Inhibit T-cell Function Research Articles

Immunosuppressive capabilities of mesenchymal stromal cells are maintained under hypoxic growth conditions and after gamma irradiation

Background aimsThe discovery of regenerative and immunosuppressive capacities of mesenchymal stromal cells (MSCs) raises hope for patients with tissue-damaging or severe, treatment-refractory autoimmune disorders. We previously presented a method to expand human MSCs in a bioreactor under standardized Good Manufacturing Practice conditions. Now we characterized the impact of critical treatment conditions on MSCs with respect to immunosuppressive capabilities and proliferation. MethodsMSC proliferation and survival after γ irradiation were determined by 5-carboxyfluorescein diacetate N-succinimidyl ester and annexinV/4′,6-diamidino-2-phenylindole (DAPI) staining, respectively. T-cell proliferation assays were used to assess the effect of γ irradiation, passaging, cryopreservation, post-thaw equilibration time and hypoxia on T-cell suppressive capacities of MSCs. Quantitative polymerase chain reaction and β-galactosidase staining served as tools to investigate differences between immunosuppressive and non-immunosuppressive MSCs. Resultsγ irradiation of MSCs abrogated their proliferation while vitality and T-cell inhibitory capacity were preserved. Passaging and long cryopreservation time decreased the T-cell suppressive function of MSCs, and postthaw equilibration time of 5 days restored this capability. Hypoxic culture markedly increased MSC proliferation without affecting their T-cell-suppressive capacity and phenotype. Furthermore, T-cell suppressive MSCs showed higher CXCL12 expression and less β-galactosidase staining than non-suppressive MSCs. DiscussionWe demonstrate that γ irradiation is an effective strategy to abrogate MSC proliferation without impairing the cells' immunosuppressive function. Hypoxia significantly enhanced MSC expansion, allowing for transplantation of MSCs with low passage number. In summary, our optimized MSC expansion protocol successfully addressed the issues of safety and preservation of immunosuppressive MSC function after ex vivo expansion for therapeutic purposes.

Pretransplantation recipient regulatory T cell suppressive function predicts delayed and slow graft function after kidney transplantation.

Delayed graft function (DGF) and slow graft function (SGF) are a continuous spectrum of ischemia-reperfusion-related acute kidney injury (AKI) that increases the risk for acute rejection and graft loss after kidney transplantation. Regulatory T cells (Tregs) are critical in transplant tolerance and attenuate murine AKI. In this prospective observational cohort study, we evaluated whether pretransplantation peripheral blood recipient Treg frequency and suppressive function are predictors of DGF and SGF after kidney transplantation. Deceased donor kidney transplant recipients (n=53) were divided into AKI (n=37; DGF, n=10; SGF, n=27) and immediate graft function (n=16) groups. Pretransplantation peripheral blood CD4CD25FoxP3 Treg frequency was quantified by flow cytometry. Regulatory T-cell suppressive function was measured by suppression of autologous effector T-cell proliferation by Treg in co-culture. Pretransplantation Treg suppressive function, but not frequency, was decreased in AKI recipients (P<0.01). In univariate and multivariate analyses accounting for the effects of cold ischemic time and donor age, Treg suppressive function discriminated DGF from immediate graft function recipients in multinomial logistic regression (odds ratio, 0.77; P<0.01), accurately predicted AKI in receiver operating characteristic curve (area under the curve, 0.82; P<0.01), and predicted 14-day estimated glomerular filtration rate in linear regression (P<0.01). Our results indicate that recipient peripheral blood Treg suppressive function is a potential independent pretransplantation predictor of DGF and SGF.

Glucocorticosteroids trigger reactivation of human cytomegalovirus from latently infected myeloid cells and increase the risk for HCMV infection in D+R+ liver transplant patients.

