Differentiation associated regulation of microRNA expression in vivo in human CD8+ T cell subsets

BACKGROUND: The differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown. METHODS: In order to explore the potential implication of microRNAs in CD8+ T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8+ T cell subsets defining the major steps of the T cell differentiation pathway. RESULTS: We found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease. CONCLUSIONS: This study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8+ T lymphocytes in vivo, which is likely to impact on specific cellular functions

MELK is an oncogenic kinase essential for mitotic progression in basal-like breast cancer cells

Despite marked advances in breast cancer therapy, basal-like breast cancer (BBC), an aggressive subtype of breast cancer usually lacking estrogen and progesterone receptors, remains difficult to treat. In this study, we report the identification of MELK as a novel oncogenic kinase from an in vivo tumorigenesis screen using a kinome-wide open reading frames (ORFs) library. Analysis of clinical data reveals a high level of MELK overexpression in BBC, a feature that is largely dependent on FoxM1, a master mitotic transcription factor that is also found to be highly overexpressed in BBC. Ablation of MELK selectively impairs proliferation of basal-like, but not luminal breast cancer cells both in vitro and in vivo. Mechanistically, depletion of MELK in BBC cells induces caspase-dependent cell death, preceded by defective mitosis. Finally, we find that Melk is not required for mouse development and physiology. Together, these data indicate that MELK is a normally non-essential kinase, but is critical for BBC and thus represents a promising selective therapeutic target for the most aggressive subtype of breast cancer. DOI: http://dx.doi.org/10.7554/eLife.01763.00

A PI3K p110β–Rac signalling loop mediates Pten-loss-induced perturbation of haematopoiesis and leukaemogenesis

The tumour suppressor PTEN, which antagonizes PI3K signalling, is frequently inactivated in haematologic malignancies. In mice, deletion of PTEN in haematopoietic stem cells (HSCs) causes perturbed haematopoiesis, myeloproliferative neoplasia (MPN) and leukaemia. Although the roles of the PI3K isoforms have been studied in PTEN-deficient tumours, their individual roles in PTEN-deficient HSCs are unknown. Here we show that when we delete PTEN in HSCs using the Mx1–Cre system, p110β ablation prevents MPN, improves HSC function and suppresses leukaemia initiation. Pharmacologic inhibition of p110β in PTEN-deficient mice recapitulates these genetic findings, but suggests involvement of both Akt-dependent and -independent pathways. Further investigation reveals that a p110β–Rac signalling loop plays a critical role in PTEN-deficient HSCs. Together, these data suggest that myeloid neoplasia driven by PTEN loss is dependent on p110β via p110β–Rac-positive-feedback loop, and that disruption of this loop may offer a new and effective therapeutic strategy for PTEN-deficient leukaemia

Exhaustion of tumor-specific CD8⁺ T cells in metastases from melanoma patients.

In chronic viral infections, CD8⁺ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8⁺ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8⁺ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy

Molecular profiling of CD8 T cells in autochthonous melanoma identifies Maf as driver of exhaustion.

T cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T cells, often termed "exhausted" T cells. We compared the transcriptome of "exhausted" CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor- and virus-induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor-exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor-specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor-induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor effector responses. We further identified TGFβ and IL-6 as main inducers of Maf expression in CD8 T cells and showed that Maf-deleted tumor-specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFβ/IL-6-mediated induction of Maf

Extended co-expression of inhibitory receptors by human CD8 T-cells depending on differentiation, antigen-specificity and anatomical localization.

Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector T-cells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair

Inhibitory receptor expression by Melan-A specific CD8 T-cells depending on vaccination.

<p>(A) Co-expression of KLRG-1, TIM-3, PD-1 and CD160, and of LAG-3, BTLA, 2B4 and CTLA-4 by Melan-A specific CD8 T-cells. Blood samples from healthy donors (HD) or from patients before immunotherapy (before vacc.) or after peptide+IFA vaccination with or without CpG-ODN 7909 were enriched for CD8 T-cells using magnetic beads. Melan-A-specific CD8 T-cells were identified by staining with CD8-specific antibody and tetramer. Positivity for inhibitory receptors was defined respective to isotype controls. n = 4 for HD; n = 3 for before vacc.; n = 9 for after vaccination without CpG-ODN and n = 11 for after vaccination with CpG-ODN. Colors of the pie arcs depict the expression of individual inhibitory receptors, while the color in the pie depicts the number of co-expressed inhibitory receptors. Co-expression was analyzed with SPICE 5.2. p-values of the permutation test are shown in tables next to the corresponding pie charts. (B) Hierarchical clustering based on co-expression data of the eight inhibitory receptors shown in A, including the four differentiation subsets (N, CM, EM, EMRA) of total CD8 T-cells. (C) Mean expression and SD of four inhibitory receptors upregulated on Melan-A-specific T-cells with vaccination. Data from HD and from patients before vaccination were pooled for the group without vaccination (no vacc.). n = 7 for no vacc.; n = 9 for vaccination with CpG-ODN.</p

Expression of ligands of inhibitory receptors in melanoma metastases and by melanoma cell lines.

<p>(A,B) Paraffin-embedded tumor sections from 16 to 18 tumors were stained by immunohistochemistry for seven inhibitory receptors and CD8. (A) Representative stainings (magnification ×200) for each ligand investigated. (B) Summary of immunohistochemical stainings represented as percent of positive samples. Low (<10%), intermediate (int; 10–50%) and high (>50%) expression is indicated in a color scale. infilt: infiltration of CD8 T-cells in tumor cell nests; sec: secreted i.e. intra- and extracellular presence of galectin-9. (C) Summary of expression by melanoma cell lines on the surface or intracellular (ic) as percent of positive cell lines.</p