Sustained treatment-free remission in chronic myeloid leukaemia is associated with an increased frequency of innate CD8(+) T-cells.

Immunological mechanisms of treatment-free remission (TFR) in chronic myeloid leukaemia (CML) are poorly defined and, to date, no correlation between successful TFR and CD8(+) T-cell subsets has been found. We analysed a new identified human subset of CD8(+) T-cells, namely innate CD8(+) T-cells, in CML patients with TFR ≥ 2 years. We demonstrated a dramatic increase of functionally active innate CD8(+) T-cells in these patients as compared to control subjects and patients in remission under tyrosine kinase inhibitors. Moreover, we found a positive correlation between frequencies of innate CD8(+) T-cells and natural killer cells, possibly representing a new innate biomarker profile of successful TFR.

The Rho-ROCK pathway as a new pathological mechanism of innate immune subversion in chronic myeloid leukaemia.

CD1d-restricted invariant natural killer T (iNKT) cells are believed to play a key role in cancer immune surveillance, and are functionally deficient in patients with chronic myeloid leukaemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDCs). Indeed, flow cytometry and confocal microscopy revealed that cell surface expression of CD1d was downregulated in CML mDCs, relative to healthy donor (HD) controls. The decreased cell surface display of CD1d could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CD1d or its mRNA transcripts, consistent with intracellular retention. In vitro treatment of CML mDCs with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 partially restored both cell surface CD1d expression and CD1d-mediated antigen presentation, whereas it had no effect on HD mDCs. An inhibitor of BCR-ABL tyrosine kinase (TK), imatinib mesylate (IM), had no such activity. Similar recovery of CD1d expression occurred with fasudil, another ROCK inhibitor that is commonly used in clinical trials. Our data support the conclusion that BCR-ABL-dependent ROCK, but not TK, is involved in CD1d downregulation. We propose that ROCK, which is most likely activated by the DH/PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CD1d expression on CML mDCs. Our study reveals the ROCK-mDC axis as a new potential target to restore immune surveillance in patients with CML, offering new therapeutic perspectives for CML treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Evidence for eomesodermin-expressing innate-like CD8(+) KIR/NKG2A(+) T cells in human adults and cord blood samples.

Polyclonal CD8(+) T cells, with a marked innate/memory phenotype, high eomesodermin (Eomes) expression, and the capacity to generate IFN-γ rapidly without prior exposure to antigen, have been described in mice. However, even though a pool of human CD8(+) T cells expressing killer Ig-like receptors (KIRs) was recently documented, the existence of a human equivalent of murine innate/memory CD8(+) T cells remains to be established. Here, we provide evidence for a population of KIR/NKG2A(+) CD8(+) T cells in healthy human adults sharing the same features, namely increased Eomes expression, prompt IFN-γ production in response to innate-like stimulation by IL-12+IL-18, and a potent antigen-independent cytotoxic activity along with a preferential terminally differentiated effector memory phenotype. None of the above functional characteristics applied to the KIR/NKG2A(-) fraction of the Eomes(+) CD8(+) T-cell population, thereby underlining the ability of KIR/NKG2A to distinguish between "innate/memory-like" and "conventional/memory" pools of CD8(+) T cells. Remarkably, KIR/NKG2A(+) Eomes(+) CD8(+) T cells with innate-like functions and a memory/terminally differentiated effector memory phenotype were also identified in human cord blood, suggesting that their development did not depend on cognate antigens. Taken together, our results support the conclusion that CD8(+) T cells co-expressing Eomes and KIR/NKG2A may represent a new, functionally distinct "innate/memory-like" subset in humans.

Innate T-αß lymphocytes as new immunological components of anti-tumoral "off-target" effects of the tyrosine kinase inhibitor dasatinib.