Graft rejection in transplant patients is managed clinically by suppressing T-cell function with immunosuppressive drugs such as prednisolone and methylprednisolone. In such immunocompromised hosts, human cytomegalovirus (HCMV) is an important opportunistic pathogen and can cause severe morbidity and mortality. Currently, the effect of glucocorticosteroids (GCSs) on the HCMV life cycle remains unclear. Previous reports showed enhanced lytic replication of HCMV in vitro in the presence of GCSs. In the present study, we explored the implications of steroid exposure on latency and reactivation. We observed a direct effect of several GCSs used in the clinic on the activation of a quiescent viral major immediate-early promoter in stably transfected THP-1 monocytic cells. This activation was prevented by the glucocorticoid receptor (GR) antagonist Ru486 and by shRNA-mediated knockdown of the GR. Consistent with this observation, prednisolone treatment of latently infected primary monocytes resulted in HCMV reactivation. Analysis of the phenotype of these cells showed that treatment with GCSs was correlated with differentiation to an anti-inflammatory macrophage-like cell type. On the basis that these observations may be pertinent to HCMV reactivation in post-transplant settings, we retrospectively evaluated the incidence, viral kinetics and viral load of HCMV in liver transplant patients in the presence or absence of GCS treatment. We observed that combination therapy of baseline prednisolone and augmented methylprednisolone, upon organ rejection, significantly increased the incidence of HCMV infection in the intermediate risk group where donor and recipient are both HCMV seropositive (D+R+) to levels comparable with the high risk D+R− group.

Helicobacter pylori γ-glutamyltranspeptidase impairs T-lymphocyte function by compromising metabolic adaption through inhibition of cMyc and IRF4 expression.

Helicobacter pylori (H. pylori) is a human-specific pathogen that has evolved to cope with the immune response elicited against the infection. We previously reported that H. pylori γ-glutamyltranspeptidase (gGT) impairs T-lymphocyte proliferation and thus might act as immune regulatory factor. In this study, we analysed the underlying mechanism and its implications for H. pylori persistence. We found that H. pylori gGT compromised T-cell proliferation, activation and effector cytokine expression by specifically depriving the extracellular space of glutamine. When assessing signalling cascades and transcription factors affected by H. pylori gGT, we found that expression of cMyc and IRF4, both required for metabolic adaptation of T-lymphocytes, was highly sensitive to extracellular glutamine levels and downregulated upon gGT treatment. Moreover, we could confirm decreased IRF4 expression in T-lymphocytes infiltrating the stomach of infected individuals. Thus, our results suggest that H. pylori gGT-mediated glutamine deprivation in the gastric mucosa may suppress T-cell function thereby contributing to bacterial persistence.

Immunomodulatory roles of lymphatic vessels in cancer progression.

Lymphatic vessels in the tumor microenvironment are known to foster tumor metastasis in many cancers, and they can undergo activation, hyperplasia, and lymphangiogenesis in the tumor microenvironment and in the tumor-draining lymph node. The mechanism underlying this correlation was originally considered as lymphatic vessels providing a physical route for tumor cell dissemination, but recent studies have highlighted new roles of the lymphatic endothelium in regulating host immunity. These include indirectly suppressing T-cell function by secreting immunosuppressive factors and inhibiting dendritic cell (DC) maturation, as well as directly driving T-cell tolerance by antigen presentation in the presence of inhibitory ligands. Furthermore, lymphatic endothelium scavenges and regulates transendothelial transport actively, controlling the sustained delivery of lymph-borne antigens from chronically inflamed tissues to draining lymph nodes where immature DCs, in the absence of danger signals, along with lymph node stromal cells present these antigens to T cells for maintenance of peripheral tolerance to self-antigens, a mechanism that may be hijacked by some tumors. This Masters of Immunology primer aims to present an overview of research in this area and highlight emerging evidence that suggests lymphatic vessels, and lymphangiogenesis, play important immunomodulatory roles in the tumor microenvironment.

Myeloid expression of angiotensin-converting enzyme facilitates myeloid maturation and inhibits the development of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells which accumulate in cancer, infection and chronic inflammation. These cells suppress T-cell function and the immune response. Angiotensin-converting enzyme (ACE) is a peptidase that is now known to regulate aspects of myelopoiesis. Here, we show that ACE expression correlates with myeloid maturation in vitro. Forced ACE overexpression in monocytic cells reduces the generation of MDSCs. In vivo, mice with a genetic change resulting in myeloid cell ACE overexpression have reduced numbers of blood and splenic MDSCs in a tumor model and in a model of chronic inflammation induced by complete Freund's adjuvant. In contrast, ACE-null mice produce large numbers of MDSCs during chronic inflammation. Macrophages from mice with myeloid ACE overexpressing are more pro-inflammatory and have more tumor-killing activity than cells from wild-type mice. Thus, manipulating myeloid ACE activity can interfere with MDSC development and the maturation of myeloid cells.

