Looking beyond Self-Protection: The Eyes Instruct Systemic Immune Tolerance Early in Life.

The eyes provide themselves with immune tolerance. Frequent skin inflammatory diseases in young blind people suggest, nonetheless, that the eyes instruct a systemic immune tolerance that benefits the whole body. We tested this premise by using delayed skin contact hypersensitivity (DSCH) as a tool to compare the inflammatory response developed by sighted (S) and birth-enucleated (BE) mice against oxazolone or dinitrofluorobenzene at the ages of 10, 30 and 60 days of life. Adult mice enucleated (AE) at 60 days of age were also assessed when they reached 120 days of life. BE mice displayed exacerbated DSCH at 60 but not at 10 or 30 days of age. AE mice, in contrast, show no exacerbated DSCH. Skin inflammation in 60-day-old BE mice was hapten exclusive and supported by distinct CD8+ lymphocytes. The number of intraepidermal T lymphocytes and migrating Langerhans cells was, however, similar between S and BE mice by the age of 60 days. Our observations support the idea that the eyes instruct systemic immune tolerance that benefits organs outside the eyes from an early age. The higher prevalence of inflammatory skin disorders reported in young people might then reflect reduced immune tolerance associated with the impaired functional morphology of the eyes.

Yin and yang roles of B lymphocytes in solid tumors: Balance between antitumor immunity and immune tolerance/immunosuppression in tumor-draining lymph nodes.

The role of B cells in antitumor immunity has been reported to be either promotive or suppressive, but the specific mechanism remains to be comprehensively understood. However, this complicated situation likely depends on the temporal and spatial relationship between the developing tumor and B cells that recognize tumor antigens. Unlike responses against microbial or pathogenic infections, tumor cells are derived from autologous cells that have mutated and become aberrant; thus, elimination by the adaptive immune system is essentially inefficient. If tumor cells can evade immune attack at an early stage, non-destructive responses, such as tolerance and immunosuppression, are established over time. In tumor-draining lymph nodes (TDLNs), tumor antigen-reactive B cells potentially acquire immunoregulatory phenotypes and contribute to an immunosuppressive microenvironment. Therefore, triggering and enhancing antitumor responses by immunotherapies require selective control of these regulatory B cell subsets in TDLNs. In contrast, B cell infiltration and formation of tertiary lymphoid structures in tumors are positively correlated with therapeutic prognosis, suggesting that tumor antigen-specific activation of B cells and antibody production are advantageous for antitumor immunity in mid- to late-stage tumors. Given that the presence of B cells in tumor tissues may reflect the ongoing antitumor response in TDLNs, therapeutic induction and enhancement of these lymphocytes are expected to increase the overall effectiveness of immunotherapy. Therefore, B cells are promising targets, but the spatiotemporal balance of the subsets that exhibit opposite characteristics, that is, the protumor or antitumor state in TDLNs, should be understood, and strategies to separately control their functions should be developed to maximize the clinical outcome.

Impact of folic acid on regulatory B lymphocytes from patients with systemic lupus erythematosus in vitro.

BACKGROUND: Epigenetic modifications of genomes are of particular interest as numerous studies indicate the correlation between DNA methylation and the development of systemic lupus. As a major methyl group donor, folic acid is an important participant in this process. The aim of this study is to determine the effect of low or high dose folate co-culturing with peripheral blood mononuclear cells (PBMCs) on the secretion of interleukin (IL)10 from regulatory cells from lupus patients or from healthy volunteers. METHODS: PBMCs from lupus patients and healthy volunteers were isolated and separated CD19+ B cell populations were cultured in the presence of 4 µg/mL or of 16 µg/mL of folic acid and the DNA methylation level as well as the percentages of B lymphocytes were measured. In another experiment, PBMCs were stimulated in vitro for IL10 production with 1 µg/mL recombinant human CD40L and with 2.5 µg/mL unmethylated CpG dinucleotides and cultured in the presence of 4 µg/mL or of 16 µg/mL of folic acid. RESULTS: Although co-culturing with low or high folic acid concentrations had no effect on the methylation level of B lymphocytes, particular patients showed an increase in the population of CD19+ IL10+ as well as of CD19- IL10+ cells. CONCLUSION: The observed increase may be a consequence of additional indirect or direct methylation of DNA in specific loci of the targeted cells. However, further analysis would clarify the exact mechanism of action of folate and would reveal its immunomodulating potential in this autoimmune disease.

