Higher Percentage Of CD4 Research Articles

IMPS-25DIFFERENTIAL TUMOR MICROENVIRONMENTS AND RESPONSE TO IMMUNE CHECKPOINT BLOCKADE IN MOLECULAR SUBTYPES OF MURINE MEDULLOBLASTOMA

PURPOSE: Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma (MB), the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and Group 3 MB for preclinical evaluation in immunocompetent C57BL/6 mice. METHODS AND RESULTS: Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid derived suppressor cells and tumor-associated macrophages in murine SHH model tumors compared with Group 3 tumors. However, murine Group 3 tumors had higher percentages of CD8+ PD-1+ T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial Group 3 tumors compared to SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. CONCLUSIONS: This is the first immunologic characterization of preclinical models of molecular subtypes of MB, and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings provide strong evidence for distinct immunophenotypes across subtypes of MB that have important value in the clinical application of relevant immunotherapeutic strategies targeting MB.

Clinical significance of high c-MYC and low MYCBP2 expression and their association with Ikaros dysfunction in adult acute lymphoblastic leukemia.

Increased expression of c-MYC is observed in both Acute Myeloid Leukemia (AML) and T-cell Acute Lymphoblastic Leukemia (T-ALL). MYC binding protein 2 (MYCBP2) is a probable E3 ubiquitin ligase and its function in leukemia is unknown. IKZF1 deletion is associated with the development and poor outcome of ALL. Here, we observed significant high c-MYC expression and low MYCBP2 expression in adult ALL patients. Patients with high c-MYC expression and/or low MYCBP2 expression had higher WBC counts and a higher percentage of CD34+ or CD33+ cells, as well as splenomegaly, liver infiltration, higher BM blasts, and lower CR rate. Ikaros bound to the regulatory regions of c-MYC and MYCBP2, suppressed c-MYC and increased MYCBP2 expression in ALL cells. Expression of c-MYC mRNA was significantly higher in patients with IKZF1 deletion; conversely MYCBP2 mRNA expression was significantly lower in those patients. A CK2 inhibitor, which acts as an Ikaros activator, also suppressed c-MYC and increased MYCBP2 expression in an Ikaros (IKZF1) dependent manner in the ALL cells. In summary, our data indicated the correlation of high c-MYC expression, low MYCBP2 expression and high c-MYC plus low MYCBP2 expression with high-risk factors and proliferation markers in adult ALL patients. Our data also revealed an oncogenic role for an Ikaros/MYCBP2/c-MYC axis in adult ALL, providing a mechanism of target therapies that activate Ikaros in adult ALL.

Hepatic lipase deficiency produces glucose intolerance, inflammation and hepatic steatosis.

Metabolic syndrome and type 2 diabetes mellitus constitute a major problem to global health, and their incidence is increasing at an alarming rate. Non-alcoholic fatty liver disease, which affects up to 90% of obese people and nearly 70% of the overweight, is commonly associated with MetS characteristics such as obesity, insulin resistance, hypertension and dyslipidemia. In the present study, we demonstrate that hepatic lipase (HL)-inactivation in mice fed with a high-fat, high-cholesterol diet produced dyslipidemia including hypercholesterolemia, hypertriglyceridemia and increased non-esterified fatty acid levels. These changes were accompanied by glucose intolerance, pancreatic and hepatic inflammation and steatosis. In addition, compared with WT mice, HL(-/-) mice exhibited enhanced circulating MCP1 levels, monocytosis and higher percentage of CD4+Th17+ cells. Consistent with increased inflammation, livers from HL(-/-) mice had augmented activation of the stress SAPK/JNK- and p38-pathways compared with the activation levels of the kinases in livers from WT mice. Analysis of HL(-/-) and WT mice fed regular chow diet showed dyslipidemia and glucose intolerance in HL(-/-) mice without any other changes in inflammation or hepatic steatosis. Altogether, these results indicate that dyslipidemia induced by HL-deficiency in combination with a high-fat, high-cholesterol diet promotes hepatic steatosis and inflammation in mice which are, at least in part, mediated by the activation of the stress SAPK/JNK- and p38-pathways. Future studies are warranted to asses the viability of therapeutic strategies based on the modulation of these kinases to reduce hepatic steatosis associated to lipase dysfunction.

