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Our results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artifacts and thus ignored in data acquisition and analysis, are the result of biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell-monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be re-visited.

Malaria is an infectious disease caused by parasites from the genus Plasmodium (P. falciparum and P. vivax are responsible for 90% of all clinical cases); it is widely distributed throughout the world’s tropical and subtropical regions. The P. vivax Pv12 protein is involved in invasion, is expressed on merozoite surface and has been recognised by antibodies from individuals exposed to the disease. In this study, B- and T-cell epitopes from Pv12 were predicted and characterised to advance in the design of a peptide-based vaccine against malaria. For evaluating the humoral response of individuals exposed to natural P. vivax infection from two endemic areas in Colombia, BepiPred-1.0 software was used for selecting B-cell epitopes. B-cell epitope 39038 displayed the greatest recognition by naturally-acquired antibodies and induced an IgG2/IgG4 response. NetMHCIIpan-3.1 prediction software was used for selecting peptides having high affinity binding for HLA-DRβ1* allele lineages and this was confirmed by in-vitro binding assays. T-epitopes 39113 and 39117 triggered a memory T-cell response (Stimulation Index2) and significant cytokine production. Combining in-silico, in-vitro and functional assays, two Pv12 protein regions (containing peptides 39038, 39040, 39113 and 39117) have thus been characterised as promising vaccine candidates against P. vivax malaria. © 2018 Yepes-Perez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The interaction over time of genetic, epigenetic and environmental factors (i.e., autoimmune ecology) increases or decreases the liability an individual would have to develop an autoimmune disease (AD) depending on the misbalance between risk and protective effects. Pathogens have been the most common antecedent events studied, but multiple other environmental factors including xenobiotic chemicals, drugs, vaccines, and nutritional factors have been implicated into the development of ADs. Three main mechanisms have been offered to explain the development of autoimmunity: molecular mimicry, epitope spreading, and bystander activation. The latter is characterized by auto-reactive B and T cells that undergo activation in an antigen-independent manner, influencing the development and course of autoimmunity. Activation occurs due to a combination of an inflammatory milieu, co-signaling ligands, and interactions with neighboring cells. In this review, we will discuss the studies performed seeking to define the role of bystander activation in systemic and organ-specific ADs. In all cases, we are cognizant of individual differences between hosts and the variable latency time for clinical expression of disease, all of which have made our understanding of the etiology of loss of immune tolerance difficult and enigmatic. © 2019 Elsevier Ltd