Immune Cell Density Evaluation Improves the Prognostic Values of Staging and p16 in Oropharyngeal Cancer.

The incidence of oropharyngeal cancers (OPSCCs) has continued to rise over the years, mainly due to human papillomavirus (HPV) infection. Although they were newly reclassified in the last TNM staging system, some groups still relapse and have poor prognoses. Based on their implication in oncogenesis, we investigated the density of cytotoxic and regulatory T cells, macrophages, and Langerhans cells in relation to p16 status, staging and survival of patients. Biopsies from 194 OPSCCs were analyzed for HPV by RT-qPCR and for p16 by immunohistochemistry, while CD8, FoxP3, CD68 and CD1a immunolabeling was performed in stromal (ST) and intratumoral (IT) compartments to establish optimal cutoff values for overall survival (OS). High levels of FoxP3 IT and CD1a ST positively correlated with OS and were observed in p16-positive and low-stage patients, respectively. Then, their associations with p16 and TNM were more efficient than the clinical parameters alone in describing patient survival. Using multivariate analyses, we demonstrated that the respective combination of FoxP3 or CD1a with p16 status or staging was an independent prognostic marker improving the outcome of OPSCC patients. These two combinations are significant prognostic signatures that may eventually be included in the staging stratification system to develop personalized treatment approaches.

Endothelial Cells Activated by Extracellular Histones Promote Foxp3+ Suppressive Treg Cells In Vitro.

Histones are widely recognized as pro-inflammatory mediators upon their release from the nucleus into the extracellular space. However, their impact on endothelial cell immunogenicity is unknown. Endothelial cells, Human Microvascular Endothelial cells 1 (HMEC1), have been exposed to recombinant histones in order to study their effect on the endothelial phenotype. We then studied the differentiation of CD4+-T lymphocytes subpopulations after three days of interaction with endothelial cells in vitro and observed that histone-treated endothelial cells differentiate a suppressive FoxP3+ T regulator subpopulation that expressed Human Leucocyte Antigen DR (HLA-DR) and Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA4). Toll-Like Receptor 4 (TLR4) inhibition significantly decreased the expansion of these Treg cells. Moreover, blockade of Interleukin (IL)-6 and Intercellular Adhesion Molecule (ICAM)-1 in cocultures significantly decreased the expansion of Tregs, suggesting an IL-6 and ICAM-1 dependent pathway. Thus, beyond their inflammatory effects, extracellular histones may induce an increase of immunosuppressive Treg population via their action on endothelial cells. Further studies are needed to evaluate the impact on immunosuppression of an increase of peripheral suppressive Treg via endothelial cell activation by histones in vivo.

Standardized approach for evaluating tumor infiltrating lymphocytes in canine mammary carcinoma: Spatial distribution and score as relevant features of tumor malignancy.

