Microbiota stimulation generates LCMV-specific memory CD8+ T cells in SPF mice and determines their TCR repertoire during LCMV infection.

Both mouse and human harbour memory phenotype CD8+ T cells specific for antigens in hosts that have not been previously exposed to these antigens. The origin and the nature of the stimuli responsible for generation of CD44hi CD8+ T cells in specific pathogen-free (SPF) mice remain controversial. It is known that microbiota plays a crucial role in the prevention and resolution of systemic infections by influencing myelopoiesis, regulating dendritic cells, inflammasome activation and promoting the production of type I and II interferons. By contrast, here we suggest that microbiota has a direct effect on generation of memory phenotype CD44hiGP33+CD8+ T cells. In SPF mice, it generates a novel GP33+CD44hiCD8+ T cell sub-population associating the properties of innate and genuine memory cells. These cells are highly enriched in the bone marrow, proliferate rapidly and express immediate effector functions. They dominate the response to LCMV and express particular TCRß chains. The sequence of these selected TCRß chains overlaps with that of GP33+CD8+ T cells directly selected by microbiota in the gut epithelium of SPF mice, demonstrating a common selection mechanism in gut and peripheral CD8+ T cell pool. Therefore microbiota has a direct role in priming T cell immunity in SPF mice and in the selection of TCRß repertoires during systemic infection. We identify a mechanism that primes T cell immunity in SPF mice and may have a major role in colonization resistance and protection from infection.

An fMRI paradigm based on Williams inhibition test to study the neural substrates of attention and inhibitory control.

The purpose of this study is to present an fMRI paradigm, based on the Williams inhibition test (WIT), to study attentional and inhibitory control and their neuroanatomical substrates. We present an index of the validity of the proposed paradigm and test whether the experimental task discriminates the behavioral performances of healthy participants from those of individuals with acquired brain injury. Stroop and Simon tests present similarities with WIT, but this latter is more demanding. We analyze the BOLD signal in 10 healthy participants performing the WIT. The dorsolateral prefrontal cortex, the inferior prefrontal cortex, the anterior cingulate cortex, and the posterior cingulate cortex were defined for specified region of interest analysis. We additionally compare behavioral data (hits, errors, reaction times) of the healthy participants with those of eight acquired brain injury patients. Data were analyzed with GLM-based random effects and Mann-Whitney tests. Results show the involvement of the defined regions and indicate that the WIT is sensitive to brain lesions. This WIT-based block design paradigm can be used as a research methodology for behavioral and neuroimaging studies of the attentional and inhibitory components of executive functions.

CD4/CD8/Dendritic cell complexes in the spleen: CD8+ T cells can directly bind CD4+ T cells and modulate their response.

CD4+ T cell help to CD8+ T cell responses requires that CD4+ and CD8+ T cells interact with the same antigen presenting dendritic cell (Ag+DC), but it remains controversial whether helper signals are delivered indirectly through a licensed DC and/or involve direct CD4+/CD8+ T cell contacts and/or the formation of ternary complexes. We here describe the first in vivo imaging of the intact spleen, aiming to evaluate the first interactions between antigen-specific CD4+, CD8+ T cells and Ag+DCs. We show that in contrast to CD4+ T cells which form transient contacts with Ag+DC, CD8+ T cells form immediate stable contacts and activate the Ag+DC, acquire fragments of the DC membranes by trogocytosis, leading to their acquisition of some of the DC properties. They express MHC class II, and become able to present the specific Marilyn peptide to naïve Marilyn CD4+ T cells, inducing their extensive division. In vivo, these CD8+ T cells form direct stable contacts with motile naïve CD4+ T cells, recruiting them to Ag+DC binding and to the formation of ternary complexes, where CD4+ and CD8+ T cells interact with the DC and with one another. The presence of CD8+ T cells during in vivo immune responses leads to the early activation and up-regulation of multiple functions by CD4+ T lymphocytes. Thus, while CD4+ T cell help is important to CD8+ T cell responses, CD8+ T cells can interact directly with naïve CD4+ T cells impacting their recruitment and differentiation.

NKp30 isoforms and NKp30 ligands are predictive biomarkers of response to imatinib mesylate in metastatic GIST patients.

