scholarly journals Differential expression of the T-cell inhibitor TIGIT in glioblastoma and MS

2020 ◽  
Vol 7 (3) ◽  
pp. e712 ◽  
Author(s):  
Liliana E. Lucca ◽  
Benjamin A. Lerner ◽  
Calvin Park ◽  
Danielle DeBartolo ◽  
Brian Harnett ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cells ◽  
Healthy Controls ◽  
Costimulatory Receptor ◽  
Healthy Donors ◽  
Circulating Lymphocytes ◽  
Immune Pathways ◽  
Novel Targets ◽  
The Brain

ObjectiveTo identify coinhibitory immune pathways important in the brain, we hypothesized that comparison of T cells in lesions from patients with MS with tumor-infiltrating T cells (TILs) from patients with glioblastoma multiforme may reveal novel targets for immunotherapy.MethodsWe collected fresh surgical resections and matched blood from patients with glioblastoma, blood and unmatched postmortem CNS tissue from patients with MS, and blood from healthy donors. The expression of TIGIT, CD226, and their shared ligand CD155 as well as PD-1 and PDL1 was assessed by both immunohistochemistry and flow cytometry.ResultsWe found that TIGIT was highly expressed on glioblastoma-infiltrating T cells, but was near-absent from MS lesions. Conversely, lymphocytic expression of PD-1/PD-L1 was comparable between the 2 diseases. Moreover, TIGIT was significantly upregulated in circulating lymphocytes of patients with glioblastoma compared with healthy controls, suggesting recirculation of TILs. Expression of CD226 was also increased in glioblastoma, but this costimulatory receptor was expressed alongside TIGIT in the majority of tumor-infiltrating T cells, suggesting functional counteraction.ConclusionsThe opposite patterns of TIGIT expression in the CNS between MS and glioblastoma reflects the divergent features of the immune response in these 2 CNS diseases. These data raise the possibility that anti-TIGIT therapy may be beneficial for patients with glioblastoma.

scholarly journals Platelet Desialylation As a Predictive Marker in Childhood Immune Thrombocytopenia (ITP)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 221-221
Author(s):  
Leona Raskova Kafkova ◽  
Diana Brokesova ◽  
Zbynek Novak ◽  
Milan Raska ◽  
Dagmar Pospisilova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cells ◽  
Transcription Factors ◽  
T Cell ◽  
Immune Thrombocytopenia ◽  
Proper Function ◽  
Healthy Controls ◽  
Platelet Surface ◽  
Surface Bond