Neoplastic cells, through immunoediting mechanisms, can establish a state of immunosuppression to evade host immune defenses. The aims of this study were: (1) to validate a standard method for assessing tumor infiltrating lymphocytes (TILs) in canine mammary carcinoma by applying international human breast cancer guidelines; (2) to investigate if the TILs population was composed of a subset of regulatory T lymphocytes (Tregs); and (3) to evaluate the relationship between the number of TILs and Tregs and the biological behavior of the tumors. One hundred and twenty-nine canine mammary tumors were retrospectively selected for this study. Histological diagnosis, grading and histological evaluation of TILs was performed on hematoxylin and eosin-stained sections. TILs were evaluated using a three-tier semiquantitative method, previously validated in human medicine, based on the percentage of TILs (0-10%, 11-40% and 41-90%). Lymphocyte immunophenotype was confirmed by CD3 and CD79, while an anti-FoxP3 antibody was used to determine the presence of Tregs. The number of stromal TILs and invasive front TILs significantly correlated with each other (P < 0.0001) and increased with increasing histological grade (P = 0.002 and P = 0.004, respectively). A subset of TILs was composed of FOXP3+ Tregs. Stromal Tregs and invasive front Tregs were associated with stromal TILs and invasive front TILs (P = 0.03; P = 0.01 and P = 0.003; P = 0.007, respectively). In conclusion, in canine mammary carcinomas, an increased number of stromal and invasive front TILs is associated with increased malignancy and significant increase of Tregs that could lead to immunosuppression and evasion of the host immune system.

Concomitant loss of regulatory T and B cells is a distinguishing immune feature of antibody-mediated rejection in kidney transplantation.

Although considerable advances have been made in understanding the cellular effector mechanisms responsible for donor-specific antibody generation leading to antibody-mediated rejection (ABMR), the identification of cellular regulators of such immune responses is lacking. To clarify this, we used high dimensional flow cytometry to concomitantly profile and track the two major subsets of regulatory lymphocytes in blood: T regulatory (TREG) and transitional B cells in a cohort of 96 kidney transplant recipients. Additionally, we established co-culture assays to address their respective capacity to suppress antibody responses in vitro. TREG and transitional B cells were found to be potent suppressors of T follicular helper-mediated B-cell differentiation into plasmablast and antibody generation. TREG and transitional B cells were both durably expanded in patients who did not develop donor-specific antibody post-transplant. However, patients who manifested donor-specific antibody and progressed to ABMR displayed a marked and persistent numerical reduction in TREG and transitional B cells. Strikingly, specific cell clusters expressing the transcription factor T-bet were selectively depleted in both TREG and transitional B-cell compartments in patients with ABMR. Importantly, the coordinated loss of these T-bet+CXCR5+TREG and T-bet+CD21- transitional B-cell clusters was correlated with increased and inflammatory donor specific antibody responses, more extensive microvascular inflammation and a higher rate of kidney allograft loss. Thus, our study identified coordinated and persistent defects in regulatory T- and B-cell responses in patients undergoing ABMR, which may contribute to their loss of humoral immune regulation, and warrant timely therapeutic interventions to replenish and sustain TREG and transitional B cells in these patients.

Protective effect of green tea and epigallocatechin-3-gallate in a LPS-induced systemic inflammation model.

Inflammation causes severe dysregulation of organ functions, via the development of oxidative stress and inflammation damage. Polyphenol compounds found in green tea (GTE), including the most important component epigallocatechin-3-gallate (EGCG), have a great therapeutic potential. Here, protective properties of GTE and EGCG against lipopolysaccharide (LPS)-induced inflammation are explored. To this end, the effects of GTE and EGCG were studied on LPS challenged macrophages. Mice received GTE (250 mg/kg/d/p.o) or EGCG (25 mg/kg/d/i.p.) for 7 d, before the inflammation shock was provoked with a single intraperitoneal injection of LPS. The frequencies of lymphocytes CD4+, CD8+, NK1-1+ and CD4+CD25highFOXP3+ (Treg), macrophages CD11b+F480+, monocytes CD11b+Ly6Clow/high, neutrophils CD11b+Ly6G+, MDSCs CD11b+Gr-1high, M2/N2-like phenotype CD206+ and M1-like phenotype CD86+ in spleen, bone marrow and peripheral blood were determined. In vitro studies revealed that GTE and EGCG significantly attenuated LPS-induced CD80 expression and increased the CD163 expression, showing a potential to reduce the macrophage inflammatory phenotype. In vivo, GTE and EGCG inhibited the inflammation, mainly by reducing M1-macrophages and increasing Treg cells in the bone marrow. In addition, GTE and EGCG increase M2-macrophages, N2-neutrophils and Tregs in the spleen and blood and block the migration of monocytes from the bone marrow to the peripheral blood. These findings indicate that EGCG and GTE prevent LPS-induced inflammatory damage contributing to restoring the immune system homeostasis.

