A high-resolution mass spectrometry based proteomic dataset of human regulatory T cells

Regulatory T cells (Tregs) play a core role in maintaining immune tolerance, homeostasis, and host health. High-resolution analysis of the Treg proteome is required to identify enriched biological processes and pathways distinct to this important immune cell lineage. We present a comprehensive proteomic dataset of Tregs paired with conventional CD4+ (Conv CD4+) T cells in healthy individuals. Tregs and Conv CD4+ T cells were sorted to high purity using dual magnetic bead-based and flow cytometry-based methodologies. Proteins were trypsin-digested and analysed using label-free data-dependent acquisition mass spectrometry (DDA-MS) followed by label free quantitation (LFQ) proteomics analysis using MaxQuant software. Approximately 4,000 T cell proteins were identified with a 1% false discovery rate, of which approximately 2,800 proteins were consistently identified and quantified in all the samples. Finally, flow cytometry with a monoclonal antibody was used to validate the elevated abundance of the protein phosphatase CD148 in Tregs. This proteomic dataset serves as a reference point for future mechanistic and clinical T cell immunology and identifies receptors, processes, and pathways distinct to Tregs. Collectively, these data will lead to a better understanding of Treg immunophysiology and potentially reveal novel leads for therapeutics seeking Treg regulation

TNF-α Is Involved in the Abnormal Thymocyte Migration during Experimental Trypanosoma cruzi Infection and Favors the Export of Immature Cells

Previous studies revealed a significant production of inflammatory cytokines together with severe thymic atrophy and thymocyte migratory disturbances during experimental Chagas disease. Migratory activity of thymocytes and mature T cells seem to be finely tuned by cytokines, chemokines and extracellular matrix (ECM) components. Systemic TNF-α is enhanced during infection and appears to be crucial in the response against the parasite. However, it also seems to be involved in disease pathology, since it is implicated in the arrival of T cells to effector sites, including the myocardium. Herein, we analyzed the role of TNF-α in the migratory activity of thymocytes in Trypanosoma cruzi (T. cruzi) acutely-infected mice. We found increased expression and deposition of TNF-α in the thymus of infected animals compared to controls, accompanied by increased co-localization of fibronectin, a cell migration-related ECM molecule, whose contents in the thymus of infected mice is also augmented. In-vivo studies showed an enhanced export of thymocytes in T. cruzi-infected mice, as ascertained by intrathymic injection of FITC alone or in combination with TNF-α. The increase of immature CD4+CD8+ T cells in secondary lymphoid organs was even more clear-cut when TNF-α was co-injected with FITC. Ex-vivo transmigration assays also revealed higher number of migrating cells when TNF-α was added onto fibronectin lattices, with higher input of all thymocyte subsets, including immature CD4+CD8+. Infected animals also exhibit enhanced levels of expression of both mRNA TNF-α receptors in the CD4+CD8+ subpopulation. Our findings suggest that in T. cruzi acute infection, when TNF-α is complexed with fibronectin, it favours the altered migration of thymocytes, promoting the release of mature and immature T cells to different compartments of the immune system. Conceptually, this work reinforces the notion that thymocyte migration is a multivectorial biological event in health and disease, and that TNF-α is a further player in the process

A primary human T-cell spectral library to facilitate large scale quantitative T-cell proteomics.

Data independent analysis (DIA) exemplified by sequential window acquisition of all theoretical mass spectra (SWATH-MS) provides robust quantitative proteomics data, but the lack of a public primary human T-cell spectral library is a current resource gap. Here, we report the generation of a high-quality spectral library containing data for 4,833 distinct proteins from human T-cells across genetically unrelated donors, covering ~24% proteins of the UniProt/SwissProt reviewed human proteome. SWATH-MS analysis of 18 primary T-cell samples using the new human T-cell spectral library reliably identified and quantified 2,850 proteins at 1% false discovery rate (FDR). In comparison, the larger Pan-human spectral library identified and quantified 2,794 T-cell proteins in the same dataset. As the libraries identified an overlapping set of proteins, combining the two libraries resulted in quantification of 4,078 human T-cell proteins. Collectively, this large data archive will be a useful public resource for human T-cell proteomic studies. The human T-cell library is available at SWATHAtlas and the data are available via ProteomeXchange (PXD019446 and PXD019542) and PeptideAtlas (PASS01587)