Background: Activated phosphoinositide 3-kinase delta syndrome (APDS) 2 (p110 delta-activating mutations causing senescent T cells, lymphadenopathy, and immunodeficiency [PASLI]-R1), a recently described primary immunodeficiency, results from autosomal dominant mutations in PIK3R1, the gene encoding the regulatory subunit (p85 alpha, p55 alpha, and p50 alpha) of class IA phosphoinositide 3-kinases. Objectives: We sought to review the clinical, immunologic, and histopathologic phenotypes of APDS2 in a genetically defined international patient cohort. Methods: The medical and biological records of 36 patients with genetically diagnosed APDS2 were collected and reviewed. Results: Mutations within splice acceptor and donor sites of exon 11 of the PIK3R1 gene lead to APDS2. Recurrent upper respiratory tract infections (100%), pneumonitis (71%), and chronic lymphoproliferation (89%, including adenopathy [75%], splenomegaly [43%], and upper respiratory tract lymphoid hyperplasia [48%]) were the most common features. Growth retardation was frequently noticed (45%). Other complications were mild neurodevelopmental delay (31%); malignant diseases (28%), most of them being B-cell lymphomas; autoimmunity (17%); bronchiectasis (18%); and chronic diarrhea (24%). Decreased serum IgA and IgG levels (87%), increased IgM levels (58%), B-cell lymphopenia (88%) associated with an increased frequency of transitional B cells (93%), and decreased numbers of naive CD4 and naive CD8 cells but increased numbers of CD8 effector/memory T cells were predominant immunologic features. The majority of patients (89%) received immunoglobulin replacement; 3 patients were treated with rituximab, and 6 were treated with rapamycin initiated after diagnosis of APDS2. Five patients died from APDS2-related complications. Conclusion: APDS2 is a combined immunodeficiency with a variable clinical phenotype. Complications are frequent, such as severe bacterial and viral infections, lymphoproliferation, and lymphoma similar to APDS1/PASLI-CD. Immunoglobulin replacement therapy, rapamycin, and, likely in the near future, selective phosphoinositide 3-kinase delta inhibitors are possible treatment options. European Union's 7th RTD Framework Programme (ERC advanced grant PID-IMMUNE) - 249816 French National Research Agency (ANR) - ANR-10-IAHU-01 Centre de Reference Deficits Immunitaires Hereditaires (CEREDIH) French National Research Agency (ANR)-European Commission - ANR-15-CE15-0020 Gebert Ruf Stiftung program "Rare Diseases-New Approaches'' - GRS-046/10 European Commission - CELL-PID HEALTH-261387 Zurich Centre for Integrative Human Physiology (ZIHP) Gottfried und Julia Bangerter-Rhyner-Stiftung Rossi Stiftung European Research Council (ERC)-European Commission - 260477 European Commission - 261441 National Institute for Health Research (NIHR) Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT)-Japan Society for the Promotion of Science Ministry of Health, Labour and Welfare, Japan Ministry of Defense Japan Agency for Medical Research and Development (AMED) National Institute for Health Research-Leeds Musculoskeletal Biomedical Research Unit (and Leeds Teaching Hospitals Charitable Foundation) National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Dublin, Ireland United States Department of Health & Human Services/National Institutes of Health (NIH) - USA/NIH National Institute of Allergy & Infectious Diseases (NIAID) Postdoctoral Research Associate (PRAT) Fellowship, National Institute of General Medical Sciences(NIGMS)/NIH EU-FP7 NET4CGD UK Research & Innovation (UKRI)/Medical Research Council UK (MRC)/European Commission - MR/M012328 - MR/M012328/2 UK Research & Innovation (UKRI)/Medical Research Council UK (MRC) - MR/M012328/2 - MR/M012328/1 Institut National de la Sante et de la Recherche Medicale (Inserm) Fondation pour la Recherche Medicale - ING20130526624 la Ligue Contre le Cancer (Comite de Paris)

Introduction: Adenosine monophosphate-activated protein kinase (AMPK), known as an enzymatic complex that regulates the energetic metabolism, is emerging as a pivotal enzyme and enzymatic pathway involved in the regulation of immune homeostatic networks. It is also involved in the molecular mechanisms underlying the pathophysiology of chronic inflammatory diseases.Areas covered: AMPK is expressed in several immune cell types including macrophages, lymphocytes, neutrophils and dendritic cells, and governs a broad array of cell functions, which include cytokine production, chemotaxis, cytotoxicity, apoptosis and proliferation. Based on its wide variety of immunoregulatory actions, the AMPK system has been targeted to reveal its impact on the course of immune-related diseases, such as atherosclerosis, psoriasis, joint inflammation and inflammatory bowel diseases.Expert opinion: The identification of AMPK subunits responsible for specific anti-inflammatory actions and the understanding of the underlying molecular mechanisms will promote the generation of novel AMPK activators, endowed with improved pharmacodynamic and pharmacokinetic profiles. These new tools will aid us to utilize AMPK pathway activation in the management of acute and chronic inflammatory diseases, while minimizing potential adverse reactions related to the effects of AMPK on metabolic energy.

The balance between antiapoptotic and proapoptotic proteins of the Bcl-2 family is critical in determining the fate of T cells in response to death stimuli. Proapoptotic genes, such as bax, are generally regulated by the p53 family of transcription factors, whereas NF-kappaB subunits can activate the transcription of antiapoptotic Bcl-2 members. Here, we show that CD28 activation protects memory T cells from irradiation-induced apoptosis by both upregulating bcl-xL and inhibiting bax gene expression. We found that p73, but not p53, binds to and trans-activates the bax gene promoter in irradiated T cells. The activation of RelA/NF-kappaB subunit in CD28 costimulated T cells and its binding onto the bax gene promoter results in suppression of bax transcription and decrease in both p73 and RNA polymerase II recruitment in vivo. RelA recruitment on the bax gene promoter is also accompanied by the lost of p300 binding and the parallel appearance of histone deacetylase-1-containing complexes. These findings identify RelA/NF-kappaB as a critical regulator of T-cell survival by affecting the balance of Bcl-2 family members.