Despite effective targeted therapy acting on KIT and PDGFRA tyrosine kinases, gastrointestinal stromal tumors (GIST) escape treatment by acquiring mutations conveying resistance to imatinib mesylate (IM). Following the identification of NKp30-based immunosurveillance of GIST and the off-target effects of IM on NK cell functions, we investigated the predictive value of NKp30 isoforms and NKp30 soluble ligands in blood for the clinical response to IM. The relative expression and the proportions of NKp30 isoforms markedly impacted both event-free and overall survival, in two independent cohorts of metastatic GIST. Phenotypes based on disbalanced NKp30B/NKp30C ratio (ΔBClow) and low expression levels of NKp30A were identified in one third of patients with dismal prognosis across molecular subtypes. This ΔBClow blood phenotype was associated with a pro-inflammatory and immunosuppressive tumor microenvironment. In addition, detectable levels of the NKp30 ligand sB7-H6 predicted a worse prognosis in metastatic GIST. Soluble BAG6, an alternate ligand for NKp30 was associated with low NKp30 transcription and had additional predictive value in GIST patients with high NKp30 expression. Such GIST microenvironments could be rescued by therapy based on rIFN-α and anti-TRAIL mAb which reinstated innate immunity.

A new mechanism shapes the naïve CD8+ T cell repertoire: the selection for full diversity.

During thymic T cell differentiation, TCR repertoires are shaped by negative, positive and agonist selection. In the thymus and in the periphery, repertoires are also shaped by strong inter-clonal and intra-clonal competition to survive death by neglect. Understanding the impact of these events on the T cell repertoire requires direct evaluation of TCR expression in peripheral naïve T cells. Several studies have evaluated TCR diversity, with contradictory results. Some of these studies had intrinsic technical limitations since they used material obtained from T cell pools, preventing the direct evaluation of clonal sizes. Indeed with these approaches, identical TCRs may correspond to different cells expressing the same receptor, or to several amplicons from the same T cell. We here overcame this limitation by evaluating TCRB expression in individual naïve CD8+ T cells. Of the 2269 Tcrb sequences we obtained from 13 mice, 99% were unique. Mathematical analysis of the data showed that the average number of naïve peripheral CD8+ T cells expressing the same TCRB is 1.1 cell. Since TCRA co-expression studies could only increase repertoire diversity, these results reveal that the number of naïve T cells with unique TCRs approaches the number of naïve cells. Since thymocytes undergo multiple rounds of divisions after TCRB rearrangement and 3-5% of thymocytes survive thymic selection events the number of cells expressing the same TCRB was expected to be much higher. Thus, these results suggest a new repertoire selection mechanism, which strongly selects for full TCRB diversity.

Correction: Depletion of Regulatory T Cells Induces High Numbers of Dendritic Cells and Unmasks a Subset of Anti-Tumour CD8+CD11c+ PD-1lo Effector T Cells.

[This corrects the article DOI: 10.1371/journal.pone.0157822.].

Study of behavioural and neural bases of visuo-spatial working memory with an fMRI paradigm based on an n-back task.

The goal of this study was to propose a new functional magnetic resonance imaging (fMRI) paradigm using a language-free adaptation of a 2-back working memory task to avoid cultural and educational bias. We additionally provide an index of the validity of the proposed paradigm and test whether the experimental task discriminates the behavioural performances of healthy participants from those of individuals with working memory deficits. Ten healthy participants and nine patients presenting working memory (WM) deficits due to acquired brain injury (ABI) performed the developed task. To inspect whether the paradigm activates brain areas typically involved in visual working memory (VWM), brain activation of the healthy participants was assessed with fMRIs. To examine the task's capacity to discriminate behavioural data, performances of the healthy participants in the task were compared with those of ABI patients. Data were analysed with GLM-based random effects procedures and t-tests. We found an increase of the BOLD signal in the specialized areas of VWM. Concerning behavioural performances, healthy participants showed the predicted pattern of more hits, less omissions and a tendency for fewer false alarms, more self-corrected responses, and faster reaction times, when compared with subjects presenting WM impairments. The results suggest that this task activates brain areas involved in VWM and discriminates behavioural performances of clinical and non-clinical groups. It can thus be used as a research methodology for behavioural and neuroimaging studies of VWM in block-design paradigms.

Depletion of Regulatory T Cells Induces High Numbers of Dendritic Cells and Unmasks a Subset of Anti-Tumour CD8+CD11c+ PD-1lo Effector T Cells.