Background and aim: Immune thrombocytopenia (ITP) is the most common bleeding condition in children. Its prognosis is mostly superior, however, severe refractory disease remains diagnostic and therapeutic challenge. Low platelet counts (<100× 109/L) are associated with increased platelet clearance by two parallel mechanisms: classical antibody-mediated pathway and a novel lectin-carbohydrate mediated pathway. The latter is based on platelet desialylation, where terminal sialic acids are cleaved from glycoconjugates, mainly glycoproteins (GPs), on the platelet surface. The loss of sialic acid enhances bond of the penultimate β-galactose to asialoglycoprotein receptors (ASGPRs, also called Ashwell-Morell receptors) on hepatocytes. Desialylated platelets are then captured and phagocytosed by ASGPR-expressing hepatocytes. Desialylation has been shown to be responsible for platelet destruction in many contexts, e.g., infection-related thrombocytopenia or clearance of senescent platelets. Loss of T-cell tolerance is another underlying mechanism in ITP; CD8+ regulatory T cells (Tregs) are able to inhibit overactive immune response and maintain immune homeostasis. Forkhead box P3 (FOXP3) and GATA3 are transcription factors crucial for development and proper function of Tregs limiting the Th2-type inflammatory response. Our aims were to distinguish contribution of the mentioned processes to ITP development and to characterize immune response in children with ITP during the course of disease (diagnosis, ongoing therapy, remission, refractory/persistent ITP). Patients and Methods: We examined 30 samples from 20 children with ITP (12 males, 8 females, age 3-17 years; 3 acute ITP, 17 chronic ITP) and 10 healthy controls (age 4-15). The degree of desialylation was determined by flow cytometry using FITC-labeled Ricinus communis agglutinin (RCA-I) specific for terminal galactose or N-acetylgalactosamine. Expression of platelet surface markers was given quantitatively as mean fluorescence intensity (MFI). Presence of platelet surface-bond antibodies (IgG, IgA and IgM) was examined by flow cytometry. Subpopulations of CD4+ and CD8+ T-cells were characterized based on intracellular expression of transcription factors T-bet (Th1 cells), GATA3 (Th2 cells), ROR gamma T (Th17 cells) and FOXP3 (for Tregs) using multicolor flow cytometry. Results: Patients with ITP showed significant increase in RCA-I reactivity in comparison with healthy controls (p<0.001). Patients with newly diagnosed ITP showed the most aberrant sialylation (i.e., maximum desialylation) of platelet surface proteins. A decrease in desialylation intensity was noticeable as soon as at three days after therapy initiation. Sialylation levels returned to normal after one month of successful treatment and were similar to healthy controls in children with ITP remission. Platelet surface-bond immunoglobulins were increased in 10 (50%) patients independently on their sialylation level. We observed significant changes in T-cell subpopulations in ITP: T lymphocytes producing T-bet were decreased within both CD4+ and CD8+ populations. Percentage of CD4+ cells expressing ROR gamma T was also reduced. Proportions of cells expressing FOXP3 and GATA3 were decreased within the CD8+ but not within the CD4+ population. Conclusion: Our results highlight the importance of Fc-independent hepatic platelet clearance in ITP. Interindividual differences in ITP pathophysiology are reflected by treatment response and may improve therapeutic management and prognostication. E.g., intravenous immunoglobulins or splenectomy will be ineffective in patients with prevalent Fc-independent mechanisms, and contrarily, possibilities for novel targeted treatment (neuraminidase inhibitors) arise. Better understanding of immune-mediated processes involved in ITP pathogenesis may reduce adverse effects of immunosuppressive therapy and considerably improve quality of life in patients with ITP. Supported by: MH CZ - DRO (FNOl, 00098892), Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868) and Ministry of Education, Youth and Sports OPVVV CEREBIT CZ.02.1.01/0.0/0.0/16_025/0007397. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential Expression of the T cell Inhibitor TIGIT in Glioblastoma and Multiple Sclerosis

2019 ◽  
Author(s):  
Liliana E. Lucca ◽  
Benjamin A. Lerner ◽  
Danielle DeBartolo ◽  
Calvin Park ◽  
Gerald Ponath ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
Differential Expression ◽  
Peripheral Lymphocytes ◽  
Immune Pathways ◽  
Novel Targets ◽  
The Brain

AbstractTo identify co-inhibitory immune pathways important in the brain, we hypothesized that comparison of T cells in lesions from patients with MS with tumor infiltrating T cells (TILs) from patients with GBM may reveal novel targets for immunotherapy. Focusing on PD-1 and TIGIT, we found that TIGIT and its ligand CD155 were highly expressed on GBM TILs but were near-absent in MS lesions, while lymphocytic expression of PD-1/PDL-1 was comparable. TIGIT was also upregulated in peripheral lymphocytes in GBM, suggesting recirculation of TILs. These data raise the possibility that anti-TIGIT therapy may be beneficial for patients with glioblastoma.

scholarly journals Insufficient Phosphorylation of STAT5 in Tregs Inhibits the Expression of BLIMP-1, Leading to a Lack of Tregs in Pediatric Aplastic Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2183-2183
Author(s):  
Lifen Huang ◽  
Junbin Huang ◽  
Chengming Zhu ◽  
Hongman Xue ◽  
Chi Kong Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Inflammatory Cytokines ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Healthy Controls ◽  
Healthy Donors ◽  
Ifn Γ ◽  
Low Expression