A review of the human vs. porcine female genital tract and associated immune system in the perspective of using minipigs as a model of human genital Chlamydia infection.

Sexually transmitted diseases constitute major health issues and their prevention and treatment continue to challenge the health care systems worldwide. Animal models are essential for a deeper understanding of the diseases and the development of safe and protective vaccines. Currently a good predictive non-rodent model is needed for the study of genital chlamydia in women. The pig has become an increasingly popular model for human diseases due to its close similarities to humans. The aim of this review is to compare the porcine and human female genital tract and associated immune system in the perspective of genital Chlamydia infection. The comparison of women and sows has shown that despite some gross anatomical differences, the structures and proportion of layers undergoing cyclic alterations are very similar. Reproductive hormonal cycles are closely related, only showing a slight difference in cycle length and source of luteolysing hormone. The epithelium and functional layers of the endometrium show similar cyclic changes. The immune system in pigs is very similar to that of humans, even though pigs have a higher percentage of CD4+/CD8+ double positive T cells. The genital immune system is also very similar in terms of the cyclic fluctuations in the mucosal antibody levels, but differs slightly regarding immune cell infiltration in the genital mucosa - predominantly due to the influx of neutrophils in the porcine endometrium during estrus. The vaginal flora in Göttingen Minipigs is not dominated by lactobacilli as in humans. The vaginal pH is around 7 in Göttingen Minipigs, compared to the more acidic vaginal pH around 3.5–5 in women. This review reveals important similarities between the human and porcine female reproductive tracts and proposes the pig as an advantageous supplementary model of human genital Chlamydia infection.

Circulating brain derived neurotrophic factor (BDNF) and frequency of BDNF positive T cells in peripheral blood in human ischemic stroke: Effect on outcome

The aim of this study was to measure the levels of circulating BDNF and the frequency of BDNF-producing T cells after acute ischaemic stroke. Serum BDNF levels were measured by ELISA. Flow cytometry was used to enumerate peripheral blood leukocytes that were labelled with antibodies against markers of T cells, T regulatory cells (Tregs), and intracellular BDNF. There was a slight increase in serum BDNF levels after stroke. There was no overall difference between stroke patients and controls in the frequency of CD4+ and CD8+ BDNF+ cells, although a subgroup of stroke patients showed high frequencies of these cells. However, there was an increase in the percentage of BDNF+ Treg cells in the CD4+ population in stroke patients compared to controls. Patients with high percentages of CD4+ BDNF+ Treg cells had a better outcome at 6months than those with lower levels. These groups did not differ in age, gender or initial stroke severity. Enhancement of BDNF production after stroke could be a useful means of improving neuroprotection and recovery after stroke.

Distinct Immune Responses in Resistant and Susceptible Strains of Mice during Neurovirulent Alphavirus Encephalomyelitis.

Susceptibility to alphavirus encephalomyelitis is dependent on a variety of factors, including the genetic background of the host. Neuroadapted Sindbis virus (NSV) causes uniformly fatal disease in adult C57BL/6 (B6) mice, but adult BALB/c (Bc) mice recover from infection. In B6 mice, fatal encephalomyelitis is immune mediated rather than a direct result of virus infection. To identify the immunological determinants of host susceptibility to fatal NSV-induced encephalomyelitis, we compared virus titers and immune responses in adult B6 and Bc mice infected intranasally with NSV. B6 mice had higher levels of virus replication, higher levels of type I interferon (IFN), and slower virus clearance than did Bc mice. B6 mice had more neuronal apoptosis, more severe neurologic disease, and higher mortality than Bc mice. B6 mice had more infiltration of inflammatory cells and higher levels of IL1b, IL-6, TNFa, Csf2, and CCL2 mRNAs and interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IFN-γ, and C-C motif ligand 2 (CCL2) protein in brains than Bc mice. However, Bc mice had more brain antibody at day 7 and a higher percentage of CD4(+) T cells. CD4(+) T cells in the brains of Bc mice included fewer Th17 cells and more regulatory T cells (Tregs) producing IL-10 than B6 mice, accompanied by higher levels of Il2 and Cxcl10 mRNAs. In the absence of IL-10, resistant Bc mice became susceptible to fatal encephalomyelitis after NSV infection. These studies demonstrate the importance of the immune response and its regulation in determining host survival during alphavirus encephalomyelitis. Mosquito-borne alphavirus infections are an important cause of encephalomyelitis in humans. The severity of disease is dependent both on the strain of the virus and on the age and genetic background of the host. A neurovirulent strain of Sindbis virus causes immune-mediated fatal encephalomyelitis in adult C57BL/6 mice but not in BALB/c mice. To determine the host-dependent immunological mechanisms underlying the differences in susceptibility between these two strains of mice, we compared their immune responses to infection. Resistance to fatal disease in BALB/c mice was associated with better antibody responses, more-rapid virus clearance, fewer Th17 cells, and more-potent regulatory T cell responses than occurred in susceptible C57BL/6 mice. In the absence of interleukin-10, a component of the regulatory immune response, resistant mice became susceptible to lethal disease. This study demonstrates the importance of the immune response and its regulation for host survival during alphavirus encephalomyelitis.