The Central Role of Arginine Catabolism in T-Cell Dysfunction and Increased Susceptibility to Infection After Physical Injury

To explore the hypothesis that decreased arginine availability by myeloid-derived suppressor cells (MDSCs) is a cause of T-cell dysfunction after physical injury (PI). Arginine is an essential amino acid for normal T-cell function whose availability becomes limited after PI. MDSCs expressing arginase 1 are induced by PI. T-cell dysfunction after PI seems to increase the risk of infection but the mechanisms that cause it are unclear. PI was created using a standard laparotomy model. Phenotypical and functional alterations in T cells were evaluated in vivo. MDSCs expressing arginase 1 were measured by flow cytometry. Infection after PI was created by intraperitoneal injection of Listeria monocytogenes. N-Hydroxy-Nor-L-arginine (Nor-NOHA) was used as an arginase inhibitor. The effect of arginine depletion on T-cell function and susceptibility to infection was assessed through adoptive transfer of MDSC or injection of arginase into noninjured mice. PI caused a decrease in intracellular arginine in T cells, loss of the T-cell receptor (TCR) CD3-ζ chain, inhibition of in vivo T-cell proliferation, memory, and cytotoxicity. PI exponentially increased bacterial growth and mortality to L. monocytogenes. T-cell dysfunction and increased infection were reversed by arginase inhibitor Nor-NOHA but were reproduced by adoptively transferring MDSC or injecting arginase 1 to noninjured mice. Arginine availability is decreased after PI coinciding with an induction of MDSC expressing arginase 1. Decreased arginine may inhibit T-cell function and increase susceptibility to infection after injury.

Activation of the PD-1 Pathway Contributes to Immune Escape in EGFR-Driven Lung Tumors

The success in lung cancer therapy with programmed death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs and increased markers of T-cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T-cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape and mechanistically link treatment response to PD-1 inhibition. We show that autochthonous EGFR-driven lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines. These findings indicate that EGFR functions as an oncogene through non-cell-autonomous mechanisms and raise the possibility that other oncogenes may drive immune escape.

Interferon‐induced programmed death‐ligand 1 (PD‐L1/B7‐H1) expression increases on human acute myeloid leukemia blast cells during treatment

While current treatment for acute myeloid leukemia is characterized by high response rates, patients' long-term outcome is still disappointing, due to frequent relapse and ineligibility of the often elderly patients for stem cell transplantation approaches. Considerable efforts have, thus, been made to incorporate immunotherapeutic approaches in the acute myeloid leukemia (AML) consolidation, with so far disappointing clinical benefit. The B7 family ligand programmed-death receptor-ligand 1 (PD-L1, B7-H1, CD274) has been recently described (with conflicting results) to be expressed on AML blast cells, and interaction with its receptor on T cells, programmed death receptor-1 (PD-1, CD279), has been shown to suppress T-cell functions and to allow survival of dormant AML cells in animal models. In this work, we analyzed freshly isolated myeloid precursor cells from healthy donors and from AML patients for PD-L1 expression with or without interferon-γ exposure at different time points during their treatment. While without IFN exposure, only minor differences were observed, we found IFN-γ-induced PD-L1 expression most prominent after initial treatment and independent of treatment outcome. Our observations support the recently suggested PD-L1-mediated adaptive immune resistance and argue for a targeting of the PD-L1/PD-1 pathway during the consolidation phase of AML treatment.

Analytical validation of a quantitative reverse transcriptase polymerase chain reaction assay for evaluation of T-cell targeted immunosuppressive therapy in the dog

Cyclosporine is an immunosuppressive agent that inhibits T-cell function by decreasing production of cytokines such as interleukin-2 (IL-2) and interferon-γ (IFN-γ). In dogs, there is currently no reliable analytical method for determining effective cyclosporine dosages in individual patients. Our laboratory has developed a quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) assay that measures IL-2 and IFN-γ gene expression, with the goal of quantifying immunosuppression in dogs treated with cyclosporine. This study focuses on analytical validation of our assay, and on the effects of sample storage conditions on cyclosporine-exposed samples. Heparinized whole blood collected from healthy adult dogs was exposed to a typical post-treatment blood concentration for cyclosporine (500ng/mL) for 1h, and then stored for 0, 24, and 48h at both room temperature and 4°C. The study was then repeated using a cyclosporine concentration of 75ng/mL, with sample storage for 0, 24, and 48h at 4°C. Cytokine gene expression was measured using RT-qPCR, and assay efficiency and inter- and intra-assay variability were determined. Storage for up to 24h at room temperature, and up to 48h at 4°C, did not significantly alter results compared to samples that were processed immediately. Validation studies showed our assay to be highly efficient and reproducible and robust enough to be feasible under standard practice submission conditions.

