Xavier Lafarge

Long-term expansion of effector/memory Vδ2− γδ T cells is a specific blood signature of CMV infection

The ability of human gammadelta T cells to develop immunologic memory is still a matter of debate. We previously demonstrated the involvement of Vdelta2- gammadelta T lymphocytes in the response of immunosuppressed organ recipients to cytomegalovirus (CMV). Here, we demonstrate their ability to mount an adaptive immune response to CMV in immunocompetent subjects. Vdelta2- gammadelta T-cell peripheral blood numbers, repertoire restriction, and cytotoxicity against CMV-infected fibroblasts were markedly increased in CMV-seropositive, compared with CMV-seronegative, healthy persons. Whereas Vdelta2- gammadelta T cells were found as naive cells in CMV- patients, they virtually all exhibited the cytotoxic effector/memory phenotype in CMV+ patients, which is also observed in transplanted patients challenged with CMV. This long-term complete remodeling of the Vdelta2- gammadelta T-cell population by CMV predicts their ability to exhibit an adaptive anti-CMV immune response. Consistent with this, we observed that the secondary response to CMV was associated with a faster gammadelta T-cell expansion and a better resolution of infection than the primary response. In conclusion, the increased level of effector-memory Vdelta2- gammadelta T cells in the peripheral blood is a specific signature of an adaptive immune response to CMV infection of both immunocompetent and immunosuppressed patients.

Interleukin-6 (IL-6) and low O2 concentration (1%) synergize to improve the maintenance of hematopoietic stem cells (pre-CFC)

Low O2 concentration (1%) favors the self‐renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL‐6 influences both stem cells and committed progenitors at 20% O2, we studied its effects in cultures at 1% O2. The pre‐CFC activity in Lin− population of mouse bone marrow was analyzed following 10 days of serum‐free culture in medium (LC1) supplemented with IL‐3 with and without IL‐6, at 20 and 1% O2 and phenotypic differentiation and proliferative history monitored. The IL‐6 receptor expression and initiation of VEGF‐A synthesis were also investigated. At 20% O2, the effects of IL‐6 on pre‐CFC were negligible but effects on CFC were apparent; conversely, at 1% O2, the IL‐6 enhances activity of pre‐CFC but not of CFC. Unlike at 20% O2, at 1% O2 a subpopulation of cells remained Lin− in spite of extensive proliferation. However, the absolute number of Lin− cells, did not correlate with pre‐CFC activity. A relative increase in VEGF transcripts at 1% O2 in presence of IL‐3 alone was enhanced by the addition of IL‐6. IL‐6 enhanced pre‐CFC activity at 1% O2 and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O2 does not exist in tissues in vivo, further studies in vitro at lower O2 concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells. J. Cell. Physiol. 212: 68–75, 2007.

Combination of low O2 concentration and Mesenchymal Stromal Cells during culture of Cord Blood CD34 + Cells Improves the Maintenance and Proliferative Capacity of Hematopoietic Stem Cells

The physiological approach suggests that an environment associating the mesenchymal stromal cells (MSC) and low O2 concentration would be most favorable for the maintenance of hematopoietic stem cells (HSCs) in course of ex vivo expansion of hematopoietic grafts. To test this hypothesis, we performed a co‐culture of cord blood CD34+ cells with or without MSC in presence of cytokines for 10 days at 20%, 5%, and 1.5% O2 and assessed the impact on total cells, CD34+ cells, committed progenitors (colony‐forming cells—CFC) and stem cells activity (pre‐CFC and Scid repopulating cells—SRC). Not surprisingly, the expansion of total cells, CD34+ cells, and CFC was higher in co‐culture and at 20% O2 compared to simple culture and low O2 concentrations, respectively. However, co‐culture at low O2 concentrations provided CD34+ cell and CFC amplification similar to classical culture at 20% O2. Interestingly, low O2 concentrations ensured a better pre‐CFC and SRC preservation/expansion in co‐culture. Indeed, SRC activity in co‐culture at 1.5% O2 was higher than in freshly isolated CD34+ cells. Interleukin‐6 production by MSC at physiologically low O2 concentrations might be one of the factors mediating this effect. Our data demonstrate that association of co‐culture and low O2 concentration not only induces sufficient expansion of committed progenitors (with respect to the classical culture), but also ensures a better maintenance/expansion of hematopoietic stem cells (HSCs), pointing to the oxygenation as a physiological regulatory factor but also as a cell engineering tool. J. Cell. Physiol. 227: 2750–2758, 2012.

Comparison of CD34+ cell collection on the CS-3000+ and Amicus blood cell separators.

BACKGROUND:  Mobilized PBPCs, detectable on the basis of CD34 expression, can be collected on various cell separators. The CD34+ cell collection efficiencies of two cell separators (CS‐3000+ and Amicus, Baxter) were tested on two comparable groups of oncology patients.