Kinase inhibitors hold great potential as targeted therapy against malignant cells. Among them, the tyrosine kinase inhibitor dasatinib is known for a number of clinically relevant off-target actions, attributed in part to effects on components of the immune system, especially conventional T-cells and natural killer (NK)-cells. Here, we have hypothesized that dasatinib also influences non-conventional T-αß cell subsets known for their potential anti-tumoral properties, namely iNKT cells and the distinct new innate CD8 T-cell subset. In mice, where the two subsets were originally characterized, an activated state of iNKT cells associated with a shift toward an iNKT cell Th1-phenotype was observed after dasatinib treatment in vivo. Despite decreased frequency of the total memory CD8 T-cell compartment, the proportion of innate-memory CD8 T-cells and their IFNγ expression in response to an innate-like stimulation increased in response to dasatinib. Lastly, in patients administered with dasatinib for the treatment of BCR-ABL-positive leukemias, we provided the proof of concept that the kinase inhibitor also influences the two innate T-cell subsets in humans, as attested by their increased frequency in the peripheral blood. These data highlight the potential immunostimulatory capacity of dasatinib on innate T-αß cells, thereby opening new opportunities for chemoimmunotherapy.

Tri-methylation of H3K79 is decreased in TGF-ß1-induced epithelial-to-mesenchymal transition in lung cancer.

BACKGROUND: The epithelial-to-mesenchymal transition (EMT) enables epithelial cancer cells to acquire mesenchymal features and contributes to metastasis and resistance to treatment. This process involves epigenetic reprogramming for gene expression. We explored global histone modifications during TGF-ß1-induced EMT in two non-small cell lung cancer (NSCLC) cell lines and tested different epigenetic treatment to modulate or partially reverse EMT. RESULTS: Loss of classical epithelial markers and gain of mesenchymal markers were verified in A549 and H358 cell lines during TGF-ß1-induced EMT. In addition, we noticed increased expression of the axonal guidance protein semaphorin 3C (SEMA3C) and PD-L1 (programmed death-ligand 1) involved in the inhibition of the immune system, suggesting that both SEMA3C and PD-L1 could be the new markers of TGF-ß1-induced EMT. H3K79me3 and H2BK120me1 were decreased in A549 and H358 cell lines after a 48-h TGF-ß1 treatment, as well as H2BK120ac in A549 cells. However, decreased H3K79me3 was not associated with expression of the histone methyltransferase DOT1L. Furthermore, H3K79me3 was decreased in tumors compared in normal tissues and not associated with cell proliferation. Associations of histone deacetylase inhibitor (SAHA) with DOT1L inhibitors (EPZ5676 or SGC0946) or BET bromodomain inhibitor (PFI-1) were efficient to partially reverse TGF-ß1 effects by decreasing expression of PD-L1, SEMA3C, and its receptor neuropilin-2 (NRP2) and by increasing epithelial markers such as E-cadherin. CONCLUSION: Histone methylation was modified during EMT, and combination of epigenetic compounds with conventional or targeted chemotherapy might contribute to reduce metastasis and to enhance clinical responses.

Phenotype of NK-Like CD8(+) T Cells with Innate Features in Humans and Their Relevance in Cancer Diseases.

Unconventional T cells are defined by their capacity to respond to signals other than the well-known complex of peptides and major histocompatibility complex proteins. Among the burgeoning family of unconventional T cells, innate-like CD8(+) T cells in the mouse were discovered in the early 2000s. This subset of CD8(+) T cells bears a memory phenotype without having encountered a foreign antigen and can respond to innate-like IL-12 + IL-18 stimulation. Although the concept of innate memory CD8(+) T cells is now well established in mice, whether an equivalent memory NK-like T-cell population exists in humans remains under debate. We recently reported that CD8(+) T cells responding to innate-like IL-12 + IL-18 stimulation and co-expressing the transcription factor Eomesodermin (Eomes) and KIR/NKG2A membrane receptors with a memory/EMRA phenotype may represent a new, functionally distinct innate T cell subset in humans. In this review, after a summary on the known innate CD8(+) T-cell features in the mouse, we propose Eomes together with KIR/NKG2A and CD49d as a signature to standardize the identification of this innate CD8(+) T-cell subset in humans. Next, we discuss IL-4 and IL-15 involvement in the generation of innate CD8(+) T cells and particularly its possible dependency on the promyelocytic leukemia zinc-finger factor expressing iNKT cells, an innate T cell subset well documented for its susceptibility to tumor immune subversion. After that, focusing on cancer diseases, we provide new insights into the potential role of these innate CD8(+) T cells in a physiopathological context in humans. Based on empirical data obtained in cases of chronic myeloid leukemia, a myeloproliferative syndrome controlled by the immune system, and in solid tumors, we observe both the possible contribution of innate CD8(+) T cells to cancer disease control and their susceptibility to tumor immune subversion. Finally, we note that during tumor progression, innate CD8(+) T lymphocytes could be controlled by immune checkpoints. This study significantly contributes to understanding of the role of NK-like CD8(+) T cells and raises the question of the possible involvement of an iNKT/innate CD8(+) T cell axis in cancer.

