Polyclonal hypergammaglobulinaemia: towards definition of a threshold.

BACKGROUND: Polyclonal hypergammaglobulinaemia (PH) represents a classic diagnosis problem in internal medicine. However, there is no consensus threshold for PH. The aim of this study was to define a threshold for PH. METHODS: We conducted a retrospective multicentric study using laboratory biological databases between 1 January 2016 and 31 December 2016 in two university hospitals and one non-university hospital. All patients 18 years old or over and with at least one serum protein electrophoresis (SPE) available in 2016 were included. Exclusion criteria were monoclonal, biclonal, or oligoclonal spikes or, in case of hypogammaglobulinaemia, proven free light chain gammopathy. The main endpoint was to define the threshold values for PH in this population. Another objective was to define the 95th percentile of the distribution. RESULTS: 20 766 SPEs were included in this cohort. The PH threshold on 95th percentile was 18.9 g/L. The threshold varied according to geographical areas. CONCLUSIONS: This is the first study to scientifically define a PH threshold. The main limitation is that our threshold is only biological. The study was not designed to associate this threshold with a clinically active disease. In conclusion, while the 19 g/L cut-off seems the most relevant threshold, but it will need to be validated by prospective studies.

Polyclonal hypergammaglobulinaemia: towards definition of a threshold.

BACKGROUND: Polyclonal hypergammaglobulinaemia (PH) represents a classic diagnosis problem in internal medicine. However, there is no consensus threshold for PH. The aim of this study was to define a threshold for PH. METHODS: We conducted a retrospective multicentric study using laboratory biological databases between 1 January 2016 and 31 December 2016 in two university hospitals and one non-university hospital. All patients 18 years old or over and with at least one serum protein electrophoresis (SPE) available in 2016 were included. Exclusion criteria were monoclonal, biclonal, or oligoclonal spikes or, in case of hypogammaglobulinaemia, proven free light chain gammopathy. The main endpoint was to define the threshold values for PH in this population. Another objective was to define the 95th percentile of the distribution. RESULTS: 20 766 SPEs were included in this cohort. The PH threshold on 95th percentile was 18.9 g/L. The threshold varied according to geographical areas. CONCLUSIONS: This is the first study to scientifically define a PH threshold. The main limitation is that our threshold is only biological. The study was not designed to associate this threshold with a clinically active disease. In conclusion, while the 19 g/L cut-off seems the most relevant threshold, but it will need to be validated by prospective studies.

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.

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.

Evidence for BCR-ABL-dependent dysfunctions of iNKT cells from chronic myeloid leukemia patients.

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.

Use of cryopreserved autologous cells for extracorporeal photochemotherapy: clinical applications.

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.

[The anti-dsDNA antibodies: validation of an original two step strategy of detection]. / Les anticorps anti-ADN natif : validation d'une stratégie originale de détection en deux temps.

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.

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.

BCR-ABL-induced deregulation of the IL-33/ST2 pathway in CD34+ progenitors from chronic myeloid leukemia 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.