Abstract Aplastic anemia (AA) is a group of bone marrow failure diseases characterized by three-line blood cell reduction and decreased myeloproliferation. It is believed that T cell immune disorder is the leading cause of the disease, especially the number and functional damage of regulatory T cells (Tregs). BLIMP-1 is a transcription factor encoded by PRDM1 gene, which is indispensable for Tregs. The expression of BLIMP-1 is mainly induced by the IL-2/STAT5 signaling pathway. However, the level of phosphorylation of STAT5 and the expression of BLIMP-1 in Tregs from patients with AA has not been revealed, and the mechanism of Tregs damage in AA has not yet been clarified. In the present study, we collected peripheral blood from 10 newly diagnosed AA children and 10 age-matched healthy donors. We observed that the ratio of Tregs/lymphocytes and Tregs/CD4 + T cells decreased significantly in AA patients, compared with healthy controls by flow cytometry. In addition, we found significantly elevated levels of inflammatory cytokines IL-2, IL-6, and IFN-γ, but decreased levels of anti-inflammatory cytokines IL-10 and TGF-β in plasma of children with AA, compared with healthy controls. Quantitative real-time PCR showed decreased transcriptional level of BLIMP-1 in peripheral blood mononuclear cells (PBMC) from children with AA, compared with healthy donors. We used flow cytometry to detect the protein level of BLIMP-1 in Tregs and found that the level of BLIMP-1 in Tregs in the peripheral blood of children with AA was significantly lower than that of healthy donors. The correlation analysis showed that the percentage of BLIMP-1 + Tregs was positively correlated with the ratio of Tregs/CD4 + T cells (r=0.829, p＜0.001), the plasma level of IL-10 (r=0.492, p=0.027), and TGF-β (r=0.482, p=0.030), suggesting that low expression level of BLIMP-1 in Tregs may lead to decreased number of Tregs in peripheral blood and declined levels of IL-10 and TGF-β in children with AA. When stimulated IL-2, the level of pSTAT5 in CD4 + T cells of children with AA was significantly reduced compared with that of healthy donors. The level of pSTAT5 in CD4 + T cells was also positively correlated with the ratio of Tregs/CD4 + T cells (r= 0.575, p= 0.008) and the expression of BLIMP-1 in Tregs (r=0.693, p＜0.0001)，suggesting that STAT5 signal is poorly activated in pediatric AA, and it may be an important cause for the low expression of BLIMP-1 in Tregs and the decrease in the number of Tregs in children with AA. Furthermore, we constructed an AA mouse model by co-administering IFN-γ and busulfan for 10 consecutive days. These mice exhibited decreased ratio of Tregs/CD4 +T cells in the spleen and lower BLIMP-1 in Tregs, compared with controls. Also, we isolated Tregs with immunomagnetic beads from spleens of mice and observed that the level of IL-2-stimulated pSTAT5 was lower in isolated Tregs from AA mice than controls. These phenotypes were partially rescued by intervention of IL-2-JES6-1, an antibody complex tends to promote the proliferation of Tregs in mice, while inhibiting the proliferation of effector T cells. IL-2-JES6-1 increased the level of pSTAT5 and the expression of BLIMP-1 in Tregs from spleen of the AA mice, and elevated the ratio of Tregs/CD4 + T cells in the spleen. In conclusion, we found that Tregs from AA patients have impaired phosphorylation of STAT5 and insufficient expression of BLIMP-1, which may contribute to the pathogenesis of AA. Disclosures No relevant conflicts of interest to declare.

scholarly journals POS0776 CORRELATION OF PERIPHERAL CD4+GRANZB+CTLS WITH DISEASE SEVERITY IN PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 641.3-642
Author(s):  
Q. Wang ◽  
L. Gu ◽  
M. Zhang
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Sjögren’S Syndrome ◽  
Mhc Class Ii ◽  
Sjögren's Syndrome ◽  
Sjogren’S Syndrome ◽  
Sjogren's Syndrome ◽  
Healthy Controls ◽  
Extraglandular Manifestations ◽  
Sjögren‘S Syndrome