Changes in B and T-cell subsets and NMO-IgG/AQP-4 levels after immunoglobulins and rituximab treatment for an acute attack of neuromyelitis optica

BackgroundThere is increasing evidence supporting that neuromyelitis optica (NMO) is an inflammatory humoral mediated disorder associated with NMO-IgG/AQP-4 antibodies. However, little is known about the subsets of B cells and T cells that contribute to the pathogenesis or therapy response. ObjectivesTo describe the clinical and immunological changes associated with intravenous immunoglobulins (IV-Igs) plus rituximab (RTX) in a patient with a severe acute attack of NMO and intrathecal synthesis of NMO-IgG/AQP-4, who previously did not respond to intravenous methylprednisolone and plasma exchange. MethodsWe sequentially analysed the levels of NMO-IgG/AQP-4 by immunohistochemistry, and B and T cells subsets by multiparametric flow-cytometry, in the CSF and peripheral blood (PB), before and after IV-Igs plus RTX therapy. ResultsIn the CSF before treatment, and compared to PB, there was a higher percentage of CD4+ T cells and a lower percentage of CD8+ T cells and CD19+ B cells. After therapy, the percentage of CD4+ T cells remained high, and that of CD8+ T cells increased. The observed decrease in the percentage of CD19+ B cells was lower than in the PB. When the CSF was compared, it was found that the percentage of effector-memory and effector CD8+ T cells had increased after therapy, and that of IgM memory B cells and switched-memory B cells decreased. The observed changes paralleled the decrease of NMO-IgG/AQP-4 results to negative and the clinical improvement. ConclusionsOur findings confirm that, besides intrathecal humoral immune response against AQP4, B and T cell subsets are involved in the modulation of inflammation within and outside the central nervous system.

Lentivirus-mediated Gene Transfer in Hematopoietic Stem Cells Is Impaired in SHIV-infected, ART-treated Nonhuman Primates

Recent studies have demonstrated that genetically modified hematopoietic stem cells (HSCs) can reduce HIV viremia. We have developed an HIV/AIDS-patient model in Simian/human immunodeficiency virus (SHIV)-infected pigtailed macaques that are stably suppressed on antiretroviral therapy (ART: raltegravir, emtricitabine and tenofovir). Following SHIV infection and ART, animals undergo autologous HSC transplantation (HSCT) with lentivirally transduced cluster of differentiation (CD)34(+) cells expressing the mC46 anti-HIV fusion protein. We show that SHIV(+), ART-treated animals had very low gene marking levels after HSCT. Pretransduction CD34(+) cells contained detectable levels of all three ART drugs, likely contributing to the low gene transfer efficiency. Following HSCT recovery and the cessation of ART, plasma viremia rebounded, indicating that myeloablative total body irradiation cannot completely eliminate viral reservoirs after autologous HSCT. The kinetics of recovery following autologous HSCT in SHIV(+), ART-treated macaques paralleled those observed following transplantation of control animals. However, T-cell subset analyses demonstrated a high percentage of C-C chemokine receptor 5 (CCR5)-expressing CD4(+) T-cells after HSCT. These data suggest that an extended ART interruption time may be required for more efficient lentiviral transduction. To avoid complications associated with ART interruption in the context of high percentages of CD4(+)CCR5(+)T-cells after HSCT, the use of vector systems not impaired by the presence of residual ART may also be beneficial.