The Hypothesis of the Human iNKT/Innate CD8(+) T-Cell Axis Applied to Cancer: Evidence for a Deficiency in Chronic Myeloid Leukemia.

We recently identified a new human subset of NK-like [KIR/NKG2A(+)] CD8(+) T cells with a marked/memory phenotype, high Eomesodermin expression, potent antigen-independent cytotoxic activity, and the capacity to generate IFN-γ rapidly after exposure to pro-inflammatory cytokines. These features support the hypothesis that this new member of the innate T cell family in humans, hereafter referred to as innate CD8(+) T cells, has a role in cancer immune surveillance analogous to invariant natural killer T (iNKT) cells. Here, we report the first quantitative and functional analysis of innate CD8(+) T cells in a physiopathological context in humans, namely chronic myeloid leukemia (CML), a well-characterized myeloproliferative disorder. We have chosen CML based on our previous report that IL-4 production by iNKT cells was deficient in CML patients at diagnosis and considering the recent evidence in mice that IL-4 promotes the generation/differentiation of innate CD8(+) T cells. We found that the pool of innate CD8(+) T cells was severely reduced in the blood of CML patients at diagnosis. Moreover, like iNKT and NK cells, innate CD8(+) T cells were functionally impaired, as attested by their loss of antigen-independent cytotoxic activity and IFN-γ production in response to innate-like stimulation with IL-12 + IL-18. Remarkably, as previously reported for IL-4 production by iNKT cells, both quantitative and functional deficiencies of innate CD8(+) T cells were at least partially corrected in patients having achieved complete cytogenetic remission following tyrosine kinase inhibitor therapy. Finally, direct correlation between the functional potential of innate CD8(+) T and iNKT cells was found when considering all healthy donors and CML patients in diagnosis and remission, in accordance with the iNKT cell-dependent generation of innate CD8(+) T cells reported in mice. All in all, our data demonstrate that CML is associated with deficiencies of innate CD8(+) T cells that are restored upon remission, thereby suggesting their possible contribution to disease control. More generally, our study strongly supports the existence of an innate iNKT/innate CD8(+) T-cell axis in humans and reveals its potential contribution to the restoration of tumor immune surveillance.

The downregulation of BAP1 expression by BCR-ABL reduces the stability of BRCA1 in chronic myeloid leukemia.

BCR-ABL induces an intrinsic genetic instability in chronic myeloid leukemia (CML). The protein breast cancer 1, early onset (BRCA1)-associated protein 1 (BAP1) is a deubiquitinase interacting with the DNA repair regulator BRCA1 and is frequently inactivated in many cancers. Here, we report that BAP1 mRNA and protein levels are downregulated in a BCR-ABL1-expressing hematopoietic cell line (UT-7/11). A decrease of BAP1 transcripts is also observed in newly diagnosed CML patients. Moreover, BAP1 protein levels are low or undetectable in CD34(+) cells from CML patients at diagnosis as compared with CD34(+) cells from normal donors. In addition, BRCA1 protein level is reduced in BCR-ABL1-expressing UT-7/11 cells. Finally, the enforced expression of BAP1 is associated with BRCA1 protein deubiquitination and restoration. These results demonstrate BAP1 as a major link with the BCR-ABL-induced downregulation of BRCA1 in CML.