; T Cells

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