Natural regulatory T (Treg) cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25+Tregs results in tumour regression in Balb/c mice, but the mechanisms underlying this process are not fully understood. Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.

A unique CD8(+) T lymphocyte signature in pediatric type 1 diabetes.

Human type 1 diabetes results from a destructive auto-reactive immune response in which CD8(+) T lymphocytes play a critical role. Given the intense ongoing efforts to develop immune intervention to prevent and/or cure the disease, biomarkers suitable for prediction of disease risk and progress, as well as for monitoring of immunotherapy are required. We undertook separate multi-parameter analyses of single naïve and activated/memory CD8(+) T lymphocytes from pediatric and adult patients, with the objective of identifying cellular profiles associated with onset of type 1 diabetes. We observe global perturbations in gene and protein expression and in the abundance of T cell populations characterizing pediatric but not adult patients, relative to age-matched healthy individuals. Pediatric diabetes is associated with a unique population of CD8(+) T lymphocytes co-expressing effector (perforin, granzyme B) and regulatory (transforming growth factor ß, interleukin-10 receptor) molecules. This population persists after metabolic normalization and is especially abundant in children with high titers of auto-antibodies to glutamic acid decarboxylase and with elevated HbA1c values. These findings highlight striking differences between pediatric and adult type 1 diabetes, indicate prolonged large-scale perturbations in the CD8(+) T cell compartment in the former, and suggest that CD8(+)CD45RA(-) T cells co-expressing effector and regulatory factors are of interest as biomarkers in pediatric type 1 diabetes.

The cyclin D1 carboxyl regulatory domain controls the division and differentiation of hematopoietic cells.

BACKGROUND: The family of D cyclins has a fundamental role in cell cycle progression, but its members (D1, D2, D3) are believed to have redundant functions. However, there is some evidence that contradicts the notion of mutual redundancy and therefore this concept is still a matter of debate. RESULTS: Our data show that the cyclin D1 is indispensable for normal hematopoiesis. Indeed, in the absence of D1, either in genetic deficient mice, or after acute ablation by RNA interference, cyclins D2 and D3 are also not expressed preventing hematopoietic cell division and differentiation at its earliest stage. This role does not depend on the cyclin box, but on the carboxyl regulatory domain of D1 coded by exons 4-5, since hematopoietic differentiation is also blocked by the conditional ablation of this region. CONCLUSION: These results demonstrate that not all functions of individual D cyclins are redundant and highlight a master role of cyclin D1 in hematopoiesis.

Thymocytes self-renewal: a major hope or a major threat?

Thymus transplants were never used to correct T-cell intrinsic deficiencies, as it is generally believed that thymocytes have short intrinsic lifespans. This notion is based on multiple thymus transplantation experiments, where it was shown that thymus-resident cells were rapidly replaced by progenitors migrating from the bone marrow (BM). This substitution occurs even when bone marrow precursors are unable to generate T cells, as in Rag1/2(-) or severe combined immunodeficiency (SCID)-deficient mice. In contrast, two groups reported that neonatal thymi transplanted into mice that cannot respond to IL-7 harbor populations with extensive capacity to self-renew, which maintain continuous thymocyte generation for several months after surgery. The consequences of this self-renewal capacity differed in these two laboratories. We found that these thymus transplants rapidly reconstitute the full diversity of peripheral T-cell repertoires 1 month after surgery, the earliest time point studied. Moreover, transplantation experiments performed across major histocompatibility barriers show that allogeneic-transplanted thymi are not rejected, and allogeneic cells do not induce graft-versus-host disease, both syngeneic and allogeneic transplants inducing rapid protection from infection. These results indicate a potential use of neonatal thymus transplants to correct T-cell intrinsic deficiencies. The other group observed that continuous thymocyte renewal from BM precursors was fundamental to prevent tumor development. In the absence of this input, thymocytes from the transplanted thymus generated tumors with all the characteristics of T-cell acute lymphoblastic leukemia (T-ALL). Moreover, they suggested that the absence of BM competition was responsible for the T-ALLs developing in X-linked severe combined immunodeficiency (SCID)-X1 patients, deficient in the expression of IL2-Rγc . These patients were treated with autologous CD34(+) cells transfected with virus vectors expressing γc in the absence of myeloablation. We here review the potential therapeutic impact of thymus transplantation and compare the results of these two laboratories aiming to find an answer to the 'Dr Jekill versus Mr. Hyde' status of thymus transplantation experiments.