Pak2 regulates hematopoietic progenitor cell proliferation, survival, and differentiation.

p21-Activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long-term hematopoiesis and lineage commitment remain unreported. Using a conditional Pak2 knockout mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short- and long-term competitive hematopoiesis than wild-type cells, it does not affect HSC self-renewal per se. Pak2 disruption decreased the survival and proliferation of multicytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by (a) a three- to sixfold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte-monocyte progenitors in mice transplanted with Pak2-disrupted bone marrow (BM); (b)Pak2-disrupted BM and c-kit(+) cells yielded higher numbers of more mature subsets of granulocyte-monocyte colonies and polymorphonuclear neutrophils, respectively, when cultured in the presence of granulocyte-macrophage colony-stimulating factor. Pak2 disruption resulted, respectively, in decreased and increased gene expression of transcription factors JunB and c-Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte-monocyte lineage commitment. Furthermore, Pak2 disruption led to (a) higher percentage of CD4(+) CD8(+) double positive T cells and lower percentages of CD4(+) CD8(-) or CD4(-) CD8(+) single positive T cells in thymus and (b) decreased numbers of mature B cells and increased numbers of Pre-Pro B cells in BM, suggesting defects in lymphopoiesis.

Phenotypic analysis of T cells infiltrating colon cancers: Correlations with oncogenetic status

Colorectal cancers (CRC) develop in the face of an important immune system associated with the intestinal mucosal tissue. The immune response against the tumor has been proposed to affect the prognosis of patients undergoing treatment for CRC. In this study T cells infiltrating the tumor were compared with T cells populating the unaffected neighboring mucosal tissue and cells from the peripheral blood. We observed that T cells from the tumor harbor an activated phenotype, with engagement of the NKG2D pathway in CD8 T cells. We show that mucosal and tumor-infiltrating T cells are enriched in NKG2D CD4 T cells, which exhibit cytotoxic functions. Finally, T cell populations in the tumor were modified according to its oncogenetic status, with higher percentages of CD8 T cells isolated from patients with microsatellite instable tumor status.

849 Gamma-Delta T-Lymphocytes As Diagnostic Criterium in Latent Celiac Disease

Introduction Flow cytometry is a validated, easy applicable methodology for the determination of intraepithelial lymphocytes (IEL) phenotype in celiac disease (CD). An increase in Gamma-Delta IEL (CD3+TCRγδ+) is a characteristic finding in CD patients yet cut-off values for CD3+TCRγδ+ IEL in CD have not been determined. A validated cut-off value might be helpful in the establishing the diagnosis of CD, especially in those patients with minimal histological abnormalities (i.e., Marsh I) or individuals with positive serology in the absence of histological abnormalities. Methods Flow cytometric immunophenotyping was performed on IELs isolated from fresh small bowel biopsy specimens obtained from histology and serology proven CD patients (n=219) (active CD: n=92, CD in remission: n=127), healthy controls without CD (n=89) and suspected CD (n=14), the latter being defined as Marsh III and/or positive serology. Thirty-one additional patients with non-celiac villous atrophy were included as a disease control group. Follow-up data were available from thirteen CD patients. Percentages of CD3+TCRγδ+ IEL from CD patients and healthy controls were used to calculate a cut-off value for CD3+TCR γδ+ IEL in suspected CD patients using a receiver operating characteristic (ROC) curve. Results A significantly higher percentage of CD3+TCRγδ+ IEL was found in biopsy and serology proven CD patients (median 19.0%, range 1-58%) compared to healthy controls (median 6.0%, range 1-15%) (p 14% CD3+TCRγδ+ IEL might aid in a diagnosis of CD. Furthermore, Flow cytometric immunophenotyping might be helpful in cases of diagnostic doubt on CD in patients compliant to a GFD without the need for a gluten-challenge.