The immune checkpoint CD96 defines a distinct lymphocyte phenotype and is highly expressed on tumor-infiltrating T cells

CD96 has recently been shown to be a potent immune checkpoint molecule in mice, but a similar role in humans is not known. In this study, we provide a detailed map of CD96 expression across human lymphocyte lineages, the kinetics of CD96 regulation on T-cell activation and co-expression with other conventional and emerging immune checkpoint molecules. We show that CD96 is predominantly expressed by T cells and has a unique lymphocyte expression profile. CD96 high T cells exhibited distinct effector functions on activation. Of note, CD96 expression was highly correlated with T-cell markers in primary and metastatic human tumors and was elevated on antigen- experienced T cells and tumor-infiltrating lymphocytes. Collectively, these data demonstrate that CD96 may be a promising immune checkpoint to enhance T-cell function against human cancer and infectious diseas

C5b-9 membrane attack complex formation and extracellular vesicle shedding in Barrett's esophagus and esophageal adenocarcinoma

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett’s Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV

Papel das interações mediadas pelos receptores de prolactina e glicocorticoide na atrofia tímica que ocorre na infecção experimental pelo Trypanosoma Cruzi

Submitted by Anderson Silva ([email protected]) on 2012-10-11T18:05:38Z No. of bitstreams: 1 ailin_l_oliveira_ioc_bcm_0048_2011.pdf: 2784238 bytes, checksum: 109591f3b041c2482398b825d1bb7d51 (MD5)Made available in DSpace on 2012-10-11T18:05:38Z (GMT). No. of bitstreams: 1 ailin_l_oliveira_ioc_bcm_0048_2011.pdf: 2784238 bytes, checksum: 109591f3b041c2482398b825d1bb7d51 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, BrasilNo curso das doenças infecciosas, os circuitos neuroendócrinos e imunes atuam em conjunto facilitando a resposta do hospedeiro. Alterações no eixo hipotálamo-hipófise- adrenal são frequentemente associadas a infecções, como as causadas pelo Trypanosoma cruzi, o agente etiológico da doença de Chagas. Durante a fase aguda da infecção por T. cruzi, a ação pró-apoptótica de glicocorticóides (GC), sistemicamente aumentados, sobre timócitos CD4+CD8+ (DP), resulta em atrofia tímica. Recentemente, nosso grupo demonstrou expressão alterada de prolactina (PRL), um outro hormônio relacionado ao estresse, durante a infecção por T. cruzi. Com base no conhecimento das ações imunomoduladoras de PRL, como a proteção de timócitos da apoptose induzida por GC, pretendemos no presente estudo investigar uma possível função da ativação cruzada entre os receptores intratímicos de PRL e GC (PRLR e GR, respectivamente) no desencadeamento da atrofia tímica induzida por T. cruzi. Observamos um circuito intratímico específico de modulação de ambos os hormônios durante a infecção experimental por T. cruzi em camundongos adultos jovens. Enquanto os níveis plasmáticos de PRL e GC apresentaram-se aumentados no início da fase aguda da infecção, quando o processo de atrofia tímica é instaurado, os níveis intratímicos desses hormônios diminuíram, restabelecendo-se posteriormente aos níveis fisiológicos do controle não-infectado. Paralelamente, variações da expressão intratímica de PRLR e GR, ocorreram em acordo com a modulação local de seus ligantes. No início do processo de atrofia tímica, quando muitas células DP estão entrando em apoptose, observamos aumento da expressão de GR e diminuição de PRLR. Em um segundo momento, durante a fase mais tardia da infecção experimental por