Background:Sjögren’s syndrome (SS) is a chronic autoimmune disorder. The major histopathologic lesion of it is a focal lymphocytic infiltrate around ductal and acinar epithelial cells, which include a majority of CD4+T. Several studies have shown that the epithelial cells in SS present diverse phenomena, such as MHC class II overexpression. CD4+T cells with cytotoxic activity (CD4 CTL) have been detected in various immune responses. They are characterized by their ability to secrete perforin and granzyme B to kill the target cells in an MHC class II-restricted fashion.Objectives:So this study was to investigate the correlation of peripheral CD4+GranzB+CTLs with disease severity and organ involvement in patients with primary Sjögren’s syndrome.Methods:We recruited 116 pSS patients and 46 healthy controls using flow cytometry to examine proportion of CD4+GranzB+CTLs in their peripheral blood, and immunofluorescence to test the expression of CD4+GranzB+CTLs in labial gland. The correlations of CD4+GranzB+CTLs and the relevant clinical data were analyzed.Results:We analyzed the percentage of CD4+GranzB+cytotoxic T cells in peripheral blood mononuclear cells (PBMCs) by flow cytometry. Frequency of peripheral CD4+GranzB+CTLs were measured in 116 patients with pSS and 46 healthy controls matched for age and sex. The percentage of CD4+GranzB+CTLs were significantly up-regulated in pSS patients than healthy controls (7.1%±4.9% vs 3.1%±1.9%, p <0.0001) and positive correlation with ESSDAI in pSS patients(r = 0.6332, p<0.001). The percentage of CD4+GranzB+CTLs were markedly higher in pSS patients with extraglandular manifestations. Moreover, CD4+GranzB+CTLs were observed in the lymphocytic foci and periductal areas of the LSGs and were elevated with increased foci index (FI). After excluding the other risk factors associated with pSS, CD4+GranzB+CTLs were still related to ESSDIA and extraglandular manifestations independently(p<0.05). ROC curve analysis indicated that the area under the curve (AUC) of CD4+GranzB+CTLs was 0.796 to predict the activity of pSS, and 0.851 to presume extraglandular manifestations. The best diagnostic cut-off point was 4.865 for pSS patients.Conclusion:In this study, We provide new evidence indicating involvement of CD4+GranzB+CTLs over activation in the disease pathophysiology of pSS, which may serve as a new biomarker to evaluate the activity and severity of pSS.References:[1]Takeuchi A, Saito T. Front Immunol. (2017) 23:194.[2]Brown DM, et al. Front Immunol. (2016) 9:93.[3]Polihronis M, et al. Clin Exp Immunol. (1998) 114:485-90.[4]Xanthou G, et al. Clin Exp Immunol. (1999) 118:154-63.[5]Maehara T, et al. Ann Rheum Dis. (2017) 76:377-385.[6]Goules AV, et al. Clin Immunol. (2017) 182:30-40.[7]Hashimoto K, et al. Proc Natl Acad Sci U S A. (2019) 116:24242-24251.[8]Croia C, et al. Arthritis Rheumatol. (2014) 66:2545-57.[9]Schmidt D,et al. J Clin Invest. (1996) 97:2027–37.[10]Pandya JM, et al. Arthritis Rheum. (2010) 62:3457–66.[11]Moosig F, et al. Clin Exp Immunol. (1998) 114:113–8.[12]Peeters LM, et al. Front Immunol. (2017) 20:1160.Table 1.Multivariate analysis of CD4+GranzB+CTLs influenced by pSS-related factorsregression coefficientstandard errort-statisticsp value95%CICD8+GranzB+CTLs(%)0.1440.0334.3346.9E-50.077, 0.211ESSDAI0.2560.1222.0950.0410.011, 0.502extraglandular manifestations2.6121.2682.0590.0450.065, 5.158Figure 1.Receiver operating characteristic (ROC) curve of the frequency of CD4+GranzB+CTLs to predict ESSDAI and extraglandular manifestations responseDisclosure of Interests:None declared

scholarly journals Early Response of CD8+ T Cells in COVID-19 Patients

2021 ◽  
Vol 11 (12) ◽  
pp. 1291
Author(s):  
Deni Ramljak ◽  
Martina Vukoja ◽  
Marina Curlin ◽  
Katarina Vukojevic ◽  
Maja Barbaric ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
T Cells ◽  
Mrna Expression ◽  
Cd8 T Cells ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Effector Memory ◽  
Healthy Controls ◽  
Ifn Γ