Immune Response to SARS-CoV-2 Vaccine in 2 Men.

INTRODUCTION: In the trials of corona virus vaccines, detailed analyses of subsets of lymphocytes were not carried out. We present perhaps the most comprehensive immunological analysis of 29 subsets of B and T cells in 2 healthy subjects receiving 2 doses of the Pfizer SARS-CoV-2 (COVID-19) vaccine. METHODS: Analyses were performed prior to vaccination, 3 weeks following the 1st dose, and 4 weeks following the 2nd dose. Total, naïve (TN), and different memory and effector subsets (TCM, TEM, and TEMRA) of CD4+ and CD8+ T cells; SARS-CoV-2 spike protein-specific tetramer+, and cytotoxic CD8+ T; subsets of T follicular cells (TFH, TFH1, TFH2, TFH1/TFH17, and TFH17); B-cell subsets (mature B cells, naive B cells, transitional B cells, marginal zone B cells, class-switched memory B cells, germinal center B cells, and CD21low B cells), and plasmablasts; and regulatory lymphocytes (CD4+ Treg, CD8+ Treg, Breg, and TFR cells) were evaluated with specific monoclonal antibodies by flow cytometry. RESULTS: A lack of COVID-19 IgG antibodies after the 1st dose in one of 2 subjects was associated with increased regulatory lymphocytes and decreased plasmablasts. Seroconversion after the 2nd dose in this subject was associated with decreased TFR cells and increased plasmablasts. In both subjects, CD4 TEM and CD8 TCM were markedly increased following the 2nd dose. TFH1 and regulatory lymphocytes were increased (except Breg) following the 1st dose. A striking increase in SARS-CoV-2-specific CD8+ T cells was observed following the 2nd dose. CONCLUSION: Our data support the need for 2nd dose of vaccine to induce strong SARS-CoV-2 CD8 T-cell specific response and generation of memory subsets of CD4+ and CD8+ T cells. Regulatory lymphocytes appear to play a role in the magnitude of response.

Immune Regulatory Cell Bias Following Alemtuzumab Treatment in Relapsing-Remitting Multiple Sclerosis.

Alemtuzumab is a highly effective treatment for relapsing-remitting multiple sclerosis. It selectively targets the CD52 antigen to induce profound lymphocyte depletion, followed by recovery of T and B cells with regulatory phenotypes. We previously showed that regulatory T cell function is restored with cellular repletion, but little is known about the functional capacity of regulatory B-cells and peripheral blood monocytes during the repletion phase. In this study (ClinicalTrials.gov ID# NCT03647722) we simultaneously analyzed the change in composition and function of both regulatory lymphocyte populations and distinct monocyte subsets in cross-sectional cohorts of MS patients prior to or 6, 12, 18, 24 or 36 months after their first course of alemtuzumab treatment. We found that the absolute number and percentage of cells with a regulatory B cell phenotype were significantly higher after treatment and were positivity correlated with regulatory T cells. In addition, B cells from treated patients secreted higher levels of IL-10 and BDNF, and inhibited the proliferation of autologous CD4+CD25- T cell targets. Though there was little change in monocytes populations overall, following the second annual course of treatment, CD14+ monocytes had a significantly increased anti-inflammatory bias in cytokine secretion patterns. These results confirmed that the immune system in alemtuzumab-treated patients is altered in favor of a regulatory milieu that involves expansion and increased functionality of multiple regulatory populations including B cells, T cells and monocytes. Here, we showed for the first time that functionally competent regulatory B cells re-appear with similar kinetics to that of regulatory T-cells, whereas the change in anti-inflammatory bias of monocytes does not occur until after the second treatment course. These findings justify future studies of all regulatory cell types following alemtuzumab treatment to reveal further insights into mechanisms of drug action, and to identify key immunological predictors of durable clinical efficacy in alemtuzumab-treated patients.