Antagonist Antibodies to PD-1 and B7-H1 (PD-L1) in the Treatment of Advanced Human Cancer—Response

The immune suppressive molecule programmed death-1 (PD-1) is upregulated in activated T lymphocytes and inhibits T-cell function upon binding to its ligands B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273). Substantial experimental data from in vitro cell culture systems and animal models, and more recently from clinical trials, indicate that PD-1/PD-1-ligand interactions are a major mechanism of immune suppression within the tumor microenvironment. Initial clinical studies of antibodies directed against PD-1 and B7-H1 showed both an encouraging safety profile and remarkable antitumor activity in subsets of patients with metastatic disease, including malignancies--such as lung cancer--which were previously thought to be unresponsive to immunotherapy. Preliminary data have suggested a correlation between tumor membrane B7-H1 expression and clinical response to anti-PD-1 antibodies. Several key challenges remain to optimize development of PD-1/B7-H1 pathway blockade, including defining the biologic significance of all potential ligand-receptor interactions in the tumor microenvironment, developing more accurate predictive biomarkers of response, determining the breadth of activity in human malignancies, and developing rational combinations of therapy that address key mechanisms involved in positive and negative regulation of antitumor immune responses.

Increased Frequency of Myeloid-derived Suppressor Cells during Active Tuberculosis and after RecentMycobacterium tuberculosisInfection Suppresses T-Cell Function

Inadequacy of T-cell responses may result in the development of tuberculosis (TB). Myeloid-derived suppressor cells (MDSCs) have been described as suppressors of T-cell function in cancer biology and recently in several infectious diseases. To explore the presence and role of MDSCs in TB. We analyzed surface markers of MDSCs in peripheral blood and at the site of disease in TB cases and in patients with lung cancer, and in peripheral blood of asymptomatic tuberculin skin test-positive individuals with recent (household) or remote exposure to Mycobacterium tuberculosis (M.tb) and in uninfected healthy control subjects. To evaluate the suppressive capacity of MDSCs, cells of household contacts infected with M.tb and TB cases were isolated and cocultured with CD3(+) T cells. Our results demonstrate an increased presence of MDSCs after recent M.tb infection and disease. We confirm their suppression of CD4(+) T-cell function, including reduced cytokine responses and inhibition of CD4(+) T-cell proliferation. Only MDSCs from TB cases reduced T-cell activation, altered T-cell trafficking, and suppressed CD8(+) T-cell functions. M.tb-expanded MDSCs were associated with significantly higher IL-1β, IL-6, IL-8, granulocyte colony-stimulating factor, and monocyte chemotactic protein-1, and reduced granulocyte-macrophage colony-stimulating factor and macrophage inflammatory protein-1 beta levels in coculture. These data reveal that innate MDSCs are induced not only during active TB at similar levels as found in cancer, but also in healthy individuals after recent exposure to M.tb. These cells diminish protective T-cell responses and may contribute to the inability of hosts to eradicate the infection and add to the subsequent development of TB disease.

Clonal expansion of renal cell carcinoma-infiltrating T lymphocytes

T lymphocytes can mediate the destruction of cancer cells by virtue of their ability to recognize tumor-derived antigenic peptides that are presented on the cell surface in complex with HLA molecules and expand. Thus, the presence of clonally expanded T cells within neoplastic lesions is an indication of ongoing HLA-restricted T cell-mediated immune responses. Multiple tumors, including renal cell carcinomas (RCCs), are often infiltrated by significant amounts of T cells, the so-called tumor-infiltrating lymphocytes (TILs). In the present study, we analyzed RCC lesions (n = 13) for the presence of expanded T-cell clonotypes using T-cell receptor clonotype mapping. Surprisingly, we found that RCCs comprise relatively low numbers of distinct expanded T-cell clonotypes as compared with melanoma lesions. The numbers of different T-cell clonotypes detected among RCC-infiltrating lymphocytes were in the range of 1–17 (median = 5), and in several patients, the number of clonotypes expanded within tumor lesions resembled that observed among autologous peripheral blood mononuclear cells. Moreover, several of these clonotypes were identical in TILs and PBMCs. Flow cytometry data demonstrated that the general differentiation status of CD8+ TILs differed from that of circulating CD8+ T cells. Furthermore, PD-1 and LAG-3 were expressed by a significantly higher percentage of CD8+ RCC-infiltrating lymphocytes as compared with PBMCs obtained from RCC patients or healthy individuals. Thus, CD8+ TILs display a differentiated phenotype and express activation markers as well as surface molecules associated with the inhibition of T-cell functions. However, TILs are characterized by a low amount of expanded T-cell clonotypes.