Healthy and controlled immune response in COVID-19 is crucial for mild forms of the disease. Although CD8+ T cells play important role in this response, there is still a lack of studies showing the gene expression profiles in those cells at the beginning of the disease as potential predictors of more severe forms after the first week. We investigated a proportion of different subpopulations of CD8+ T cells and their gene expression patterns for cytotoxic proteins (perforin-1 (PRF1), granulysin (GNLY), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK)), cytokine interferon-γ (IFN-γ), and apoptotic protein Fas ligand (FASL) in CD8+ T cells from peripheral blood in first weeks of SARS-CoV-2 infection. Sixteen COVID-19 patients and nine healthy controls were included. The absolute counts of total lymphocytes (p = 0.007), CD3+ (p = 0.05), and CD8+ T cells (p = 0.01) in COVID-19 patients were significantly decreased compared to healthy controls. In COVID-19 patients in CD8+ T cell compartment, we observed lower frequency effector memory 1 (EM1) (p = 0.06) and effector memory 4 (EM4) (p < 0.001) CD8+ T cells. Higher mRNA expression of PRF1 (p = 0.05) and lower mRNA expression of FASL (p = 0.05) at the fifth day of the disease were found in COVID-19 patients compared to healthy controls. mRNA expression of PRF1 (p < 0.001) and IFN-γ (p < 0.001) was significantly downregulated in the first week of disease in COVID-19 patients who progressed to moderate and severe forms after the first week, compared to patients with mild symptoms during the entire disease course. GZMK (p < 0.01) and FASL (p < 0.01) mRNA expression was downregulated in all COVID-19 patients compared to healthy controls. Our results can lead to a better understanding of the inappropriate immune response of CD8+ T cells in SARS-CoV2 with the faster progression of the disease.

scholarly journals Different Profile of Interleukin-10 Production in Circulating T Cells from Atopic Asthmatics Compared with Healthy Subjects

2004 ◽  
Vol 11 (1) ◽  
pp. 33-38 ◽  
Author(s):  
K Matsumoto ◽  
S Narita ◽  
T Rerecich ◽  
DP Snider ◽  
PM O'Byrne
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Healthy Subjects ◽  
Mononuclear Cells ◽  
Interleukin 10 ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Circulating T Cells ◽  
Normal Controls

BACKGROUND:Interleukin (IL)-10 is a pleiotropic cytokine released from various cells, including T cells. Although IL-10 is suggested to inhibit allergic responses, its role in asthma remains uncertain. The purpose of the present study was to compare the profile of IL-10 in circulating T cells from stable atopic asthmatics, atopic nonasthmatics and healthy controls.METHODS:Peripheral blood mononuclear cells were isolated, stained with anti-CD3 and CD4/CD8 antibodies, and then processed for intracellular IL-10 detection by flow cytometry.RESULTS:A kinetic study in healthy controls showed that stimulation with phorbol 12-myristate 13-acetate and ionomycin significantly increased the frequencies of IL-10-producing CD3+, CD4+and CD8+cells. Without stimulation, the frequencies of IL-10-producing CD3+, CD4+and CD8+cells were significantly higher in asthmatics than in healthy controls, while a similar trend was observed in atopic nonasthmatics. Stimulation for 24 h significantly increased IL-10-producing CD3+, CD4+and CD8+cells in healthy controls and atopic nonasthmatics, but not in asthmatics.CONCLUSIONS:The frequency of IL-10-producing T cells is increased in the circulation of stable atopic asthmatics compared with normal controls. The lack of enhancement in their frequency by phorbol 12-myristate 13-acetate and ionomycin in asthmatics suggests that the circulating T cells of asthmatic subjects are maximally stimulated with regards to IL-10 production; alternatively, IL-10 production by T cells from asthmatics may be regulated differently than T cells from other subjects.

scholarly journals Zika virus infects human blood mononuclear cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Carolina V. Messias ◽  
Julia P. Lemos ◽  
Daniela P. Cunha ◽  
Zilton Vasconcelos ◽  
Lidiane M. S. Raphael ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nervous System ◽  
Zika Virus ◽  
Mononuclear Cells ◽  
Health Concern ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Blood Mononuclear Cells ◽  
The Central Nervous System