T. cruzi, houve restabelecimento da expressão gênica de ambos os receptores nessas células. Nessa fase, quando o processo de morte celular encontra-se estabilizado e somente algumas células aparentemente mais resistentes à ação pró-apoptótica de GC sobreviveram, observamos aumento da proteína Bcl-xl, um importante fator anti apoptótico induzido por PRL. Em acordo com esses achados, utilizando-se um modelo de indução de apoptose in vitro, verificamos maior Suscetibilidade de timócitos à morte induzida por GC nos períodos iniciais da infecção, bem como aos efeitos protetores de PRL, o que não foi observado na fase mais tardia da infecção. Tomados em conjunto, nossos dados apontam para uma função cruzada (cross-talk) entre PRLR e GR intratímicos, e que seria responsável pela manutenção da homeostasia tímica. Nesse sentido, alterações desse circuito podem contribuir para o processo de atrofia tímica característico da fase aguda da infecção pelo T. cruzi.During infectious diseases, neuroendocrine and immune networks act in concert, facilitating host response. Disorders in the hypothalamic-pituitary-adrenal axis are frequently observed associated to infections. It has been demonstrated that increased level of circulating glucocorticoids (GC) systemic levels during the acute phase of Trypanosoma cruzi infection, the etiological agent of Chaga ́s disease, results in thymus atrophy. Such steroid can trigger apoptosis on cortical thymocytes bearing the phenotype CD4+CD8+ (DP) via a specific glucocorticoid receptor (GR). Changes in thymocyte development may have an important consequence for the disease, i.e., further autoimmune reactions. Recently our group demonstrated altered expression of prolactin (PRL), another hormone related to stress, during T. cruzi infection. Based on studies showing expression of PRL receptors (PRLR) on lymphocytes, and its effects on growth, differentiation, and apoptosis in lymphoid cells, this hormone is supposed to functions as an immunomodulator. Furthermore, prolactin protects T cells from GC- induced apoptosis both in vivo and in vitro, which corroborates to this hypothesis. Based on these data, here we aim to investigate the possible role of intrathymic GR- PRLR cross-talk to the outcome of T. cruzi induced thymus atrophy. Our results demonstrate altered intrathymic PRL and corticosterone levels during the acute phase of T. cruzi infection. When the same hormones were evaluated on plasma, a different modulation was detected, suggesting an independence of intrathymic and systemic circuits that may be related to thymus atrophy. Furthermore, we detected the variation of PRLR and GR intrathymic expression, that ocurred in accordance to the modulation of their locally produced ligands. The analysis of DP cells that survived to the apoptosis process at the late phase of acute infection, demonstrated increased PRLR expression in parallel to the decrease of GR levels, This receptor expression profile courses with an increase of anti apoptotic proteins and the stabilization of the thymus atrophy process and seems an important factor for the survival of these cells. Using an in vitro model of thymocyte apoptosis induction, we observed that such alterations result in different GC-induced cell death susceptibility as well as an altered answer to prolactin anti -apoptotic effects. Taken together our results point to an relevant intrathymic PRLR-GR cross-talk involved in thymus homeostasis. In this way, changes of this circuit may contribute to the outcome of the thymus atrophy characteristic of the acute phase of T. cruzi infection