Etiology and pathogenesis of basal cell carcinoma

Abstract Basal cell carcinoma (BCC) is the most common cancer among Caucasians. It generally occurs on sun-exposed areas of the body, mostly on the head and neck (80%), trunk (15%), rarely on arms and legs. Basal cell carcinoma is a good example of a disease caused by a combination of genetic and environmental factors. Ultraviolet (UV) radiation plays a dual role in the development of BCC: it causes DNA damage and immunosuppression. UVA and UVB rays damage the DNA via various mechanisms. UVB radiation directly damages DNA within skin cells, causing cytosine → thymine mutations at dipyrimidine sites, whereas UVA radiation is 10.000 times less mutagenic, but it is significantly more present in the natural UV radiation. Also, UVA photons have lower energy than UVB photons and do not induce mutations. UV radiation exerts immune suppression by decreasing the antigen presenting cells ability and by producing immunosuppressive cytokines, such as interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α). Mediators of UV-induced immunosuppression are DNA and cis-urocanic acid. Several studies showed a significant association between the development of BCC and sun-exposure during childhood and adolescence, and a strong relation with family history of skin cancer. Exposure to ionizing radiation increases the risk of nonmelanoma skin cancers by three times, while the risk is proportional to the radiation dose. Chemical carcinogens, such as arsenic, tar, psoralen, and pesticides, increase risks for nonmelanoma skin cancers, predominantly for squamous cell carcinoma (SCC). Regarding genetic predisposition, there is glutathione S-transferase (GST) as an important part of cellular defense against endogenous and exogenous chemicals. Several polymorphisms in GST family members have been associated with impaired detoxification, thus influencing the risk for some cancers, including nonmelanoma skin cancers. Cytochrome P450 enzymes are involved in detoxification of photosensitizing agents, and thus involved in BCC carcinogenesis. PTCH is a tumor suppressor gene first identified in patients with Gorlin syndrome. Abnormal activation of this gene and its pathways result in various types of tumorigenesis. BCC is associated with homozygous PTCH gene deletion. With regard to acquired genetic mutations, it was found that aggressive BCCs are significantly associated with increased p53 protein expression, probably representing the mutated form, although that assertion could not be established with certainty. Considering the apparently limited contribution of DNA damage and chromosome instability to the expression of BCC phenotype, the relevance of p53 mutations for BCC growth remains to be demonstrated. Data on the role of Bcl-2 gene family in the development of BCC are scarce. It is unclear whether Bcl-2 has a functional role in the development of BCC, or it only indicates the level of gene expression in tumor stem cells. Activation of Ras gene may play an important role during early stages in the development of nonmelanoma skin cancers, and it is often found on UV-exposed skin in BCC, actinic keratosis and SCC. Concerning immunologic factors, studies have shown that tumor necrosis factor-α (TNF-α) is the critical mast cell product involved in ultraviolet-induced immunosuppression: mast cells contain high quantities of TNF-α which is released after activation; the level of TNF-α is increased in the skin exposed to UV radiation disrupting the morphology and function of Langerhans cells, the principal antigen-presenting cells of the skin. An animal study suggests that the degree of susceptibility to ultraviolet-B-induced local immunosuppression depends on TNF-α level within the epidermis after UVB. It has been established that mast cell-derived histamine stimulates prostaglandin E2 (PGE2) production from keratinocytes. PGE2 alters the cytokine balance in favor of the immunosuppressive interleukin-10 (IL-10) against the immunostimulatory IL-12; histamine also increases suppressor T-cell function by binding to the H2 receptors, which in turn release higher levels of immune suppressive cytokines including IL-10 and induce apoptosis of antigen-presenting cells. All this results in a shift of the immune response from T helper 1 (Th1) cytokine profile to T helper 2 (Th2) cytokine profile, inhibiting antigen-presenting cells to induce antitumor activity.