Abstract Background Zika virus (ZIKV) infection gained public health concern after the 2015 outbreak in Brazil, when microcephaly rates increased in babies born from infected mothers. It was demonstrated that ZIKV causes a congenital Zika virus syndrome, including various alterations in the development of the central nervous system. Although the infection of cells from the nervous system has been well documented, less is known in respect of ZIKV ability to infect immune cells. Herein, we investigated if peripheral blood mononuclear cells (PBMCs), freshly-isolated from healthy donors, could be infected by ZIKV. Methods PBMCs from healthy donors were isolated and cultured in medium with ZIKV strain Rio-U1 (MOI = 0.1). Infection was analyzed by RT-qPCR and flow cytometry. Results We detected the ZIKV RNA in PBMCs from all donors by RT-qPCR analysis. The detection of viral antigens by flow cytometry revealed that PBMC from more than 50% the donors were infected by ZIKV, with CD3+CD4+ T cells, CD3−CD19+ B cells and CD3+CD8+ T cells being, respectively, the most frequently infected subpopulations, followed by CD14+ monocytes. Additionally, we observed high variability in PBMC infection rates among different donors, either by numbers or type infected cells. Conclusions These findings raise the hypothesis that PBMCs can act as a reservoir of the virus, which may facilitate viral dissemination to different organs, including immune-privileged sites.

Iron Overload Diminishes the Effectiveness of the Innate Immune Response in Thalassemia Major: a Possible Mechanism for Increased Infection Risk.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4071-4071
Author(s):  
Patrick B Walter ◽  
Paul R Harmatz ◽  
Annie Higa ◽  
David Killilea ◽  
Nancy Sweeters ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Iron Overload ◽  
Board Of Directors ◽  
Innate Immune Response ◽  
Research Funding ◽  
Innate Immune ◽  
Healthy Controls ◽  
Advisory Committees ◽  
Fluorescent Intensity

Abstract Abstract 4071 Poster Board III-1006 Introduction Infection is the second most common cause of death in thalassemia. The innate immune system provides a first line of defense against infection and specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) are important for transducing the signal for bacterial Lipopolysaccharide (LPS), resulting not only in cytokine production, but also in the control of extracellular iron levels through production of neutrophil gelatinase associated Lipocalin (NGAL). However, the exact role that NGAL plays and the expression level of PRRs are unknown in thalassemia. Thus, the goal in these studies is to investigate the relationship of iron overload to the innate immune cell expression of PRRs and NGAL in thalassemia. Patients and Methods Fifteen transfusion dependent thalassemia patients (11 – 29 yrs old) participating in the combination trial of deferasirox (an oral iron chelator) and deferoxamine were enrolled (Novartis sponsored CICL670AUS24T). Fasting blood samples were obtained i) at baseline after a 72 hr washout of chelator, and ii) at 6 and 12 months on study. Five healthy controls (13 - 18 yrs old) were also enrolled. Fresh monocytes were isolated using antibody-linked magnetic microbeads (Miltenyi Biotec Inc). Highly enriched populations of CD14+ monocytes were verified by flow cytometry. The expression of TLR4, also examined by flow cytometry is reported as the mean fluorescent intensity (MFI). In patients with thalassemia, liver iron concentration (LIC) was analyzed by biomagnetic susceptibility (“SQUID”, Ferritometer®). The plasma levels of NGAL were analyzed by ELISA. Results At baseline the expression of monocyte TLR4 (mean 18.8 ± 3.5 MFI) was reduced 30% compared to the healthy controls (mean 26.9 ± 7.6 MFI, p<0.05). The expression of TLR4 over the follow-up period of 52 weeks in patients receiving intensive combination chelator therapy significantly increased 27% / year (7 MFI / year, p=0.005). Interestingly the expression of monocyte TLR4 was negatively correlated with LIC (r=-0.6, p=0.04). Finally, thalassemia patients at baseline have significantly higher levels of NGAL (80 ± 20 ng/ml) compared to controls (42 ± 15 ng/ml, p=0.01). Conclusions These preliminary studies support the hypothesis that iron burden has a negative impact on the innate immune response in thalassemia as demonstrated by the decreased expression of TLR4. After intensive chelation, the levels of TLR4 increased, indicating that decreased iron overload with chelation may improve innate immune responsiveness. Finally, the iron transport protein NGAL is significantly elevated in thalassemia possibly acting to prevent essential iron uptake by pathogenic bacteria. Disclosures: Harmatz: Novartis: Research Funding; Apotex : Membership on an entity's Board of Directors or advisory committees; Ferrokin: Membership on an entity's Board of Directors or advisory committees. Vichinsky:Novartis: Consultancy, Research Funding.