LBP24: MULTI-PARAMETER FLOW CYTOMETRY OF T-CELL SUBPOPULATIONS AND LINEAR MIXED EFFECTS MODEL TO CHARACTERIZE MULTIORGAN DYSFUNCTION SYNDROME AFTER MECHANICAL CIRCULATORY SUPPORT DEVICE

Aim A main cause of death in Advanced Heart Failure (AdHF) after Mechanical Circulatory Support Device (MCSD) is the Multiorgan Dysfunction Syndrome (MOD), mediated by an aberrant immune response. Since implantation of MCSD has immune modulating effects that may influence patient outcome, we hypothesized that T cell immunophenotyping would reflect assessment of patient risk before and after MCSD implant. Methods Peripheral blood was collected from 11 consented patients at days −1, 1, 3, 5 and 7 before and after MCSD surgery, and at 5 corresponding time points from 4 healthy controls. Multi-parameter flow cytometry was performed to measure CD4 and CD8 T cell maturation state as well as experienced and exhausted T cell subpopulations. Clinical risk was calculated using a modified Sequential Organ Failure Assessment (SOFA) score and the MELD-XI score. Results Multi-parameter immunophenotyping generated 34 CD4 and CD8 subpopulations. High scored MELD samples, obtained from higher risk patients, exhibited higher percentage of both effector and central memory CD8 cells (p = 0.0071 and p = 0.0001) and higher percentage of effector memory CD4 cells (p = 0.0074). There was a trend towards association with higher percentage of CD8+KLRG1+CD28 (p = 0.06) and lower %PD1+CD57+CD4+cells (p = 0.0785) compared with lower MELD scored patient samples. We further analyzed the data using stepwise and penalized regression methods. We reduced the number of predictors down to 3 T-cell subpopulations with immunophenotyping profile of chronically activated T cells: CD8+CD38+CD57+, CD4+CD38+CD57+ and CD4+CD38+KLRG1+. We used these subpopulations as the inputs to a linear mixed effect model to successfully predict the SOFA scores of patients’ samples. The overall model was found to be statistically significant (p = 0.006), and the regression coefficient for CD8+CD38+CD57 was statistically significant (p Conclussion Statistical modeling of T-cell subpopulation frequencies in peripheral blood of MOD patients can provide significant information predictive of their clinical phenotype, warranting further study for developing models to better predict patient outcomes.

CD4+ T cells apoptosis in Plasmodium vivax infection is mediated by activation of both intrinsic and extrinsic pathways.

BackgroundReduction in the number of circulating blood lymphocytes (lymphocytopaenia) has been reported during clinical episodes of malaria and is normalized after treatment with anti-malaria drugs. While this phenomenon is well established in malaria infection, the underlying mechanisms are still not fully elucidated. In the present study, the occurrence of apoptosis and its pathways in CD4+ T cells was investigated in naturally Plasmodium vivax-infected individuals from a Brazilian endemic area (Porto Velho – RO).MethodsBlood samples were collected from P. vivax-infected individuals and healthy donors. The apoptosis was characterized by cell staining with Annexin V/FITC and propidium iodide and the apoptosis-associated gene expression profile was carried out using RT2 Profiler PCR Array–Human Apoptosis. The plasma TNF level was determined by ELISA. The unpaired t-test or Mann–Whitney test was applied according to the data distribution.ResultsPlasmodium vivax-infected individuals present low number of leukocytes and lymphocytes with a higher percentage of CD4+ T cells in early and/or late apoptosis. Increased gene expression was observed for TNFRSF1B and Bid, associated with a reduction of Bcl-2, in individuals with P. vivax malaria. Furthermore, these individuals showed increased plasma levels of TNF compared to malaria-naive donors.ConclusionsThe results of the present study suggest that P. vivax infection induces apoptosis of CD4+ T cells mediated by two types of signaling: by activation of the TNFR1 death receptor (extrinsic pathway), which is amplified by Bid, and by decreased expression of the anti-apoptotic protein Bcl-2 (intrinsic pathway). The T lymphocytes apoptosis could reflect a strategy of immune evasion triggered by the parasite, enabling their persistence but also limiting the occurrence of immunopathology.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-14-5) contains supplementary material, which is available to authorized users.