Elevated CD3+ and CD8+ tumor-infiltrating immune cells correlate with prolonged survival in glioblastoma patients despite integrated immunosuppressive mechanisms in the tumor microenvironment and at the systemic level

We characterized GBM patients' tumor and systemic immune contexture with aim to reveal the mechanisms of immunological escape, their impact on patient outcome, and identify targets for immunotherapy. Increased CD3+ T-cell infiltration was associated with prolonged survival independent of age, MGMT promoter methylation and post-operative treatment that implies potential for immunotherapy for GBM. Several mechanisms of escape were identified: within the tumor microenvironment: induced CD8+CD28−Foxp3+ Tregs that may tolerize antigen presenting cells, elevated CD73 and CD39 ectonucleotidases that suppress T-cell function, and at the systemic level: elevated IL-10 levels in serum, diminished helper T-cell counts, and upregulated inhibitory CTLA-4.

Negative Regulation of Type I IFN Expression by OASL1 Permits Chronic Viral Infection and CD8+ T-Cell Exhaustion

The type I interferons (IFN-Is) are critical not only in early viral control but also in prolonged T-cell immune responses. However, chronic viral infections such as those of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in humans and lymphocytic choriomeningitis virus (LCMV) in mice overcome this early IFN-I barrier and induce viral persistence and exhaustion of T-cell function. Although various T-cell-intrinsic and -extrinsic factors are known to contribute to induction of chronic conditions, the roles of IFN-I negative regulators in chronic viral infections have been largely unexplored. Herein, we explored whether 2′–5′ oligoadenylate synthetase-like 1 (OASL1), a recently defined IFN-I negative regulator, plays a key role in the virus-specific T-cell response and viral defense against chronic LCMV. To this end, we infected Oasl1 knockout and wild-type mice with LCMV CL-13 (a chronic virus) and monitored T-cell responses, serum cytokine levels, and viral titers. LCMV CL-13-infected Oasl1 KO mice displayed a sustained level of serum IFN-I, which was primarily produced by splenic plasmacytoid dendritic cells, during the very early phase of infection (2–3 days post-infection). Oasl1 deficiency also led to the accelerated elimination of viremia and induction of a functional antiviral CD8 T-cell response, which critically depended on IFN-I receptor signaling. Together, these results demonstrate that OASL1-mediated negative regulation of IFN-I production at an early phase of infection permits viral persistence and suppresses T-cell function, suggesting that IFN-I negative regulators, including OASL1, could be exciting new targets for preventing chronic viral infection.

Melanoma-Educated CD14+ Cells Acquire a Myeloid-Derived Suppressor Cell Phenotype through COX-2–Dependent Mechanisms

Tumors can suppress the host immune system by employing a variety of cellular immune modulators, such as regulatory T cells, tumor-associated macrophages, and myeloid-derived suppressor cells (MDSC). In the peripheral blood of patients with advanced stage melanoma, there is an accumulation of CD14(+)HLA-DR(lo/-) MDSC that suppress autologous T cells ex vivo in a STAT-3-dependent manner. However, a precise mechanistic basis underlying this effect is unclear, particularly with regard to whether the MDSC induction mechanism relies on cell-cell contact of melanoma cells with CD14(+) cells. Here, we show that early-passage human melanoma cells induce phenotypic changes in CD14(+) monocytes, leading them to resemble MDSCs characterized in patients with advanced stage melanoma. These MDSC-like cells potently suppress autologous T-cell proliferation and IFN-γ production. Notably, induction of myeloid-suppressive functions requires contact or close proximity between monocytes and tumor cells. Further, this induction is largely dependent on production of cyclooxygenase-2 (COX-2) because its inhibition in these MDSC-like cells limits their ability to suppress T-cell function. We confirmed our findings with CD14(+) cells isolated from patients with advanced stage melanoma, which inhibited autologous T cells in a manner relying up prostaglandin E2 (PGE2), STAT-3, and superoxide. Indeed, PGE2 was sufficient to confer to monocytes the ability to suppress proliferation and IFN-γ production by autologous T cells ex vivo. In summary, our results reveal how immune suppression by MDSC can be initiated in the tumor microenvironment of human melanoma.