TGFß-dependent expression of PD-1 and PD-L1 controls CD8(+) T cell anergy in transplant tolerance.

CD8(+) T cell anergy is a critical mechanism of peripheral tolerance, poorly investigated in response to immunotherapy. Here, using a pancreatic islet allograft model and CD3 antibody therapy, we showed, by single cell gene profiling, that intragraft CD8(+) lymphocytes coexpressing granzyme B and perforin were selectively depleted through the Fas/FasL pathway. This step led to long-standing anergy of the remaining CD8(+) T cells marked by the absence of cytotoxic/inflammatory gene expression also confirmed by transcriptome analysis. This sustained unresponsiveness required the presence of the alloantigens. Furthermore, tissue-resident CD8(+) lymphocytes produced TGFß and expressed the inhibitory receptors PD-1 and PD-L1. Blockade of TGFß downregulated PD-1 and PD-L1 expression and precipitated graft rejection. Neutralizing PD-1, PD-L1 or TGFßRII signaling in T cells also abrogated CD3 antibody-induced tolerance. These studies unravel novel mechanisms underlying CD8(+) T cell anergy and reveal a cell intrinsic regulatory link between the TGFß and the PD-1/PD-L1 pathways.

CD8 Memory Cells Develop Unique DNA Repair Mechanisms Favoring Productive Division.

Immune responses are efficient because the rare antigen-specific naïve cells are able to proliferate extensively and accumulate upon antigen stimulation. Moreover, differentiation into memory cells actually increases T cell accumulation, indicating improved productive division in secondary immune responses. These properties raise an important paradox: how T cells may survive the DNA lesions necessarily induced during their extensive division without undergoing transformation. We here present the first data addressing the DNA damage responses (DDRs) of CD8 T cells in vivo during exponential expansion in primary and secondary responses in mice. We show that during exponential division CD8 T cells engage unique DDRs, which are not present in other exponentially dividing cells, in T lymphocytes after UV or X irradiation or in non-metastatic tumor cells. While in other cell types a single DDR pathway is affected, all DDR pathways and cell cycle checkpoints are affected in dividing CD8 T cells. All DDR pathways collapse in secondary responses in the absence of CD4 help. CD8 T cells are driven to compulsive suicidal divisions preventing the propagation of DNA lesions. In contrast, in the presence of CD4 help all the DDR pathways are up regulated, resembling those present in metastatic tumors. However, this up regulation is present only during the expansion phase; i.e., their dependence on antigen stimulation prevents CD8 transformation. These results explain how CD8 T cells maintain genome integrity in spite of their extensive division, and highlight the fundamental role of DDRs in the efficiency of CD8 immune responses.

Single-cell analysis of thymocyte differentiation: identification of transcription factor interactions and a major stochastic component in αß-lineage commitment.

T cell commitment and αß/γδ lineage specification in the thymus involves interactions between many different genes. Characterization of these interactions thus requires a multiparameter analysis of individual thymocytes. We developed two efficient single-cell methods: (i) the quantitative evaluation of the co-expression levels of nine different genes, with a plating efficiency of 99-100% and a detection limit of 2 mRNA molecules/cell; and (ii) single-cell differentiation cultures, in the presence of OP9 cells transfected with the thymus Notch1 ligand DeltaL4. We show that during T cell commitment, Gata3 has a fundamental, dose-dependent role in maintaining Notch1 expression, with thymocytes becoming T-cell-committed when they co-express Notch1, Gata3 and Bc11b. Of the transcription factor expression patterns studied here, only that of Bcl11b was suggestive of a role in Pu1 down-regulation. Individual thymocytes became αß/γδ lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not αß/γδ-lineage committed and TN4 cells comprise two main subpopulations with different degrees of maturity. The existence of a correlation between differentiation potential and expression of the pre-TCR showed that 83% of αß-committed cells do not express the pre-TCR and revealed a major stochastic component in αß-lineage specification.

Cognate antigen stimulation generates potent CD8(+) inflammatory effector T cells.

