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PDF file - 208KB, Xenotransplant experiments in immunodeficient NOG mice (A); Mouse model of CML-like induced syndrome (B-C).

PDF file - 105KB, CML is associated with high circulating levels of soluble ST2: reversion after IM therapy.

PDF file - 192KB, CML-CP CD34(+) cells proliferate in response to IL-33.

PDF file - 145KB, IL-33 is as efficient as SCF in inducing proliferation of CML-CP CD34(+) cells.

PDF file - 200KB, The kinase activity of BCR-ABL induces upregulation of ST2 in UT7 cells.

PDF file - 154KB, CML-CP CD34(+) cells proliferate in response to IL-33 independently from GM-CSF.

PDF file - 118KB, IL-33 does not modulate apoptosis of CD34(+) cells from CML-CP patients.

Although it is generally acknowledged that cytokines regulate normal hematopoiesis in an autocrine/paracrine fashion, their possible role in chronic myelogenous leukemia (CML) and resistance to imatinib mesylate treatment remain poorly investigated. Here, we report that CD34(+) progenitors from patients with CML at diagnosis are selectively targeted by the cytokine/alarmin interleukin (IL)-33. Indeed, CML CD34(+) progenitors upregulate their cell surface expression of the IL-33–specific receptor chain ST2, proliferate and produce cytokines in response to IL-33, conversely to CD34(+) cells from healthy individuals. Moreover, ST2 overexpression is normalized following imatinib mesylate therapy, whereas IL-33 counteracts in vitro imatinib mesylate-induced growth arrest in CML CD34(+) progenitors via reactivation of the STAT5 pathway, thus supporting the notion that IL-33 may impede the antiproliferative effects of imatinib mesylate on CD34(+) progenitors in CML. Clinically, the levels of circulating soluble ST2, commonly considered a functional signature of IL-33 signaling in vivo, correlate with disease burden. Indeed, these elevated peripheral concentrations associated with a high Sokal score predictive of therapeutic outcome are normalized in patients in molecular remission. Finally, we evidenced a facilitating effect of IL-33 on in vivo maintenance of CD34(+) progenitors from patients with CML by using xenotransplant experiments in immunodeficient NOG mice, and we showed that engraftment of mouse BCR-ABL–transfected bone marrow progenitors was less efficient in IL-33–deficient mice compared with wild-type recipients. Taken together, our results provide evidence that IL-33/ST2 signaling may represent a novel cytokine-mediated mechanism contributing to CML progenitor growth and support a role for this pathway in CML maintenance and imatinib mesylate resistance. Cancer Res; 74(10); 2669–76. ©2014 AACR.

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.

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.

Although it is generally acknowledged that cytokines regulate normal hematopoiesis in an autocrine/paracrine fashion, their possible role in chronic myelogenous leukemia (CML) and resistance to imatinib mesylate treatment remain poorly investigated. Here, we report that CD34(+) progenitors from patients with CML at diagnosis are selectively targeted by the cytokine/alarmin interleukin (IL)-33. Indeed, CML CD34(+) progenitors upregulate their cell surface expression of the IL-33–specific receptor chain ST2, proliferate and produce cytokines in response to IL-33, conversely to CD34(+) cells from healthy individuals. Moreover, ST2 overexpression is normalized following imatinib mesylate therapy, whereas IL-33 counteracts in vitro imatinib mesylate-induced growth arrest in CML CD34(+) progenitors via reactivation of the STAT5 pathway, thus supporting the notion that IL-33 may impede the antiproliferative effects of imatinib mesylate on CD34(+) progenitors in CML. Clinically, the levels of circulating soluble ST2, commonly considered a functional signature of IL-33 signaling in vivo, correlate with disease burden. Indeed, these elevated peripheral concentrations associated with a high Sokal score predictive of therapeutic outcome are normalized in patients in molecular remission. Finally, we evidenced a facilitating effect of IL-33 on in vivo maintenance of CD34(+) progenitors from patients with CML by using xenotransplant experiments in immunodeficient NOG mice, and we showed that engraftment of mouse BCR-ABL–transfected bone marrow progenitors was less efficient in IL-33–deficient mice compared with wild-type recipients. Taken together, our results provide evidence that IL-33/ST2 signaling may represent a novel cytokine-mediated mechanism contributing to CML progenitor growth and support a role for this pathway in CML maintenance and imatinib mesylate resistance. Cancer Res; 74(10); 2669–76. ©2014 AACR.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem-cell malignancy characterized by the presence of the chimeric BCR-ABL oncoprotein with deregulated tyrosine-kinase (TK) activity. Although conventional T cells are acknowledged as important players in the control of CML, a possible modification of invariant NKT (iNKT) cells, known for their antitumoral activity, has not been established as yet. Here, we showed that the expression of perforin, CD95L, and promyelocytic leukemia zinc finger, a transcription factor required for maintenance of iNKT cell functions, was reduced or suppressed in CML patients at diagnosis, as compared with healthy individuals. The proliferation rate of blood iNKT cells in response to their cognate ligand was likewise diminished. These functional deficiencies were corrected in patients having achieved complete cytogenetic remission following TK inhibitor or IFN-α therapy. iNKT cells from CML patients in the chronic phase did not display increased TK activity, which argued against a direct autonomous action of BCR-ABL. Instead, we found that their anergic status originated from both intrinsic and APC-dependent dysfunctions. Our data demonstrate that chronic phase CML is associated with functional deficiencies of iNKT cells that are restored upon remission. These results suggest a possible contribution to disease control by TK inhibitor therapies.