Searching for prognostic factors in Common Variable Immunodeficiency based on epidemiological and immunological parameters

Searching for prognostic factors in Common Variable Immunodeficiency based on epidemiological and immunological parameters

Alpha-Synuclein Deficiency Is Associated with Defective Th2 Differentiation and Enhanced Regulatory T Cell Development

Synucleins (including α-, β- and γ-synucleins) are a group of proteins that are highly expressed in the central nervous system. Alpha-synuclein, in particular, has been implicated in the pathogenesis of neurodegenerative disorders known as synucleinopathies. The function of these proteins in other organ systems is largely unknown. Some studies have demonstrated expression of α-synuclein on peripheral blood mononuclear cells (PBMC), including B and T lymphocytes, NK cells and monocytes; and its expression has been shown to be higher in PBMCs of individuals with Parkinson’s compared to healthy controls. We have recently shown that α-synuclein-deficiency is associated with marked defect in development of mature B and T lymphocytes. In particular, we showed enhanced negative selection of developing thymic T cells in the absence α-synuclein. Furthermore, we demonstrated that α-synuclein-deficiency is associated with an impaired IgG response to T cell-dependent antigens.Here we used age and sex-matched α-synuclein knock-out (KO) and wild type (WT) mice to further investigate the lineage differentiation and activation of T cells. We found that few splenic T cells that develop in KO mice contain a higher percentage of CD8+ T cell expressing early activation markers CD69 (7.6 ± 0.09 for KO vs. 5.3 ± 0.23 for WT, p=0.005, figure 1A) and CD49d (12.97 ± 0.3 for KO vs. 7.32 ± 0.6 for WT, p=0.006, figure 1A). A similar trend was noted for CD4+ CD49d+ T cells, although the difference did not reach statistical significance (23.90 ± 3.48 for KO vs.13.23 ± 0.73 for WT, p=0.086, figure 1A). No difference was noted in the expression of late activation marker CD44, and lymph node homing marker CD62L. This was associated with significantly increased IL-2 production from KO CD4+ T cells (OD 2.70 ± 0.12 for KO vs.1.05 ± 0.39 for WT, p=0.002, figure 1B) and a trend for increased IFN-γ production from KO CD4+ T cells (OD 2.89 ± 0.33 for KO vs.2.12 ± 0.59 for WT, p=0.12) after in vitro activation with anti-CD3/anti-CD28 beads. Interestingly, In vitro activation of splenic CD4+ T cells resulted in significantly reduced IL-4 production from KO T cells (OD 0.20 ± 0.14 for KO, vs. 0.74 ± 0.31 for WT, p=0.05, Figure 2) suggesting a defective Th2 differentiation in KO CD4+ T cells.Further flow cytometric analysis of T cells showed that while thymic Foxp3+ CD4+ regulatory T cells are significantly reduced in KO mice (3.07 ±0.35 for KO vs. 5.00 ± 0.87 for WT, p=0.02, Figure 3), the percentage of splenic Foxp3+ CD4+ T cells is higher in the KO mice compared to WT mice (18.33 ± 4.90 for KO vs.10.33 ± 1.39 for WT, p=0.05, Figure 3). No difference was noted among NK cells from KO and WT mice.In summary, we demonstrate a role for α-synuclein in lineage differentiation and function of T cells. While α-synuclein-deficiency leads to a significant defect in development of mature T cells, the small population of cells that do mature, express higher levels of early activation markers, and produce higher levels of IL-2 upon antigenic stimulation. Of interest, these cells are defective in IL-4 production. Additionally, we also show that α-synuclein-deficiency is associated with a higher percentage of peripheral CD4+ Foxp3+ T cells, a finding that might be explained by higher levels of IL-2 production by α-synuclein-deficient CD4+ T cells, although a direct effect of α-synuclein on the survival of regulatory T cells cannot be excluded. The underlying mechanism for the function α-synuclein in development and function of T cells is subject of future studies. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparative Immune Phenotypic Analysis of Cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in Immune-Competent Individuals: Proportional Representation of CD8+ T-Cells but Not FoxP3+ Regulatory T-Cells Is Associated with Disease Stage

Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure. SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC). Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear. This study aimed to define whether IEC and SCC are associated with distinct immune profiles. We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest. We found that IEC lesions contain higher percentages of CD3+ T-cells than photo-damaged skin, however, the abundance of CD3−CD56+ Natural Killer (NK) cells, CD11c+HLA-DR+ conventional Dendritic Cells (cDC), BDCA-2+HLA-DR+ plasmacytoid DC (pDC), FoxP3+ Regulatory T-cells (T-reg), Vα24+Vβ11+ invariant NKT-cells, and γδ Tcells did not alter with disease stage. Within the total T-cell population, high percentages of CD4+ T-cells were associated with SCC, yet CD8+ T-cells were less abundant in SCC compared with IEC. Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8+ T-cells than IEC. We propose that differences in CD8+ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments. Our study also suggests that a high ratio of CD4+ T-cells to CD8+ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.