Inflammatory reactions are believed to be triggered by innate signals and have a major protective role by recruiting innate immunity cells, favoring lymphocyte activation and differentiation, and thus contributing to the sequestration and elimination of the injurious stimuli. Although certain lymphocyte types such as TH17 cells co-participate in inflammatory reactions, their generation from the naïve pool requires the pre-existence of an inflammatory milieu. In this context, inflammation is always regarded as beginning with an innate response that may be eventually perpetuated and amplified by certain lymphocyte types. In contrast, we here show that even in sterile immunizations or in MyD88-deficient mice, CD8 T cells produce a burst of pro-inflammatory cytokines and chemokines. These functions follow opposite rules to the classic CD8 effector functions since they are generated prior to cell expansion and decline before antigen elimination. As few as 56 CD8(+) inflammatory effector cells in a lymph node can mobilize 10(7) cells in 24 h, including lymphocytes, natural killer cells, and several accessory cell types involved in inflammatory reactions. Thus, although inflammation modulates cognate responses, CD8 cognate responses also initiate local inflammatory reactions.

Thymocytes may persist and differentiate without any input from bone marrow progenitors.

Thymus transplants can correct deficiencies of the thymus epithelium caused by the complete DiGeorge syndrome or FOXN1 mutations. However, thymus transplants were never used to correct T cell-intrinsic deficiencies because it is generally believed that thymocytes have short intrinsic lifespans. This notion is based on thymus transplantation experiments where it was shown that thymus-resident cells were rapidly replaced by progenitors originating in the bone marrow. In contrast, here we show that neonatal thymi transplanted into interleukin 7 receptor-deficient hosts harbor populations with extensive capacity to self-renew, and maintain continuous thymocyte generation and export. These thymus transplants reconstitute the full diversity of peripheral T cell repertoires one month after surgery, which is the earliest time point studied. Moreover, transplantation experiments performed across major histocompatibility barriers show that allogeneic transplanted thymi are not rejected, and allogeneic cells do not induce graft-versus-host disease; transplants induced partial or total protection to infection. These results challenge the current dogma that thymocytes cannot self-renew, and indicate a potential use of neonatal thymus transplants to correct T cell-intrinsic deficiencies. Finally, as found with mature T cells, they show that thymocyte survival is determined by the competition between incoming progenitors and resident cells.

Chronic HIV infection affects the expression of the 2 transcription factors required for CD8 T-cell differentiation into cytolytic effectors.

CD8 T cells lose the capacity to control HIV infection, but the extent of the impairment of CD8 T-cell functions and the mechanisms that underlie it remain controversial. Here we report an extensive ex vivo analysis of HIV-specific CD8 T cells, covering the expression of 16 different molecules involved in CD8 function or differentiation. This approach gave remarkably homogeneous readouts in different donors and showed that CD8 dysfunction in chronic HIV infection was much more severe than described previously: some Ifng transcription was observed, but most cells lost the expression of all cytolytic molecules and Eomesodermin and T-bet by chronic infection. These results reveal a cellular mechanism explaining the dysfunction of CD8 T cells during chronic HIV infection, as CD8 T cells are known to maintain some functionality when either of these transcription factors is present, but to lose all cytotoxic activity when both are not expressed. Surprisingly, they also show that chronic HIV and lymphocytic choriomeningitis virus infections have a very different impact on fundamental T-cell functions, "exhausted" lymphocytic choriomeningitis virus-specific cells losing the capacity to secrete IFN-γ but maintaining some cytotoxic activity as granzyme B and FasL are overexpressed and, while down-regulating T-bet, up-regulating Eomesodermin expression.

O investimento na resolutividade e proficiência da atenção às pessoas com úlceras crônicas e pés diabéticos na coordenadoria regional de saúde Sul/SMS/PSP: o desafio de promover o cuidado humano com segurança e qualidade / Investment in solving proficiency and attention to people with chronic ulcers and diabetic foot in the south regional health / SMS / PSP: the challenge of promoting human care with safety and quality

Este relato descreve a singular experiência da Responsável Técnica pela Interlocução de Curativos da COORDENADORIA REGIONAL de SAÚDE SUL, da SECRETARIA MUNICIPAL de SAÚDE de SÃO PAULO, na organização e implantação de uma Rede Assistencial, resolutiva e proficiente, para oferecer acolhimento e assistência às pessoas com úlceras crônicas e pés diabéticos, dentro de uma proposta de atenção humanitária e com qualidade para o SISTEMA ÚNICO de SAÚDE, na região sul da cidade de São Paulo.