Interpreting Immunoregulation in Lung Fibrosis: A New Branch of the Immune Model.

Immunostimulation is recognized as an important contribution in lung fibrosis in some animal models and patient subsets. With this review, we illustrate an additional scenario covering the possible implication of immunoregulation during fibrogenesis. Available animal and human data indicate that pulmonary fibrosis also includes diverse and discrete immunoregulating populations comprising regulatory lymphocytes (T and B regs) and myeloid cells (immunosuppressive macrophages and myeloid-derived suppressive cells; MDSC). They are initially recruited to limit the establishment of deleterious inflammation but participate in the development of lung fibrosis by producing immunoregulatory mediators (mainly TGF-ß1 and IL-10) that directly or indirectly stimulate fibroblasts and matrix protein deposition. The existence of this silent immunoregulatory environment sustains an alternative mechanism of fibrosis that explains why in some conditions neither pro-inflammatory cytokine deficiency nor steroid and immunosuppressive therapies limit lung fibrosis. Therefore, the persistent presence of immunoregulation is an important parameter to consider for refining therapeutical strategies in lung fibrotic disorders under non-immunostimulatory conditions.

Altered eutopic endometrial T-regulatory and T-helper 17 lymphocyte ratio in women with unexplained subfertility.

PROBLEM: Perturbations in T-helper lymphocyte profiles have previously been associated with endometriosis related subfertility and conception failure. Hence a retrospective in vitro study was conducted to evaluate the relationship between T-regulatory (Treg) and T-helper 17 (Th17) lymphocytes in the eutopic endometrium of women with unexplained subfertility and correlate these profiles to their conception status. METHOD OF STUDY: Eutopic endometrial biopsies were collected during the mid-secretory phase of the menstrual cycle, from women with unexplained subfertility. These samples were evaluated immunohistochemically for Treg and Th17 lymphocytes as well as the related proinflammatory cytokine, Interleukin-17 (IL-17). These eutopic endometrial T lymphocyte subpopulations were compared to the patients' conception status in subsequent cycles. RESULTS: Though Treg cells were not indicative of conception success in subsequent cycles, patients who maintained their subfertile (no conception) status were observed to have a higher Th17 cell count in their eutopic endometrium. The ratio of Treg:Th17 cell counts was significantly correlated to patient conception status as well. These trends stayed consistent irrespective of concurrent endometriosis. CONCLUSION: Patients with a high proinflammatory Th17 lymphocyte profile and low Treg:Th17 ratio in their eutopic endometrium during the secretory phase of their menstrual cycle are more likely to not conceive in subsequent cycles.

Telomerase-based GX301 cancer vaccine in patients with metastatic castration-resistant prostate cancer: a randomized phase II trial.