CD4+T (helper) cells migrate in huge numbers through lymphoid organs. However, little is known about traffic routes and kinetics of CD4+T-cell subsets within different organ compartments. Such information is important because there are indications that CD4+T cells may influence the function of microenvironments depending on their developmental stage. Therefore, we investigated the migration of resting (naïve), activated, and recently activated (memory) CD4+T cells through the different compartments of the spleen. Resting and recently activated CD4+T cells were separated from thoracic duct lymph and activated CD4+T cells were generated in vitro by cross-linking the T-cell receptor and CD28. The present study shows that all three CD4+T-cell subsets selectively accumulate in the T-cell zone of the spleen. However, only activated T cells induce the formation of germinal centers (GCs) and autoantibodies in rats and mice. Our results suggest that in a two-step process they first activate B cells independent of the T-cell receptor repertoire and CD40 ligand (CD154) expression. The activated B cells then form GCs whereby CD154-dependend T-cell help is needed. Thus, activated T cells may contribute to the development of autoimmune diseases by activating autoreactive B cells in an Ag-independent manner. European Journal of Immunology published by Wiley-VCH Verlag GmbH & Co. KGaA Weinheim.

CD4+T (helper) cells migrate in huge numbers through lymphoid organs. However, little is known about traffic routes and kinetics of CD4+T-cell subsets within different organ compartments. Such information is important because there are indications that CD4+T cells may influence the function of microenvironments depending on their developmental stage. Therefore, we investigated the migration of resting (naïve), activated, and recently activated (memory) CD4+T cells through the different compartments of the spleen. Resting and recently activated CD4+T cells were separated from thoracic duct lymph and activated CD4+T cells were generated in vitro by cross-linking the T-cell receptor and CD28. The present study shows that all three CD4+T-cell subsets selectively accumulate in the T-cell zone of the spleen. However, only activated T cells induce the formation of germinal centers (GCs) and autoantibodies in rats and mice. Our results suggest that in a two-step process they first activate B cells independent of the T-cell receptor repertoire and CD40 ligand (CD154) expression. The activated B cells then form GCs whereby CD154-dependend T-cell help is needed. Thus, activated T cells may contribute to the development of autoimmune diseases by activating autoreactive B cells in an Ag-independent manner. European Journal of Immunology published by Wiley-VCH Verlag GmbH & Co. KGaA Weinheim.

CD8+ T cells undergo rapid expansion during infection with intracellular pathogens, which is followed by swift and massive culling of primed CD8+ T cells. The mechanisms that govern the massive contraction and maintenance of primed CD8+ T cells are not clear. We show in this study that the transcription factor, FoxO3a, does not influence Ag presentation and the consequent expansion of CD8+ T cell response during Listeria monocytogenes infection, but plays a key role in the maintenance of memory CD8+ T cells. The effector function of primed CD8+ T cells as revealed by cytokine secretion and CD107a degranulation was not influenced by inactivation of FoxO3a. Interestingly, FoxO3a-deficient CD8+ T cells displayed reduced expression of proapoptotic molecules BIM and PUMA during the various phases of response, and underwent reduced apoptosis in comparison with wild-type cells. A higher number of memory precursor effector cells and memory subsets was detectable in FoxO3a-deficient mice compared with wild-type mice. Furthermore, FoxO3a-deficient memory CD8+ T cells upon transfer into normal or RAG1-deficient mice displayed enhanced survival. These results suggest that FoxO3a acts in a cell-intrinsic manner to regulate the survival of primed CD8+ T cells. Copyright © 2013 by The American Association of Immunologists, Inc.