Potent Graft-versus-Leukemia Effect after Reduced-Intensity Allogeneic SCT for Intermediate-Risk AML with FLT3-ITD or Wild-Type NPM1 and CEBPA without FLT3-ITD

To investigate the role of reduced-intensity allogeneic (RIC-allo) stem cell transplant (SCT) as postremission therapy in adult intermediate-risk patients with acute myelogenous leukemia (AML) with FLT3-ITD or wild-type NPM1 and CEBPA without FLT3-ITD, we conducted a single-center retrospective study between January 2001 and December 2010. Sixty-six patients were included: 37 treated with RIC-alloSCT and 29 with nonallogeneic SCT therapies. Both groups were comparable concerning age, WBC count at diagnosis, gender, karyotype, genotype, and number of courses of chemotherapy to reach complete remission (CR1). Median follow-up after CR1 was 37 months (range, 11-112 months) and 48 months (range, 9-83 months) in the allo and no-allo groups, respectively. In the allo versus no-allo groups, the 3-year cumulative incidence of relapse (CIR) rates were 25% ± 8% versus 61% ± 9%; P = .005. The 3-year nonrelapse mortality (NRM), overall survival (OS), and relapse-free survival (RFS) were 22% ± 7% versus 4% ± 4% (P = .005), 52% ± 9% versus 44% ± 10% (P = .75), and 53% ± 9% versus 35% ± 9% (P = .28), respectively. Multivariate analysis indicated that CIR was reduced by allo (hazard ratio [HR], 0.32; P = .01). A landmark analysis performed at day 185 after CR1 confirmed a lower CIR after allo. RIC-allo reduces the risk of relapse, suggesting a potent graft-versus-leukemia (GVL) effect in these patients at a high risk of relapse.

Expression of MHC class I receptors confers functional intraclonal heterogeneity to a reactive expansion of γδ T cells

NK cell receptors for MHC class I molecules (MHC‐NKR) can be expressed by T cell subsets. The restricted repertoire and phenotypic characteristics of MHC‐NKR+ T cells indicate that expression of MHC‐NKR is acquired upon antigenic challenge and might promote expansion of T cells. Previous studies performed on in vitro generated αβ T cell clones concluded that MHC‐NKR expression was not a clonal attribute. Here, we examined a massive monoclonal expansion of a non‐leukemic γδ T cell population found in the peripheral blood of a lung‐transplanted patient who suffered from a cytomegalovirus infection. Despite their monoclonality, these T cells displayed a heterogeneous and stable in vivo Ig‐ and lectin‐like MHC‐NKR phenotype. Twenty percent of the cells displayed a CD94+NKG2A+ phenotype, and 10% were labeled with an anti‐CD158b1/b2/j monoclonal antibody. A CD158b/j+ γδ T cell clone derived in vitro from patients peripheral blood lymphocytes was shown to express the activating form CD158j (KIR2DS2), which once cross‐linked stimulated the clone cytolytic function and costimulated the TCR‐induced production of cytokines, independently of the killer‐activating receptor‐associated protein (KARAP). In conclusion, heterogeneity of MHC‐NKR expression confers a functional intraclonal diversity that may participate to induction of specific γδ T cell effector functions or proliferation upon pathogen challenge.

CD34+ cells obtained from “good mobilizers” are more activated and exhibit lower ex vivo expansion efficiency than their counterparts from “poor mobilizers”

BACKGROUND: The classification of patients into “good” or “poor” mobilizers is based on CD34+ cell count in their peripheral blood (PB) after granulocyte–colony‐stimulating factor (G‐CSF) injection. We hypothesized that, apart from their mobilization from marrow to the blood, the response to G‐CSF of CD34+ cells also includes activation of proliferation, metabolic activity, and proliferative capacity.

Whole-blood leukodepletion filters as a source of CD34+ progenitors potentially usable in cell therapy

BACKGROUND:  Used leukodepletion filters (LDFs), containing billions of white blood cells (WBCs), are discarded. Because the steady‐state blood contains low quantities of stem and progenitor cells that are retained in LDFs, the viability and the functional properties of mononuclear cells (MNCs) and CD34+ cells recovered from LDFs were investigated.

Gamma‐delta T cell expansion is closely associated with cytomegalovirus infection in all solid organ transplant recipients

We read with interest the manuscript published by Puig-Pey et al. entitled ‘‘Characterisation of gammadelta T cells in organ transplantation’’ [1]. For a better reading of this paper, we would like to add several comments on the basis of our experience in cd T cell immunomonitoring in kidney transplant recipients (KTR), to enlighten their role in the context of cytomegalovirus (CMV) infection. Indeed, in KTR, we demonstrated, 10 years ago, that CMV infection is the sole parameter independently associated with a persistent

Thrombopoietin to replace megakaryocyte-derived growth factor: impact on stem and progenitor cells during ex vivo expansion of CD34+ cells mobilized in peripheral blood

BACKGROUND: The first protocol of ex vivo expansion that enabled almost total abrogation of postmyeloablative chemotherapy neutropenia was based on a three‐cytokine cocktail (stem cell factor [SCF], granulocyte–colony‐stimulating factor [G‐CSF], pegylated‐megakaryocyte growth and development factor [PEG‐MGDF]) in a serum‐free medium. Since the clinical‐grade molecule MGDF is no longer available on the market, we evaluated its substitution by thrombopoietin (TPO).