Debate is around the optimal immunization regimen for cancer vaccines since too intense vaccination schedules may exhaust reactive lymphocytes. GX301 is a telomerase-based cancer vaccine whose safety and immunological effects were tested in a phase I trial applying an eight administrations schedule. Main objective of this study was to comparatively analyse safety and immunological response to three GX301 regimens in metastatic castration-resistant prostate cancer patients with response/disease stability after docetaxel chemotherapy. This was a multicentre, randomized, parallel-group, open-label trial registered with EudraCT (2014-000095-26) and ClinicalTrials.gov (NCT02293707, 2014). Ninety-eight patients were randomized to receive either eight (regimen 1), four (regimen 2) or two (regimen 3) vaccine administrations. Sixty-three patients were assessable for the primary immunological end-point. Vaccine-specific immune responses were evaluated by intracellular staining for IFN, elispot and cytotoxic assay at 90 and 180 days from baseline. No major side effects were recorded. A 54% overall immune responder rate was observed with 95% of patients showing at least one vaccine-specific immune response. Rate of immunological responders and number of immunizations were proportionally related, suggesting superiority of regimens 1 and 2 over regimen 3. Overall survival did not differ among regimens in both immunological responders and non-responders and was inversely associated (P = 0.002) with increase in the number of circulating CD8 + T regulatory cells at 180 days. These data indicate that GX301 cancer vaccine is safe and immunogenic in metastatic castration-resistant prostate cancer patients. Schedules with high number of administrations should be preferred in future studies due to their better immunological outcome.

Reduced Expression of Autophagy Markers and Expansion of Myeloid-Derived Suppressor Cells Correlate With Poor T Cell Response in Severe COVID-19 Patients.

Widespread coronavirus disease (COVID)-19 is causing pneumonia, respiratory and multiorgan failure in susceptible individuals. Dysregulated immune response marks severe COVID-19, but the immunological mechanisms driving COVID-19 pathogenesis are still largely unknown, which is hampering the development of efficient treatments. Here we analyzed ~140 parameters of cellular and humoral immune response in peripheral blood of 41 COVID-19 patients and 16 age/gender-matched healthy donors by flow-cytometry, quantitative PCR, western blot and ELISA, followed by integrated correlation analyses with ~30 common clinical and laboratory parameters. We found that lymphocytopenia in severe COVID-19 patients (n=20) strongly affects T, NK and NKT cells, but not B cells and antibody production. Unlike increased activation of ICOS-1+ CD4+ T cells in mild COVID-19 patients (n=21), T cells in severe patients showed impaired activation, low IFN-γ production and high functional exhaustion, which correlated with significantly down-regulated HLA-DR expression in monocytes, dendritic cells and B cells. The latter phenomenon was followed by lower interferon responsive factor (IRF)-8 and autophagy-related genes expressions, and the expansion of myeloid derived suppressor cells (MDSC). Intriguingly, PD-L1-, ILT-3-, and IDO-1-expressing monocytic MDSC were the dominant producers of IL-6 and IL-10, which correlated with the increased inflammation and accumulation of regulatory B and T cell subsets in severe COVID-19 patients. Overall, down-regulated IRF-8 and autophagy-related genes expression, and the expansion of MDSC subsets could play critical roles in dysregulating T cell response in COVID-19, which could have large implications in diagnostics and design of novel therapeutics for this disease.

SARS-CoV-2-Associated T-Cell Responses in the Presence of Humoral Immunodeficiency.

We report perhaps the most comprehensive study of subsets of CD4+ and CD8+ and subsets of B cells in a mild symptomatic SARS-CoV-2+ immunocompetent patient and a common variable immunodeficiency disease (CVID) patient who had normal absolute lymphocyte counts and remained negative for SARS-CoV-2 IgG antibodies. Naïve (TN), central memory (TCM), effector memory (TEM), and terminally differentiated effector memory (TEMRA) subsets of CD4+ and CD8+ T cells, subsets of T follicular helper cells (cTFH, TFH1, TFH2, TFH17, TFH1/TFH17, and TFR), CD4 Treg, CD8 Treg, mature B cells, transitional B cells, marginal zone B cells, germinal center (GC) B cells, CD21low B cells, antibody-secreting cells (plasmablasts), and Breg cells were examined in patients and age-matched controls with appropriate monoclonal antibodies and isotype controls using multicolor flow cytometry. Different patterns of abnormalities (often contrasting) were observed in the subsets of CD4+ T, CD8+ T, B-cell subsets, and regulatory lymphocytes among the immunocompetent patient and CVID patient as compared to corresponding healthy controls. Furthermore, when data were analyzed between the 2 patients, the immunocompetent patient demonstrated greater changes in various subsets as compared to the CVID patient. These data demonstrate different immunological responses to SARS-CoV-2 infection in an immunocompetent patient and the CVID patient. A marked decrease in GC B cells and plasmablasts may be responsible for failure to make SARS-CoV-2 antibodies. The lack of SARS-CoV-2 antibodies with mild clinical disease suggests an important role of T-cell response in defense against SARS-CoV-2 infection.