Introduction: Adenosine monophosphate-activated protein kinase (AMPK), known as an enzymatic complex that regulates the energetic metabolism, is emerging as a pivotal enzyme and enzymatic pathway involved in the regulation of immune homeostatic networks. It is also involved in the molecular mechanisms underlying the pathophysiology of chronic inflammatory diseases.Areas covered: AMPK is expressed in several immune cell types including macrophages, lymphocytes, neutrophils and dendritic cells, and governs a broad array of cell functions, which include cytokine production, chemotaxis, cytotoxicity, apoptosis and proliferation. Based on its wide variety of immunoregulatory actions, the AMPK system has been targeted to reveal its impact on the course of immune-related diseases, such as atherosclerosis, psoriasis, joint inflammation and inflammatory bowel diseases.Expert opinion: The identification of AMPK subunits responsible for specific anti-inflammatory actions and the understanding of the underlying molecular mechanisms will promote the generation of novel AMPK activators, endowed with improved pharmacodynamic and pharmacokinetic profiles. These new tools will aid us to utilize AMPK pathway activation in the management of acute and chronic inflammatory diseases, while minimizing potential adverse reactions related to the effects of AMPK on metabolic energy.

Background/Purpose: Human FoxP3+T-cells are functionally heterogeneous, and can be classified into three phenotypically distinct subpopulations based on the expression levels of FoxP3 and the memory T-cell marker CD45RO. These three subpopulations can be defined as: activated suppressor TRegs (FoxP3HighCD45RO+; ASTReg), resting suppressor TRegs (FoxP3Low CD45RO-; RSTReg), and cytokine-secreting non-suppressor TRegs (FoxP3Low CD45RO+; NONTReg). This study aimed to evaluate the identity of the elevated frequency of FoxP3+T-cells in patients with granulomatosis with polyangiitis (GPA). Methods: Peripheral blood mononuclear cells were isolated from 46 GPApatients (27 ANCA-positief and 19 ANCA-negative at the time of inclusion) in remission and from 22 age- and sex-matched HCs. Expression of CD4 CD45RO, and FoxP3 was determined by flow cytometric analysis. The expression levels of FoxP3 and CD45RO were used for distinction between ASTReg, RSTReg, and NONTRegFoxP3+T-cells. Next, frequencies of intracellular production of IL-17 within the FoxP3+ cell subpopulations were analyzed by flow cytometry in peripheral blood of ANCA-positive (n=10) and ANCA-negatieve (n=9) GPApatients and matched HCs (n=12) upon ex vivo stimulation with phorbolmyristate- acetate and calcium ionophore in the presence of brefeldin A. Results: A significant increase in the frequency of FoxP3+TRegcells was observed in GPA-patients as compared with HCs. No differences were detected in RSTReg- and ASTRegcells between GPA-patients and HCs, whereas the NONTRegcells were significantly increased in GPA-patients. The distribution of RSTReg- and NONTRegcells did not differ between ANCA-negative and ANCApositive patients, whereas lower percentages of ASTRegcells were observed in ANCA-positive patients as compared to ANCA-negative patients and HCs. In addition, frequencies of IL-17+ T-cells were significantly increased within the NONTRegsubset from ANCA-positive patients in comparison with ANCA-negative patients and HCs, whereas no such difference was found between ANCA-negative patients and HCs. Conclusion: Increased FoxP3 expression in CD4+T-cells from GPA-patients is related to an increase in a subset of non-suppressive T-cells that produce higher levels of IL-17 and display low expression of FoxP3. Plasticity of CD4+T-cells in GPA points towards FoxP3+IL-17+ effector cells and decrease in suppressive TRegcells in relation to ANCA production.

Introduction: Immunotherapeutic strategies have been developed and tested in ovarian carcinoma patients, but no single approach has proven clinical effective yet. These disappointing clinical results were explained by immune escape mechanisms such as the immune suppressive pathway of the programmed death-1 (PD-1) receptor, and its ligands PD-L1 and PD-L2. Objective: We determined the prognostic importance of immune inhibitory ligands PD-L1 and PD-L2, in a well-defined homogeneous subgroup of epithelial ovarian cancer (EOC). Methods: PD-L1/L2 expression was evaluated in primary advanced serous EOC tissue of 127 patients by immunohistochemistry using tissue microarrays (TMAs). PD-L1/L2 expression, was correlated with the number of tumor infiltrating lymphocytes (TIL) and TIL-subsets data, i.e. CD8+ cytotoxic- (CTL), FoxP3+ regulatory- (Tregs) and CD45R0+ memory T-lymphocytes. Results: PD-L1 and PD-L2 expression was observed in 65 (61.9%) and 77 (73.3%) of 127 tumor samples, respectively. Prognosis was not influenced by the expression of PD-L1 or PD-L2 in advanced serous EOC patients. Moreover, PD-L1 expression did not correlate with the number of one of the TIL subsets. PD-L2 expression correlated negatively with the number of memory T-cells [odds ratio (OR), 0.2; 95% confidence interval (95% CI), 0.1-0.5; P = 0.001] and positively with the number of CTL (OR, 3.2; 95% CI, 1.1-9.5, P = 0.037). Conclusion: Despite high expression rates of PD-L1/L2, PD-Ls have no prognostic impact in advanced serous EOC. Blockade of the PD-1/PD-Ls pathway may not provide a beneficial effect for immunotherapeutic treatment of advanced serous EOC.