Uniform-sized water-in-oil vaccine formulations enhance immune response against Newcastle disease and avian influenza in chickens.

Water-in-oil (W/O) emulsion is one of the best "one-shot" delivery system for antigen to generate high and durable antibody response. Here a facile method using premixed Shirasu Porous Glass (SPG) membrane emulsification technique was employed to fabricate uniform-sized emulsion and mechanical stirring was employed to prepare non-uniform-sized emulsion as control. In release kinetics studies, uniform-sized emulsion showed low initial release, and during the followed 17 days, uniform-sized emulsions released antigen faster than emulsions of non-uniform size. In addition, vaccination studies against Newcastle disease virus (NDV) and avian Influenza virus (AIV) demonstrated that emulsion of uniform size induced higher HI antibodies and antigen-specific IgG titers. Furthermore, chickens vaccinated with emulsion of uniform size had a significantly greater ratio of CD8+ T cells to CD4+ T cells and a higher percentage of CD8+CD4+ T cells. Taken together, these results indicated that emulsion of uniform size induced a more potent immune response than emulsion prepared by mechanical stirring, and warranted the use of SPG membrane emulsification technique in generating new avian vaccines.

Immune development in late preterm neonates

Several recent studies have underlined that late preterm infants have a significantly increased risk of infection and sepsis, [1-3] mainly related to problems of adaptation from intra- to extra-uterine life of the immune defense mechanisms. Indeed, both the innate (natural, non specific) and the adaptive (acquired, specific) immune systems are incompletely developed at birth, the more preterm the neonate, the more severe and prolonged the immunodeficiency.[4,5] T lymphocytes response to mitogens is poor, and T and B lymphocytes are immature: higher percentage of CD4+ T lymphocytes and lower of CD8+ cells, with a gradual decline with age of the CD4+/CD8+ ratio, and predominant naive phenotype with elevated percentages of CD4+/CD45RA+ T cells; in addition, cytokine production is reduced and Th1-like response inadequate. The immaturity of lymphocytes and of antigen presenting cells are responsible for the marked deficiency of antibody production; also, levels of IgG are low in late preterm infants because transplacental passage from the mother mostly occurs during the last trimester of gestation; therefore, these neonates may lack the protection ensured by maternal derived pathogen-specific IgG. The inability to produce adequate amounts of hematopoietic growth factors, particularly G- and GM-CSF, and the reduced neutrophil, complement and natural killer cell activity, may further amplify the neonatal impairment of immune defenses. The combined neonatal deficiency of immunoglobulin, complement and neutrophil activity results in increased susceptibility to systemic infections from encapsulated pathogens, such as Group B Streptococcus, Staphylococci and Klebsiella, that require opsonization for efficient phagocytosis and killing. The immaturity of pattern recognition receptors (PRR) response to pathogen-associated molecular patterns (PAMP), in particular the impaired TLR4 (Toll Like Receptor) signaling, [1] may contribute to the late preterm vulnerability to Gram-negative bacteria.[6] It should be noted, however, that neonatal T cells are capable to raise type 1 and 2 immune responses upon appropriate stimulus. Neonatal immunization does not generally lead to rapid antibody responses, however, it may result in an efficient immunologic priming which can act as a basis for future responses. It is therefore possible to induce early protection by immunization at birth.[7] Finally, to mitigate detrimental consequences of immunodeficiency in late preterm infants, it is of paramount importance to maintain the mother-newborn protective immunological link by ensuring the host of protective components provided by human milk.[8]

Abstract 3030: Correlation of circulating tumor cells (CTCs) expressing stemness and EMT phenotypes with immunosuppressive cells in metastatic breast cancer patients