Dysregulated Mucosal Immunity and Associated Pathogeneses in Preterm Neonates.

Many functions of the immune system are impaired in neonates, allowing vulnerability to serious bacterial, viral and fungal infections which would otherwise not be pathogenic to mature individuals. This vulnerability is exacerbated in compromised newborns such as premature neonates and those who have undergone surgery or who require care in an intensive care unit. Higher susceptibility of preterm neonates to infections is associated with delayed immune system maturation, with deficiencies present in both the innate and adaptive immune components. Here, we review recent insights into early life immunity, and highlight features associated with compromised newborns, given the challenges of studying neonatal immunity in compromised neonates due to the transient nature of this period of life, and logistical and ethical obstacles posed by undertaking studies newborns and infants. Finally, we highlight how the unique immunological characteristics of the premature host play key roles in the pathogenesis of diseases that are unique to this population, including necrotizing enterocolitis and the associated sequalae of lung and brain injury.

Pathophysiology of Alloimmunization.

INTRODUCTION: Alloimmunization is caused by exposure to erythrocytes from a donor that expresses blood group antigens other than those of the recipient and is related to processes that alter the balance of the immune system. Knowing the pathophysiology of alloimmunization process is essential to understand clinical complications associated with this process. PATIENTS AND METHODS: From October 2016 to April 2017, irregular antibody screening was performed in 1,434 polytransfused (compatible with the ABO and D system) patients by means of agglutination techniques using erythrocytes of a known phenotype of 44 patients with a positive alloantibody screening. Non-alloimmunized (control) subjects were matched for age, gender, pathology, and treatment group with alloimmunized patients. The subsets of B, T, and Treg lymphocytes were determined by flow cytometry. RESULTS: The results of screening for alloantibodies in patients by specificity of antibodies were as follows: nonspecific (30%), followed by anti-Dia (13%), anti-e (9%), anti-S (9%), anti-I (7%), anti-K (7%), and anti-P (7%). A lower percentage of CD4+ T lymphocytes and an increase of CD8+ T lymphocytes were observed in alloimmunized patients, as well as a low CD4/CD8 ratio (0.7 vs. 1.6, p = 0.003), a higher percentage of B lymphocytes versus the control group (30 vs. 20%, p = 0.003), and a decrease of Treg CD4+ lymphocytes versus the control group (3 vs. 12 cells/µL, p = 0.043). These observations suggest that alloimmunized patients have important alterations in the number of some lymphocyte subsets that can be translated into clinical immune dysregulation. CONCLUSION: A decreased CD4/CD8 ratio, increased B lymphocytes, and Treg lymphocyte deficiency are the most significant changes observed in alloimmunized patients.

Blockade of CD73 delays glioblastoma growth by modulating the immune environment.