Ativação de células progenitoras epiteliais tímicas após castração

Made available in DSpace on 2016-04-12T12:39:34Z (GMT). No. of bitstreams: 2 ailin_oliveira_ioc_dout_2015.pdf: 4901825 bytes, checksum: 4248ae90175b5218e570617bea7ee462 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilA involução do timo associada ao envelhecimento é acompanhada por redução do desenvolvimento e exportação de células T para a periferia do sistema imune, o que afeta a competência imunológica. A relação causal entre hormônios sexuais e atrofia tímica senil já está bem estabelecida. Embora a depleção de hormônios sexuais tenha sido demonstrada em regenerar o timo, restabelecendo o compartimento linfóide e promovendo a recuperação de células T, pouco é conhecido sobre os fatores desencadeadores desse processo e as alterações promovidas no microambiente tímico. O presente estudo demonstra que embora a castração afete globalmente as diferentes subpopulações de células epiteliais tímicas (TEC) e linfóides, o processo regenerativo do timo em camundongos envelhecidos é iniciado especificamente a partir da diferenciação de TEC corticais expressando baixos níveis de MHCII (cTEC lo). Observamos que a depleção de hormônios sexuais em camundongos envelhecidos (9 - 11 meses) resulta em redução de células cTEC lo seguida do aumento de cTEC hi e TEC medulares, tanto com baixa quanto alta expressão de MHCII (mTEC lo e mTEC hi, respectivamente) Ainda, observamos a recuperação do compartimento epitelial ocorre simultaneamente ao aumento da expressão dos fatores de diferenciação e ativação de TEC, FoxN1, Wnt4 e DLL4, além de aumento dos receptores de activina acvr1a, acvr2a e acvr2b em cTEC lo no dia 4 pós castração, ao mesmo tempo em que a biodisponibilidade de activina no está aumentada no timo desses animais. A adição de activina em sistema de cultura celular primária 3D gerou aumento da diferenciação de mTEC a partir de cTEC lo obtidas do timo senil, enquanto ensaios in vitro e in vivo demonstraram que, por outro lado, a inibição da sinalização de activina no timo jovem gera um perfil de TEC semelhante ao de animais envelhecidos. Através do transplante de cultura de timo fetal (FTOC) na capsula renal de camundongos atímicos demonstramos que a produção de activina pelo microambiente tímico é necessária pra o desenvolvimento de células T CD4-CD8-, CD4+ e CD8+. Por ultimo, através do tratamento de animais envelhecidos castrados com folistatina, antagonista fisiológico de activina, demonstramos que o aumento da sinalização de activina pós castração é essencial para recuperação das diferentes subpopulações de TEC e de timócitos CD4-CD8- e CD4+, além de restabelecer a expressão de DLL4 no compartimento cortical de TEC Juntos, esses dados apontam activina vii como um hormônio essencial para o restabelecimento da atrofia tímica senil tanto diretamente por induzir o restabelecimento do compartimento epitelial, como indiretamente por restabelecer a sinalização de DLL4-Notch, necessária para o desenvolvimento das diferentes populações de timócitos a partir de progenitores hematopoiéticosThe involution associated with aging is accompanied by reduction in thymus development and export of T cells towards the periphery of the immune system, which affects the immune competence. The causal relationship between sex hormones and senile thymic atrophy is already well established. Althou gh the depletion of sex hormones have been shown to regenerate the thymus restoring lymphoid compartment and promoting the recovery of T cells, little is known about the factors that trigger this process and the changes induced in th e thymic microenvironment. The present study demonstrates that although castration generally affects the different subpopulations of thymic epithelial cell s (TEC) and lymphoid cells the regenerative process of the thymus in age d mice is initiated specifically from cortical TEC expressing low levels of differentiation MHCII (cTEC lo) . We note that the depletion of sex hormones in aged mice (9 - 11 months) results in reduction in cTEC lo cells followed by the increase of cTEC hi and medullary TEC, both with low and high expression of MHCII (mTEC lo and mTEC hi, respectively) . Also, we observe the recovery of the epithelial compartment occurs simultaneously with the increase in the expression of TEC differentiation and activation factors, Foxn1, Wnt4 and Dll4, and increased activin receptors acvr1a, acvr2a and acvr2b in cTEC lo at day 4 post castration, at the same time the activin bioavailability is increased in the thymus of the animals. The addition of activin in primary 3D cell culture system generated increased differentiation mTEC from cTEC lo obtained from senile thymus, while in vitro and in vivo assays showed that, on the other hand , inhibition of activin signaling in the young thymus generates a TEC profile similar to that of aged animals. Through fetal thymus transplantation cultur e (FTOC) in renal capsule of athymic mice we demonstrate that the production of activin in thymic microenvironment is required for the development of CD4 - CD8 - T cells, CD4 + and CD8 + . Finally, by treatment of aged castrated animals with follistatin, physiological antagonist of activin, we demonstrate that the increase in activin signaling post castration is essential for the recovery of different subpopulations of TEC and thymocytes CD4 - CD8 - and CD4 + , and reestablish the expression of DLL 4 in cTEC. Together, these data indicate activin as a hormone essential for the restoration of the senile thymic atrophy either dir ectly inducing the restoration of TEC and indirectly by restoring DII4-Notch signaling necessary for the growth of different populations of thymocytes from hematopoietic progenitor