Introduction: Immunotherapeutic strategies have been developed and tested in ovarian carcinoma patients, but no single approach has proven clinical effective yet. These disappointing clinical results were explained by immune escape mechanisms such as the immune suppressive pathway of the programmed death-1 (PD-1) receptor, and its ligands PD-L1 and PD-L2. Objective: We determined the prognostic importance of immune inhibitory ligands PD-L1 and PD-L2, in a well-defined homogeneous subgroup of epithelial ovarian cancer (EOC). Methods: PD-L1/L2 expression was evaluated in primary advanced serous EOC tissue of 127 patients by immunohistochemistry using tissue microarrays (TMAs). PD-L1/L2 expression, was correlated with the number of tumor infiltrating lymphocytes (TIL) and TIL-subsets data, i.e. CD8+ cytotoxic- (CTL), FoxP3+ regulatory- (Tregs) and CD45R0+ memory T-lymphocytes. Results: PD-L1 and PD-L2 expression was observed in 65 (61.9%) and 77 (73.3%) of 127 tumor samples, respectively. Prognosis was not influenced by the expression of PD-L1 or PD-L2 in advanced serous EOC patients. Moreover, PD-L1 expression did not correlate with the number of one of the TIL subsets. PD-L2 expression correlated negatively with the number of memory T-cells [odds ratio (OR), 0.2; 95% confidence interval (95% CI), 0.1-0.5; P = 0.001] and positively with the number of CTL (OR, 3.2; 95% CI, 1.1-9.5, P = 0.037). Conclusion: Despite high expression rates of PD-L1/L2, PD-Ls have no prognostic impact in advanced serous EOC. Blockade of the PD-1/PD-Ls pathway may not provide a beneficial effect for immunotherapeutic treatment of advanced serous EOC.

Trypanosoma cruzi, the agent of Chagas disease contains a major cysteine proteinase, cruzipain (Cz), with an unusual carboxyl-terminal extension (C-T). We have previously reported the presence of sulfate groups in the N-linked oligosaccharide chains of this domain. In order to evaluate the immune responses to sulfated moieties on Cz, BALB/c mice were immunized with purified Cz and C-T prior and after desulfation treatment. The humoral immune response to sulfates on Cz or C-T was mainly IgG2b. Interestingly, the abolishment of IgG2b reactivity when desulfated antigens were used as immunogens demonstrates that esterified sulfate groups are absolutely required for eliciting IgG2b response to Cz. Sera from chronically T. cruzi -infected subjects with mild disease displayed higher levels of total IgG and IgG2 antibodies specific for sulfated epitopes compared with those in more severe forms of the disease. A significant reduction of C-T-specific delayed-type hypersensitivity reaction in C-T-immunized mice was observed when desulfated C-T was challenged, suggesting the involvement of sulfate groups in the generation of memory T-cell responses. Moreover, immunization with C-T in the absence of infection elicited ultrastructural abnormalities in heart tissue. Surprisingly, hearts from sulfate-depleted C-T-immunized mice did not present pathological alterations. This is the first report showing that sulfate-bearing glycoproteins from trypanosomatids are able to elicit specific humoral and cellular immune responses and appeared to be involved in the generation of heart tissue damage. These results represent a further step in the understanding of the role of Cz in the course of T. cruzi infection. © The Japanese Society for Immunology. 2008. All rights reserved. Fil:Laucella, S.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Couto, A.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Major problems in the analysis of CD4+ effector cell and regulatory T cell (Treg) populations in an activated immune system are caused by the facts that both cell types can express CD25 and that the discriminatory marker forkhead box p3 can only be analyzed in nonviable (permeabilized) cells. Here, we show that CD134 (OX40) can be used as a discriminatory marker combined with CD25 to isolate and characterize viable CD4+ effector cells and Tregs. Before and during adjuvant arthritis in rats, coexpression of CD134 and CD25 identified activated Tregs consistently, as these T cells proliferated poorly to disease-associated antigens and were suppressive in vitro and in vivo. Depending on the time of isolation and location, CD4+ T cell populations expressing CD134 or CD25 contained effector/memory T cells. Analysis of the function, phenotype, and amount of the CD4+ T cell subsets in different lymph node stations revealed spatiotemporal differences in effector cell and Treg compartments during experimental arthritis. © Society for Leukocyte Biology.