Abstract Background: The detection of circulating tumor cells (CTCs) in peripheral blood (PB) of breast cancer (BC) patients is an unfavourable prognostic factor; however further characterization of these cells is required to understand their biological role. The presence of stemness and epithelial-to-mesenchymal transition (EMT) markers in CTCs could be an important marker of enhanced metastatic capacity. On the other hand, myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) play an essential role in tumor-immune evasion, by suppressing the normal function of both CD4+and CD8+ T cells. Increased levels of MDSCs and Tregs have been correlated with advanced disease and poor prognosis. Recent findings in preclinical models and primary tumours indicate that immune cells can induce EMT and stemness characteristics of cancer cells; however this has not been demonstrated in circulation. The purpose of this study was to evaluate the correlation of CTCs expressing stemness and EMT phenotypes with immunosuppressive cells in BC patients. Patients and Methods: PB was obtained from 38 metastatic BC patients prior to treatment. Triple immunofluorescence was performed on PBMCs cytospin preparations using antibodies against putative markers for epithelial cells (anti-pancytokeratin, A45-B/B3), stemness (anti-ALDH1) and EMT (anti-TWIST) phenotypes. Quantification of ALDH1 expression levels and characterization of TWIST subcellular localization was performed using the ARIOL system. The frequency of different immune cell subpopulations was also evaluated in PB using flow cytometry and was correlated to the presence of distinct CTC phenotypes. Results: Cytokeratin+ CTCs were detected in 16/38 (42%) patients. Patients with detectable CTCs had a higher percentage of CD4+ Tregs compared to those without CTCs (p=0.022). Moreover, elevated levels of CD4+ Tregs were associated with detectable stem cell-like CTCs (ALDH1high) (p=0.023), while in patients with non-stem CTCs (ALDH1low/-) an increased frequency of MDSCs [CD15+CD14-HLA-DR-Lin-CD33+CD11b+] occurred (p=0.020). The detection of EMT-like CTCs was not associated with the incidence of MDSCs and Tregs. Conclusions: The detection of CTCs using ARIOL system was associated with a higher incidence of CD4+ Tregs and MDSCs in metastatic BC patients. However, different immunosuppressive cell subpopulations were correlated with phenotypically different CTCs according to EMT and stemness characteristics. These findings indicate that the immune system may be differentially involved in regulating CTCs with different biological behaviour or reversely, CTCs with different characteristics might induce specific subpopulations of immunosuppressive cells. Citation Format: Despoina Aggouraki, Maria Papadaki, Eleni Kyriaki Vetsika, Anna Koutoulaki, Athanasios Kotsakis, Galatea Kallergi, Sofia Agelaki, Dimitris Mavroudis, Vassilis Georgoulias. Correlation of circulating tumor cells (CTCs) expressing stemness and EMT phenotypes with immunosuppressive cells in metastatic breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3030. doi:10.1158/1538-7445.AM2014-3030

Intrabronchial infection of rhesus macaques with simian varicella virus results in a robust immune response in the lungs.

Varicella-zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (shingles). Primary VZV infection is believed to occur via the inhalation of virus either in respiratory droplets or from shedding varicella lesions or by direct contact with infectious vesicular fluid. However, the ensuing immune response in the lungs remains incompletely understood. We have shown that intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we performed an in-depth analysis of the host immune response to acute SVV infection in the lungs and peripheral blood. We report that acute SVV infection results in a robust innate immune response in the lungs, characterized by the production of inflammatory cytokines, chemokines, and growth factors as well as an increased frequency of plasmacytoid dendritic cells (DCs) that corresponded with alpha interferon (IFN-α) production and a rapid decrease in viral loads in the lungs. This is followed by T and B cell proliferation, antibody production, T cell differentiation, and cytokine production, which correlate with the complete cessation of viral replication. Although terminally differentiated CD8 T cells became the predominant T cell population in bronchoalveolar lavage cells, a higher percentage of CD4 T cells were SVV specific, which suggests a critical role for these cells in the resolution of primary SVV infection in the lungs. Given the homology between SVV and VZV, our data provide insight into the immune response to VZV within the lung. Although primary VZV infection occurs primarily via the respiratory route, the host response in the lungs and its contribution to the cessation of viral replication and establishment of latency remain poorly understood. The difficulty in accessing lung tissue and washes from individuals infected with VZV has hampered efforts to address this knowledge gap. SVV infection of rhesus macaques is an important model of VZV infection of humans; therefore, we utilized this animal model to gain a comprehensive view of the kinetics of the immune response to SVV in the lung and its relationship to the resolution of acute infection in respiratory tissues. These data not only advance our understanding of host immunity to VZV, a critical step in developing new vaccines, but also provide additional insight into immunity to respiratory pathogens.