Stress related hormonal circuitry in chagas disease

During stressful processes, for example infectious diseases, neuroendocrine and immune networks act multi-directionally facilitating the host response. However in exacerbated settings, this homeostatic mechanism may be lost. Recent findings unravelled an imbalance of the immunoneuroendocrine network during Chagas disease, the infection caused by the protozoan Trypanosoma cruzi. During the acute immune response against T. cruzi, inflammatory and neuroendocrine responses become dysregulated with harmful effects for the host. One target organ is the thymus. In acutely-infected mice, it undergoes a severe atrophy, with massive depletion of immature double positive CD4+CD8+ (DP) thymocytes, which seems to be linked to a systemic and intrathymic cytokine/hormonal imbalance, involving TNF-α, glucocorticoids and prolactin. In addition, there is an abnormal export of potentially autoreactive DP cells to the periphery of the immune system, which is apparently regulated by the prolactin levels. Furthermore, TNF-α is able to differentially modulate the hypothalamus-pituitary-adrenal (HPA) axis: while having stimulatory effects at the HP unit at the adrenal it is inhibitory. Interestingly, chronically infected humans with chagasic myocardiopathy also showed alterations in HPA axis. Understanding of how T. cruzi infection lead to neuroendocrine immune-associated disturbances will provide important clues to better dissect the mechanisms underlying the pathophysiology of Chagas disease.Fil: Lepletier, Ailin. Instituto Oswaldo Cruz; BrasilFil: Villar, Silvina Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología; ArgentinaFil: Perez, Ana Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología; ArgentinaFil: Morrot, Alexandre. Universidade Federal do Rio de Janeiro; BrasilFil: Savino, Wilson. Instituto Oswaldo Cruz; Brasi

Tumor Necrosis Factor-α Regulates Glucocorticoid Synthesis in the Adrenal Glands of Trypanosoma cruzi Acutely-Infected Mice. The Role of TNF-R1

Adrenal steroidogenesis is under a complex regulation involving extrinsic and intrinsic adrenal factors. TNF-α is an inflammatory cytokine produced in response to tissue injury and several other stimuli. We have previously demonstrated that TNF-R1 knockout (TNF-R1−/−) mice have a dysregulated synthesis of glucocorticoids (GCs) during Trypanosoma cruzi acute infection. Since TNF-α may influence GCs production, not only through the hypothalamus-pituitary axis, but also at the adrenal level, we now investigated the role of this cytokine on the adrenal GCs production. Wild type (WT) and TNF-R1−/− mice undergoing acute infection (Tc-WT and Tc-TNF-R1−/− groups), displayed adrenal hyperplasia together with increased GCs levels. Notably, systemic ACTH remained unchanged in Tc-WT and Tc-TNF-R1−/− compared with uninfected mice, suggesting some degree of ACTH-independence of GCs synthesis. TNF-α expression was increased within the adrenal gland from both infected mouse groups, with Tc-WT mice showing an augmented TNF-R1 expression. Tc-WT mice showed increased levels of P-p38 and P-ERK compared to uninfected WT animals, whereas Tc-TNF-R1−/− mice had increased p38 and JNK phosphorylation respect to Tc-WT mice. Strikingly, adrenal NF-κB and AP-1 activation during infection was blunted in Tc-TNF-R1−/− mice. The accumulation of mRNAs for steroidogenic acute regulatory protein and cytochrome P450 were significantly increased in both Tc-WT and Tc-TNF-R1−/− mice; being much more augmented in the latter group, which also had remarkably increased GCs levels. TNF-α emerges as a potent modulator of steroidogenesis in adrenocortical cells during T. cruzi infection in which MAPK pathways, NF-κB and AP-1 seem to play a role in the adrenal synthesis of pro-inflammatory cytokines and enzymes regulating GCs synthesis. These results suggest the existence of an intrinsic immune-adrenal interaction involved in the dysregulated synthesis of GCs during murine Chagas disease.Fil. Villar, Silvina R. Instituto de Inmunología, Facultad de Ciencias Médicas, Universidad Nacional de Rosario; ArgentinaFil. Ronco, María Teresa. Instituto de Fisiología Experimental (IFISE-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario; Argentin