CD4+ctla-4+ t Lymphocytes Inversely Correlate With CD4+CD127+ T Cells In Brazilian Patients With Classical Hodgkin Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4240-4240
Author(s):  
Joyce M. K. Silva ◽  
Adriana M. Damasco Penna ◽  
Maria Mirtes Sales ◽  
Elma Maria Chaves ◽  
Priscilla Brito Silva ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Lymphocytes ◽  
Hodgkin Lymphoma ◽  
Cd4 T Cells ◽  
Surface Expression ◽  
Post Treatment ◽  
Healthy Controls ◽  
Classical Hodgkin Lymphoma ◽  
Epstein Barr

Abstract Introduction CD4+CTLA-4+ T lymphocytes has long been recognized as regulatory T cells, potentially decreasing antitumor immune response. Augmentation of the immune response via blockade of CTLA-4 has shown an improvement in survival for patients with metastatic melanoma, which prompted the Food and Drug Administration (FDA) approval of the CTLA-4 function blocking antibody Ipilimumab for this disease. CD4+CD127+ T lymphocytes also participate in immune homeostasis and T-cell development. The increased expression of this marker on CD4+ T cells is associated with a effector phenotype. CD127-mediated signaling in human leukemia T-cells that may be of therapeutic value, namely regarding the potential use of PI3K and mTOR pharmacological inhibitors. Increased frequencies of regulatory CD4+ cells, together with decreased effector CD4+ cells in the tumor microenvironment and peripheral blood have been proposed as one of the mechanisms for the immunosuppression state observed in classical Hodgkin lymphoma (cHL) patients. However, little is known about CD4+ T cells subsets in patients with classical Hodgkin lymphoma (cHL) and how treatment can modify these cells. Objective The aim of the study was to evaluate the surface expression of CTLA-4 and CD127 on CD4+ T cells in peripheral blood mononuclear cells (PBMC) of patients with classical Hodgkin lymphoma (cHL) at diagnosis and post-treatment and correlate these findings with clinical and epidemiological aspects. Material and Methods This is an open multicentric study and, so far, we included 54 patients from december 2009 to July 2013. Thirty-four patients have completed therapy until July 2012 and were included in this study. Blood was drawn at diagnosis and post-treatment (1 to 4 months after completion of therapy). The T cell phenotype was evaluated by flow cytometry using CD3, CD4, CD8, CTLA-4 and CD127 and correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. Nineteen healthy blood donors volunteers were recruited as controls. In this study, only cHL patients whose histology could be confirmed and Epstein-Barr (EBV) association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. Results From the 34 cHL patients recruited for this study, 17 (50%) were male, 16 (47%) had Epstein-Barr virus (EBV) related cHL, 27 (79%) patients presented with B symptoms and 18 (53%) patients had advanced diseases at diagnosis. The percentage of CD4+ T cells with CTLA-4 surface expression was significantly increased in patients with cHL at diagnosis compared with healthy controls (median 8.7 (0.8 - 30.3) vs 2.5 (0.7 - 11.2); P<0.001). Additionally, CD4+CTLA-4+ T lymphocytes significantly decreased following treatment (8.7 (0.8 - 30.3) vs 3.9 (0.8 - 10.3); p=0.01), with values similar to healthy controls (3.9 vs 2.5; p=0.42). By contrast, CD4+CD127+ T lymphocytes were decreased at diagnosis, with values increasing after therapy (41.2 (3.3 – 75.7) vs 54.9 (17.1 – 81.3); p=0.002), similar to healthy controls (54.9 (17.1 – 81.3) vs 58.2 (41.2 – 89.8); p=0.21). The expression of CD127 on CD4+ T cells negative correlated with the expression of CTLA-4 (p<0.001). In this study, these CD4+ T cells subpopulations were neither associated with treatment response nor relapse. The frequencies of these cells were not correlated with age, gender, disease stage, erythrocyte sedimentation rate (ESR), albumin levels and EBV status. Conclusions In this study we showed a negative correlation between CTLA-4 expression on CD4+ T cells with the expression of CD127 at diagnosis of patients with cHL. These results suggest a role of CTLA-4 and CD127 on Hodgkin lymphomagenesis, possibly negatively regulating host anti-tumor immune response. Further studies investigating these CD4+ T lymphocytes subpopulations with functional assays are warranted. The promising immunotherapy regimen targeting CTLA-4 and the use of drugs that alter CD127 signaling might be beneficial in classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

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