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.

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.

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.

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.

CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response.

To study in vivo CD8 T cell differentiation, we quantified the coexpression of multiple genes in single cells throughout immune responses. After in vitro activation, CD8 T cells rapidly express effector molecules and cease their expression when the antigen is removed. Gene behavior after in vivo activation, in contrast, was quite heterogeneous. Different mRNAs were induced at very different time points of the response, were transcribed during different time periods, and could decline or persist independently of the antigen load. Consequently, distinct gene coexpression patterns/different cell types were generated at the various phases of the immune responses. During primary stimulation, inflammatory molecules were induced and down-regulated shortly after activation, generating early cells that only mediated inflammation. Cytotoxic T cells were generated at the peak of the primary response, when individual cells simultaneously expressed multiple killer molecules, whereas memory cells lost killer capacity because they no longer coexpressed killer genes. Surprisingly, during secondary responses gene transcription became permanent. Secondary cells recovered after antigen elimination were more efficient killers than cytotoxic T cells present at the peak of the primary response. Thus, primary responses produced two transient effector types. However, after boosting, CD8 T cells differentiate into long-lived killer cells that persist in vivo in the absence of antigen.

Gamma(c) deficiency precludes CD8+ T cell memory despite formation of potent T cell effectors.

Several cytokines (including IL-2, IL-7, IL-15, and IL-21) that signal through receptors sharing the common gamma chain (gamma(c)) are critical for the generation and peripheral homeostasis of naive and memory T cells. Recently, we demonstrated that effector functions fail to develop in CD4(+) T cells that differentiate in the absence of gamma(c). To assess the role of gamma(c) cytokines in cell-fate decisions that condition effector versus memory CD8(+) T cell generation, we compared the response of CD8(+) T cells from gamma(c)(+) or gamma(c)(-) P14 TCR transgenic mice after challenge with lymphocytic choriomeningitis virus. The intrinsic IL-7-dependent survival defect of gamma(c)(-) naive CD8(+) T cells was corrected by transgenic expression of human Bcl-2. We demonstrated that although gamma(c)-dependent signals are dispensable for the initial expansion and the acquisition of cytotoxic functions following antigenic stimulation, they condition the terminal proliferation and differentiation of CD8(+) effector T cells (i.e., KLRG1(high) CD127(low) short-lived effector T cells) via the transcription factor, T-bet. Moreover, the gamma(c)-dependent signals that are critical for memory T cell formation are not rescued by Bcl2 overexpression. Together, these data reveal an unexpected divergence in the requirement for gamma(c) cytokines in the differentiation of CD4(+) versus CD8(+) cytotoxic T lymphocytes.

Characterization of T cell differentiation in the murine gut.

Gut intraepithelial CD8 T lymphocytes (T-IEL) are distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. The intermediate stages of differentiation between CP and mature T-IEL were not identified, and the local differentiation process was not characterized. We identified and characterized six phenotypically distinct lineage-negative populations in the CP and the gut epithelium: (a) we determined the kinetics of their generation from bone marrow precursors; (b) we quantified CD3-epsilon, recombination activating gene (Rag)-1, and pre-Talpha mRNAs expression at single cell level; (c) we characterized TCR-beta, -gamma, and -alpha locus rearrangements; and (d) we studied the impact of different mutations on the local differentiation. These data allowed us to establish a sequence of T cell precursor differentiation in the gut. We also observed that the gut differentiation varied from that of the thymus by a very low frequency of pre-Talpha chain mRNA expression, a different kinetics of Rag-1 mRNA expression, and a much higher impact of CD3 epsilon/delta and pre-Talpha deficiencies. Finally, only 3% of CP cells were clearly involved in T cell differentiation, suggesting that these structures may have additional physiological roles in the gut.

Major T cell progenitor activity in bone marrow-derived spleen colonies.

Common lymphoid progenitors (CLP) are generated in adult bone marrow (BM), but the intermediate steps leading to T cell commitment are unknown, and so is the site at which this commitment occurs. Here, we show that colonies arising in the spleen 12 days after BM injection harbor T cell precursors that are undetectable in BM. These precursors did not generate myeloid cells in vivo but repopulated the thymus and the peripheral T cell compartment much faster than did CLP. Two lineage negative (Lin(-)) subpopulations were distinguished, namely CD44(+) Thy1(-) cells still capable of natural killer generation and transient low-level B cell generation, and T cell-restricted CD44(-) Thy1(+) cells. At a molecular level, frequency of CD3epsilon and preTalpha mRNA was very different in each subset. Furthermore, only the CD44(-) Thy1(+) subset have initiated rearrangements in the T cell receptor beta locus. Thus, this study identifies extramedullary T cell progenitors and will allow easy approach to T cell commitment studies.

Epitope specificity and relative clonal abundance do not affect CD8 differentiation patterns during lymphocytic choriomeningitis virus infection.

To evaluate the impact of immunodominance on CD8 T-cell properties, we compared the functional properties of dominant and subdominant populations in the response to lymphocytic choriomeningitis virus (LCMV). To improve functional discrimination, in addition to the usual tests of phenotype and function, we used a sensitive technique that allows the screening of all CD8 effector genes simultaneously in single cells. Surprisingly, these methods failed to reveal a major impact of clonal dominance in CD8 properties throughout the response. Aiming to increase clonal dominance, we examined high-frequency transferred P14 T-cell receptor transgenic (TCR Tg) cells. Under these conditions LCMV is cleared faster, and accordingly we found an accelerated response. However, when Tg and endogenous cells were studied in the same mice, where they should be subjected to the same antigen load, they showed overlapping properties, and the presence of P14 cells did not modify endogenous responses to other LCMV epitopes or a perturbed immunodominance hierarchy in the memory phase. Using allotype-labeled Tg cells, we found that during acute infection up to 80% downregulated their TCR and were undetectable by tetramer binding, and that tetramer-negative and tetramer-positive cells had very different features. Since Tg cells are not available to evaluate immune responses in humans and, in many cases, are not available from the mouse, the tetramer-based evaluation of early immune responses in most situations of high viremia may be incomplete and biased.

Transcriptional regulation during CD8 T-cell immune responses.

Naïve CD8 T cells differentiate in response to antigen stimulation. They acquire the capacity to express multiple effector molecules and mediate effector functions that contribute to infection control. Once antigen loads are reduced they revert progressively to a less activated status and eventually reach a steady-state referred to as "memory" that is very different from that of naive cells. Indeed, these "memory" cells are "ready-to-go" populations that acquired the capacity to respond more efficiently to antigen stimulation. They modify their cell cycle machinery in order to divide faster; they likely improve DNA repair and other cell survival mechanisms in order to survive during division and thus to generate much larger clones of effector cells; finally, they also mediate effector functions much faster. These modifications are the consequence of changes in the expression of multiple genes, i.e., on the utilization of a new transcription program.

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.

Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses.

Whereas the definition of B-cell memory is based on well-known cellular properties and differentiation steps, the process of T-cell memory generation was, until recently, less well understood. A series of recent reports, however, have drastically modified our notion of CD8(+) memory T cells. They show that, in addition to division, the generation of efficient memory cells requires a previously unknown differentiation process. As a whole, the generation of CD8(+) memory T cells appears to mimic the generation of memory B cells. Both processes depend on the help of CD4(+) T cells, they are irreversible, they have the same mechanism, and they occur progressively during the late expansion phase of the primary immune response.

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.

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.

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.

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.