Major problems in the analysis of CD4+ effector cell and regulatory T cell (Treg) populations in an activated immune system are caused by the facts that both cell types can express CD25 and that the discriminatory marker forkhead box p3 can only be analyzed in nonviable (permeabilized) cells. Here, we show that CD134 (OX40) can be used as a discriminatory marker combined with CD25 to isolate and characterize viable CD4+ effector cells and Tregs. Before and during adjuvant arthritis in rats, coexpression of CD134 and CD25 identified activated Tregs consistently, as these T cells proliferated poorly to disease-associated antigens and were suppressive in vitro and in vivo. Depending on the time of isolation and location, CD4+ T cell populations expressing CD134 or CD25 contained effector/memory T cells. Analysis of the function, phenotype, and amount of the CD4+ T cell subsets in different lymph node stations revealed spatiotemporal differences in effector cell and Treg compartments during experimental arthritis. © Society for Leukocyte Biology.

Background/Purpose: Human FoxP3+T-cells are functionally heterogeneous, and can be classified into three phenotypically distinct subpopulations based on the expression levels of FoxP3 and the memory T-cell marker CD45RO. These three subpopulations can be defined as: activated suppressor TRegs (FoxP3HighCD45RO+; ASTReg), resting suppressor TRegs (FoxP3Low CD45RO-; RSTReg), and cytokine-secreting non-suppressor TRegs (FoxP3Low CD45RO+; NONTReg). This study aimed to evaluate the identity of the elevated frequency of FoxP3+T-cells in patients with granulomatosis with polyangiitis (GPA). Methods: Peripheral blood mononuclear cells were isolated from 46 GPApatients (27 ANCA-positief and 19 ANCA-negative at the time of inclusion) in remission and from 22 age- and sex-matched HCs. Expression of CD4 CD45RO, and FoxP3 was determined by flow cytometric analysis. The expression levels of FoxP3 and CD45RO were used for distinction between ASTReg, RSTReg, and NONTRegFoxP3+T-cells. Next, frequencies of intracellular production of IL-17 within the FoxP3+ cell subpopulations were analyzed by flow cytometry in peripheral blood of ANCA-positive (n=10) and ANCA-negatieve (n=9) GPApatients and matched HCs (n=12) upon ex vivo stimulation with phorbolmyristate- acetate and calcium ionophore in the presence of brefeldin A. Results: A significant increase in the frequency of FoxP3+TRegcells was observed in GPA-patients as compared with HCs. No differences were detected in RSTReg- and ASTRegcells between GPA-patients and HCs, whereas the NONTRegcells were significantly increased in GPA-patients. The distribution of RSTReg- and NONTRegcells did not differ between ANCA-negative and ANCApositive patients, whereas lower percentages of ASTRegcells were observed in ANCA-positive patients as compared to ANCA-negative patients and HCs. In addition, frequencies of IL-17+ T-cells were significantly increased within the NONTRegsubset from ANCA-positive patients in comparison with ANCA-negative patients and HCs, whereas no such difference was found between ANCA-negative patients and HCs. Conclusion: Increased FoxP3 expression in CD4+T-cells from GPA-patients is related to an increase in a subset of non-suppressive T-cells that produce higher levels of IL-17 and display low expression of FoxP3. Plasticity of CD4+T-cells in GPA points towards FoxP3+IL-17+ effector cells and decrease in suppressive TRegcells in relation to ANCA production.