Immunotherapy as an approach for cancer treatment is clinically promising. CD73, which is the enzyme that produces extracellular adenosine, favors cancer progression and protects the tumor from immune surveillance. While CD73 has recently been demonstrated to be a potential target for glioma treatment, its role in regulating the inflammatory tumor microenvironment has not yet been investigated. Thus, this study explores the immunotherapeutic value of the CD73 blockade in glioblastoma. The immuno-therapeutic value of the CD73 blockade was evaluated in vivo in immunocompetent pre-clinical glioblastoma model. As such, glioblastoma-bearing rats were nasally treated for 15 days with a siRNA CD73-loaded cationic-nanoemulsion (NE-siRNA CD73R). Apoptosis was determined by flow cytometry using Annexin-V staining and cell proliferation was analyzed by Ki67 expression by immunohistochemistry. The frequencies of the CD4+, CD8+, and CD4+CD25highCD39+ (Treg) T lymphocytes; CD11b+CD45high macrophages; CD11b+CD45low-microglia; and CD206+-M2-like phenotypes, along with expression levels of CD39 and CD73 in tumor and tumor-associated immune cells, were determined using flow cytometry, while inflammatory markers associated with tumor progression were evaluated using RT-qPCR. The CD73 blockade by NE-siRNA CD73 was found to induce tumor cell apoptosis. Meanwhile, the population of Tregs, microglia, and macrophages was significantly reduced in the tumor microenvironment, though IL-6, CCL17, and CCL22 increased. The treatment selectively decreased CD73 expression in the GB cells as well as in the tumor-associated-macrophages/microglia. This study indicates that CD73 knockdown using a nanotechnological approach to perform nasal delivery of siRNA-CD73 to CNS can potentially regulate the glioblastoma immune microenvironment and delay tumor growth by inducing apoptosis.

Tumor infiltrating lymphocytes in malignant melanoma - allies or foes?

This is an overview of current problematics regarding the role of tumor infiltrating lymphocytes (TILs) in malignant melanomas. Various and often conflicting data have been published, correlating tumor type, stage, prognosis, as well as sex and age of patients. This is partly due to heterogeneity in scaling systems and unstandardized TILs grading but also due to changes of tumor-host interactions. Melanomas are an immunologically heterogeneous group with variability of TILs, where distinct gene expression patterns were found in tumors with absent, and/or non- brisk TIL grade versus brisk TIL grade. However, the presence of TILs alone appears to be inadequate for implicating them as immunologically functional. Further characterisation of TIL phenotype and function is warranted. This especially concerns, evaluation of TILs of the suppressor phenotype but rather than as a prognostic factor, more for prediction of targeted immunotherapy.

Clinical parameters and biomarkers predicting spontaneous operational tolerance after liver transplantation: a scoping review protocol.

Objective: This scoping review aims at systematically mapping reported prognostic factors for spontaneous immunosuppression (IS) free allograft tolerance (operational tolerance, OT) in non-viral hepatitis and non-autoimmune disease liver transplant (LT) recipients who are undergoing immunosuppression withdrawal (ISW). The results may inform the subsequent conduct of a systematic review with a more specific review question. Background: LT is currently the most effective treatment for end-stage liver diseases. Whereas the short-term outcomes after LT have dramatically improved over the last decades, the long-term outcomes remain unsatisfactory, mainly because of side effects of lifelong IS, such as infections, cardiovascular diseases, malignancies, and nephrotoxicity. ISW studies have shown that OT can be achieved by a subset of LT recipients and recent research has identified biomarkers of OT in these patients. However, an evidence-based selection algorithm for patients that can predictably benefit from ISW is not available to date. The planned review will, therefore, map existing knowledge on prognostic clinical parameters and biomarkers for OT. Inclusion criteria: We will consider studies that record any clinical parameter or biomarker before the initiation of ISW in paediatric or adult non-viral hepatitis and non-autoimmune disease LT recipients and analyse their possible association with ISW outcomes (OT or non-tolerance). Studies addressing the effectiveness of OT-inducing treatments will be excluded. Methods: Embase, MEDLINE, and Cochrane Library will be searched for relevant articles or conference abstracts. Full-texts of selected abstracts will be independently screened for inclusion by two reviewers. References and citing articles of included records will be screened for additional relevant records. Clinical trial registries will be searched for ongoing studies, and their investigators contacted for the sharing of unpublished data. Data from included records will be independently extracted by two reviewers using a prespecified data extraction table and presented in both tabular and narrative form.