Pathogenesis, prophylaxis and treatment of infections in patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) patients are at high risk for infections. The pathogenesis of infection in patients with this leukemia is complex and multifactorial. Patients with CLL have a number of immune system defects, including disordered B-cell function with decreased production of normal B-cells and abnormal production of immunoglobulins, suppressed T-cell function and neutropenia. Other immune abnormalities present in CLL patients include neutrophil dysfunction, and complement deficiencies. In addition, further perturbations in immune function are related to the antileukemic therapies. Immune disturbance might be common prior to CLL diagnosis and infectious agents could trigger CLL development. Current chemotherapy-based regimens are not curative and often worsen this immune suppression. The introduction of new effective therapeutic agents such as the purine analogues and monoclonal antibodies has influenced the spectrum of infections diagnosed in CLL patients. Some conditions increase the risk for the development of infections including advanced age, decreased levels of immunoglobulins, advanced Binet stage, neutropenia and treatment with more than one line of chemotherapy. Until now it is debatable whether and when antibacterial prophylaxis could be useful in CLL patients. The prevention of infection includes antimicrobial prophylaxis, as well as immunoglobulin replacement and vaccination. Antibacterial prophylaxis should be given to CLL patients with previous severe and/or relapsing bacterial infections. This article reviews the immune defects in CLL and discusses strategies aimed at prophylaxis and treatment of infections in patients with CLL.

CD4⁺CD73⁺ T cells are associated with lower T-cell activation and C reactive protein levels and are depleted in HIV-1 infection regardless of viral suppression.

The role of the adenosine (ADO) suppression pathway, specifically CD39-expressing and CD73-expressing CD4⁺ T cells in HIV-1 infection is unclear. We evaluated the frequency and numbers of CD4⁺CD39⁺ and CD4⁺CD73⁺ T cells, activated T cells, and plasma C reactive protein (CRP) levels in 36 HIV-1-positive individuals and 10 normal controls (NC). Low-level plasma viremia was evaluated using single copy assay. Mass spectrometry was used to measure hydrolysis of ATP by ectoenzyme-expressing CD4⁺ T cells, whereas cyclic adenosine monophosphate (cAMP) levels were measured using enzyme immunoassay. Suppression of T-cell function by exogenous ADO and CD4⁺CD73⁺ T cells was tested by flow cytometry. CD39 and CD73 are expressed in different CD4⁺ T-cell subsets. CD4⁺CD73⁺ T cells do not express CD25 and FOXP3, and their frequency and numbers were lower in HIV-1-positive individuals regardless of virologic suppression (P=0.005 and P<0.001, respectively). CD4⁺CD73⁺ numbers inversely correlated with CD4⁺CD38⁺DR⁺ (P=0.002), CD8⁺CD38⁺DR⁺ T-cell frequency (P=0.05), and plasma CRP levels (P=0.01). Both subsets are required for hydrolysis of exogenous ATP to ADO and can increase CD4⁺ T-cell cAMP levels when incubated with exogenous ATP. Low-level viremia did not correlate with activated T-cell frequency. In vitro, ADO suppressed T-cell activation and cytokine expression. CD4⁺CD7⁺ T cells suppressed T-cell proliferation only in the presence of exogenous 5'-AMP. The ADO-producing CD4⁺CD73⁺ subset of T cells is depleted in HIV-1-positive individuals regardless of viral suppression and may play a key role in controlling HIV-1-associated immune activation.

Intrahepatic IL-8 producing Foxp3+CD4+ regulatory T cells and fibrogenesis in chronic hepatitis C

Regulatory CD4(+) T cells (Tregs) are considered to affect outcomes of HCV infection, because they increase in number during chronic hepatitis C and can suppress T-cell functions. Using microarray analysis, in situ immunofluorescence, ELISA, and flowcytometry, we characterised functional differentiation and localisation of adaptive Tregs in patients with chronic hepatitis C. We found substantial upregulation of IL-8 in Foxp3(+)CD4(+) Tregs from chronic hepatitis C. Activated GARP-positive IL-8(+) Tregs were particularly enriched in livers of patients with chronic hepatitis C in close proximity to areas of fibrosis and their numbers were correlated with the stage of fibrosis. Moreover, Tregs induced upregulation of profibrogenic markers TIMP1, MMP2, TGF-beta1, alpha-SMA, collagen, and CCL2 in primary human hepatic stellate cells (HSC). HSC activation, but not Treg suppressor function, was blocked by adding a neutralizing IL-8 antibody. Our studies identified Foxp3(+)CD4(+) Tregs as an additional intrahepatic source of IL-8 in chronic hepatitis C acting on HSC. Thus, Foxp3(+)CD4(+) Tregs in chronic hepatitis C have acquired differentiation as regulators of fibrogenesis in addition to suppressing local immune responses.