BACKGROUND: Using autologous cryopreserved mononuclear cells (MNCs) for extracorporeal photochemotherapy (ECP) offers several advantages, such as treating patients from geographically distant care centers or maintaining ECP schedule while dramatically reducing number of apheresis sessions. We previously reported that cryopreserved cells retain their immunomodulatory properties when exposed to UVA and psoralen. To date, there are no clinical data on the use of cryopreserved MNCs for ECP (“cryo-ECP”). CASE REPORTS: Three patients were treated by cryo-ECP for refractory dermatomyositis, juvenile localized scleroderma, and acute graft-versus-host disease. For the first two patients, cryo-ECP aimed to reduce the number of apheresis sessions. Each cell product was split into three equal fractions: one was infused, and the other two were frozen for later infusion. The third patient was referred to our center from a hospital 700 km away. Fifteen apheresis procedures were performed during his stay: 12 were immediately treated and infused while three were cryopreserved. After discharge, the three cryopreserved bags were thawed, ECP-treated, and then sent back to the patient. CONCLUSION: In all three patients, cryo-ECP was safe and feasible. These cases illustrate promising clinical applications of the technique, opening perspectives for making ECP much more acceptable to patients while extending its indications.

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.

Among the burgeoning family of unconventional T cells, innate-like CD8(+) T cells bear 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 (IM) 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. Here, we proposed Eomes together with KIR/NKG2A and CD49d as a signature to standardize the identification of this innate CD8(+) T-cell subset in humans. Focusing on cancer diseases, we have provided new insights into the potential role of these IM 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 have observed both the possible contribution of IM CD8(+) T cells to cancer disease control and their susceptibility to tumor immune subversion. Finally, we have investigated the involvement of IL-4 and IL-15 in the generation of the IM CD8(+) T cell pool and its dependency on promyelocytic leukemia zinc finger (PLZF)-expressing invariant Natural Killer T (iNKT) cells. All in all, this study significantly contributes to understanding of the physiopathological role of NK-like CD8(+) T cells in humans and raises the question of the possible involvement of an iNKT/IM CD8(+) T cell axis in cancer.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem-cell malignancy characterized by the presence of the chimeric BCR-ABL oncoprotein with deregulated tyrosine-kinase (TK) activity. Although conventional T cells are acknowledged as important players in the control of CML, a possible modification of invariant NKT (iNKT) cells, known for their antitumoral activity, has not been established as yet. Here, we showed that the expression of perforin, CD95L, and promyelocytic leukemia zinc finger, a transcription factor required for maintenance of iNKT cell functions, was reduced or suppressed in CML patients at diagnosis, as compared with healthy individuals. The proliferation rate of blood iNKT cells in response to their cognate ligand was likewise diminished. These functional deficiencies were corrected in patients having achieved complete cytogenetic remission following TK inhibitor or IFN-α therapy. iNKT cells from CML patients in the chronic phase did not display increased TK activity, which argued against a direct autonomous action of BCR-ABL. Instead, we found that their anergic status originated from both intrinsic and APC-dependent dysfunctions. Our data demonstrate that chronic phase CML is associated with functional deficiencies of iNKT cells that are restored upon remission. These results suggest a possible contribution to disease control by TK inhibitor therapies.