Introduction: Immunotherapeutic strategies have been developed and tested in ovarian carcinoma patients, but no single approach has proven clinical effective yet. These disappointing clinical results were explained by immune escape mechanisms such as the immune suppressive pathway of the programmed death-1 (PD-1) receptor, and its ligands PD-L1 and PD-L2. Objective: We determined the prognostic importance of immune inhibitory ligands PD-L1 and PD-L2, in a well-defined homogeneous subgroup of epithelial ovarian cancer (EOC). Methods: PD-L1/L2 expression was evaluated in primary advanced serous EOC tissue of 127 patients by immunohistochemistry using tissue microarrays (TMAs). PD-L1/L2 expression, was correlated with the number of tumor infiltrating lymphocytes (TIL) and TIL-subsets data, i.e. CD8+ cytotoxic- (CTL), FoxP3+ regulatory- (Tregs) and CD45R0+ memory T-lymphocytes. Results: PD-L1 and PD-L2 expression was observed in 65 (61.9%) and 77 (73.3%) of 127 tumor samples, respectively. Prognosis was not influenced by the expression of PD-L1 or PD-L2 in advanced serous EOC patients. Moreover, PD-L1 expression did not correlate with the number of one of the TIL subsets. PD-L2 expression correlated negatively with the number of memory T-cells [odds ratio (OR), 0.2; 95% confidence interval (95% CI), 0.1-0.5; P = 0.001] and positively with the number of CTL (OR, 3.2; 95% CI, 1.1-9.5, P = 0.037). Conclusion: Despite high expression rates of PD-L1/L2, PD-Ls have no prognostic impact in advanced serous EOC. Blockade of the PD-1/PD-Ls pathway may not provide a beneficial effect for immunotherapeutic treatment of advanced serous EOC.

Background/Purpose: Human FoxP3+T-cells are functionally heterogeneous, and can be classified into three phenotypically distinct subpopulations based on the expression levels of FoxP3 and the memory T-cell marker CD45RO. These three subpopulations can be defined as: activated suppressor TRegs (FoxP3HighCD45RO+; ASTReg), resting suppressor TRegs (FoxP3Low CD45RO-; RSTReg), and cytokine-secreting non-suppressor TRegs (FoxP3Low CD45RO+; NONTReg). This study aimed to evaluate the identity of the elevated frequency of FoxP3+T-cells in patients with granulomatosis with polyangiitis (GPA). Methods: Peripheral blood mononuclear cells were isolated from 46 GPApatients (27 ANCA-positief and 19 ANCA-negative at the time of inclusion) in remission and from 22 age- and sex-matched HCs. Expression of CD4 CD45RO, and FoxP3 was determined by flow cytometric analysis. The expression levels of FoxP3 and CD45RO were used for distinction between ASTReg, RSTReg, and NONTRegFoxP3+T-cells. Next, frequencies of intracellular production of IL-17 within the FoxP3+ cell subpopulations were analyzed by flow cytometry in peripheral blood of ANCA-positive (n=10) and ANCA-negatieve (n=9) GPApatients and matched HCs (n=12) upon ex vivo stimulation with phorbolmyristate- acetate and calcium ionophore in the presence of brefeldin A. Results: A significant increase in the frequency of FoxP3+TRegcells was observed in GPA-patients as compared with HCs. No differences were detected in RSTReg- and ASTRegcells between GPA-patients and HCs, whereas the NONTRegcells were significantly increased in GPA-patients. The distribution of RSTReg- and NONTRegcells did not differ between ANCA-negative and ANCApositive patients, whereas lower percentages of ASTRegcells were observed in ANCA-positive patients as compared to ANCA-negative patients and HCs. In addition, frequencies of IL-17+ T-cells were significantly increased within the NONTRegsubset from ANCA-positive patients in comparison with ANCA-negative patients and HCs, whereas no such difference was found between ANCA-negative patients and HCs. Conclusion: Increased FoxP3 expression in CD4+T-cells from GPA-patients is related to an increase in a subset of non-suppressive T-cells that produce higher levels of IL-17 and display low expression of FoxP3. Plasticity of CD4+T-cells in GPA points towards FoxP3+IL-17+ effector cells and decrease in suppressive TRegcells in relation to ANCA production.