Detection of anti-dsDNA antibodies is one of the major biological criteria of use in the diagnosis of systemic lupus erythematosus. Sensitivity and specificity vary greatly between existing techniques, and differ largely from one study to another. The aim of our prospective study was to evaluate a new strategy of detection comprising two steps, first, the use of a sensitive automated technique, ELISA Phadia EliA™, and second, if necessary, a more specific technique: the Crithidia luciliae immunofluorescence test (CLIFT). The latter was used in case of discrepancy with previous laboratory findings or according to the available clinical data. During the study period of 18 months, 1729 tests were requested of which 96 were finally assayed using CLIFT. Analysis of 53 discordant results showed 14 cases of lupus identified only with ELISA, and 3 only by Crithidia. In addition, 35 likely false positives of ELISA were evidenced by negative CLIFT results. These data show a clear gain in sensitivity without any loss of specificity due to the use of a second technique. Thus, this strategy was validated in our lab; it can be useful by any medical laboratory because the cost of few Crithidia luciliae slides is very low.

Innate-like CD8(+) T cells have been recently described in mice. These cells express NK cell markers with high levels of the transcription factor Eomesodermin (Eomes(br)) and rapidly produce IFN-γ in response to IL-12 and IL-18 stimulation without previous exposure to antigen. To date, only a single study in human has investigated Eomes(br) innate-like T cells which were found in small numbers in fetal spleen and cord blood. In mice, their generation depends on IL-4 expressing invariant NKT (iNKT) cells. Here, we identified a similar subset of T cells, mainly CD8(+) T cells, in PBMC from healthy adult individuals that have increased expression of Eomes. They harbor a marked innate phenotype as attested by a higher frequency of perforin(+) and NK-like (CD158(+) and/or CD159(+)) cells, as compared with total T cells. Likewise, they preferentially produce IFN-γ in response to stimulation by IL-12 and IL-18. Remarkably, in patients with chronic myeloid leukemia (CML), a disease known to be associated with a deficiency in IL-4 expression by iNKT cells, the size of Eomes(br) innate-like T cells is severely impaired. and they have lost the capacity to produce IFN-γ after stimulation with IL-12 and IL-18. These impairments together with the deficiency in IL-4 expression by iNKT cells are corrected in CML patients having achieved complete remission. Altogether, these results demonstrate the presence of Eomes expressing innate-like T cells in adult human peripheral blood.

Although it is generally acknowledged that cytokines regulate normal hematopoiesis in an autocrine/paracrine fashion, their possible role in chronic myeloid leukemia (CML) and resistance to imatinib mesylate (IM) treatment remain poorly investigated. Here, we report that CD34(+) progenitors from CML patients at diagnosis are selectively targeted by the cytokine/alarmin IL-33. Indeed, CML CD34(+) progenitors up-regulate their cell surface expression of the IL-33-specific receptor chain ST2, proliferate and produce cytokines in response to IL-33, conversely to CD34(+) cells from healthy individuals. Moreover, ST2 overexpression is normalized following IM therapy, while IL-33 counteracts in-vitro IM-induced growth arrest in CML CD34(+) progenitors via re-activation of the STAT5 pathway. Clinically, CML is associated with high circulating levels of soluble ST2, commonly used as a functional signature of IL-33 signaling in vivo. Taken together, our results support the hypothesis that the IL-33/ST2 pathway facilitates Bcr/Abl-induced leukemogenesis and contributes to IM resistance in CML patients.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder, caused by the presence of the Philadelphia chromosome and the consecutive chimerical BCR-ABL oncoprotein, with deregulated tyrosine kinase (TK) activity. Whether invariant NKT (iNKT) cells, which are documented for their anti-tumoral activity, are affected during the disease remains unknown. Here, we addressed this issue by showing that proliferation rate of blood iNKT cells in response to their cognate ligand α-galactosylceramide is altered in CML patients at diagnosis, as compared to healthy subjects. This anergic state of iNKT cells was associated with both intrinsic and APC-dependent alterations. Furthermore, expression of perforin and CD95-L as well as that of PLZF, a transcription factor that is requisite for maintenance of functions of iNKT cells, were also decreased in patients at diagnosis. Importantly, all these functional impairments were corrected in patients who achieved a complete cytogenetic remission with the TK inhibitor imatinib mesylate (IM) or IFN-γ therapy. Together, these data demonstrate that chronic phase CML is associated with an acquired but potentially reversible defect in iNKT cells. Given the critical role of iNKT cells in tumor surveillance, it is tempting to propose that iNKT cell dysfunctions associated with CML thus could be one way used by tumors to escape from the anti-CML immune response while IM and IFN-γ exert their anti-leukemic effects in part in targeting iNKT cells.