Danièle Bensoussan

Natural-killer cell amplification for adoptive leukemia relapse immunotherapy: Comparison of three cytokines, IL-2, IL-15, or IL-7 and impact on NKG2D, KIR2DL1, and KIR2DL2 expression

OBJECTIVE Natural killer (NK) cells are a lymphocyte subset that, in a hematopoietic stem cell transplantation setting, mediates a graft-vs-leukemia effect without any graft-vs-host disease. We aimed to evaluate an isolation method that can be used with Good Manufacturing Practices-grade reagents and to compare three cytokines for expansion in order to design future clinical protocols based on donor NK-cell infusions to cure relapse after allograft. MATERIALS AND METHODS NK cells were enriched using a CD3/CD19 depletion method and expanded for 13 days in the presence of 2, 10, and 50 ng/mL interleukin (IL)-2, IL-15, or IL-7. NK-cell cytotoxicity was evaluated after isolation and culture. Expression of NKG2D, KIR2DL2, and KIR2DL1 was monitored during expansion. RESULTS Highly T- and B-cell-depleted NK cells were obtained and enriched 2.6-fold. The optimal cytokine concentration for expansion was 10 ng/mL for IL-2 or 50 ng/mL for IL-15. NK-cell cytotoxicity was significantly improved after an overnight incubation with 10 or 50 ng/mL IL-2 or with 2, 10, or 50 ng/mL IL-15, and after 13 days with 50 ng/mL IL-15. The use of a combination of IL-2 and IL-15 showed no additional benefit and negative results were obtained with IL-7. The three NK cell receptors were significantly upregulated after culture, mainly with IL-2 or IL-15. CONCLUSION In our study, 10 ng/mL IL-2 or 50 ng/mL IL-15 were the optimal concentrations for expansion and were equivalent in significantly enhancing cytotoxicity and modifying NK-cell receptor expression patterns.

Hematologic recovery after autologous PBPC transplantation: importance of the number of postthaw CD34+ cells†

BACKGROUND: The implementation of a quality‐assurance program is a major requirement to ensure quality and safety of the final PBPC components intended for clinical use. It is not clear whether the quantification of CFU‐GM and CD34+ cells should be done on fresh components and after cryopreservation, which better represents the actual composition of the graft.

Rapid generation of full clinical-grade human antiadenovirus cytotoxic T cells for adoptive immunotherapy.

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-γ (IFN-γ) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01±0.84×106 total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56%±20.8%, yield=51%±32.4%) but also CD8+ (mean=42%±27%, yield=56%±39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-γ and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

T-cell immune constitution after peripheral blood mononuclear cell transplantation in complete DiGeorge syndrome

Summary.  Complete DiGeorge syndrome (cDGS) is a congenital disorder characterized by typical facies, thymic aplasia, susceptibility to infections, hypoparathyroidism and conotruncal cardiac defect. Fetal thymus or post‐natal thymus tissue transplantations and human leucocyte antigen (HLA)‐genoidentical bone marrow transplantations were followed in a few cases by immune reconstitution. More recently, a peripheral blood mononuclear cell transplantation (PBMCT) was performed with an HLA‐genoidentical donor and followed by a partial T‐cell engraftment and immune reconstitution. We report a boy with cDGS, without cardiac defect, who suffered recurrent severe infections. At the age of 4 years, he underwent PBMCT from his HLA‐genoidentical sister. He received no conditioning regimen, but graft‐versus‐host disease (GVHD) prophylaxis was with oral cyclosporin A and mycophenolate mofetil. Toxicity was mild, with grade I acute GVHD. The patient is currently 2·5 years post‐PBMCT with excellent clinical performances. Mixed chimaerism can only be observed on the T‐cell population (50% donor T cells). T‐lymphocyte count fluctuated (CD3 more than 400 × 106/l at d 84 and CD4 more than 200 × 106/l at d 46). Exclusive memory phenotype T cells and absence of new thymic emigrants suggest expansion of infused T cells. T‐cell mitogen and tetanus antigen responses normalized a few months after transplantation. After immunizations, specific antibodies were produced. PBMCT from an HLA identical sibling could be an efficient treatment of immune deficiency in cDGS.

Residual viability is a predictor of the perfusion enhancement obtained with the cell therapy of chronic myocardial infarction: a pilot multimodal imaging study.

Purpose Up to now, there has been limited investigation into cell therapy in the chronic phase of severe myocardial infarction (MI), and many questions remain concerning the contribution of the engrafted cells and especially their impact on the reperfusion of MI areas, when assessed by objective quantitative imaging techniques. This randomized pilot SPECT, PET, and MRI study was aimed at assessing the effects of bone marrow mononuclear cells (BMNCs) when implanted in areas of severe and chronic MI. Materials and Methods Fourteen patients, who were referred for coronary artery bypass grafting (CABG) and in whom a screening MIBI-SPECT revealed severely damaged myocardium (<50% uptake under nitrate), were randomized between a cell therapy group (n = 7; CABG and injection of BMNCs within MI areas) and a control group (n = 7; CABG alone). Results The MI areas exhibited a posttherapeutic enhancement in the rest–uptake of MIBI in the cell therapy group [difference between 6-month control and baseline: +6.8% (5.4%), P = 0.03] but not in the control group [+1.0% (4.3%)]. However, in a per-patient analysis, this improvement was significant (> +9%) in only 3 cell therapy patients, whose MI areas before therapy had a higher FDG uptake [59% (9%) vs 38% (8%), P = 0.03] and a lower transmural extent at MRI [40% (6%) vs 73% (18%), P = 0.03] when compared with the other cell therapy patients. Conclusions Perfusion enhancement, obtained with BMNCs in areas of chronic MI, might require an intermediate level of viability documented with FDG-PET and MRI and that totally necrotic MI seems refractory to this cell therapy technique.

Mesenchymal stem cells derived from Wharton's jelly: Comparative phenotype analysis between tissue and in vitro expansion

Mesenchymal stem cells (MSCs) are useful multipotent stem cells that are found in many tissues. While MSCs can usually be isolated from adults via bone marrow aspiration (BM-MSCs), MSCs derived from the discarded umbilical cord, more precisely from Whartons jelly (WJ), offer a low-cost and pain-free collection method of MSCs that may be cryogenically stored, and are considered extremely favorable for tissue engineering purpose. The aim of this study was to analyze the harvested number of cells per centimeter of human umbilical cord (UC) and carry out the phenotype of these WJ-MSCs after explant or enzymatic methods. Fresh UCs were obtained from full-term births, and processed within 6 hours from partum to obtain the WJ-MSCs. UC sections were analyzed in confocal microscopy to analyze cells phenotype in situ. Others UC components were treated either by enzymatic method or by explant method to obtain isolated cells and to analyze cells phenotype until the end of the first passage. We have successfully generated MSCs from UC by using explant and enzymatic methods. Using microscopy confocal, we identified the expression of some MSCs markers in situ of Whartons jelly tissue as well as in perivascular region. Our comparative study, between explant and enzymatic digestion, indicated, that WJ expressed most of MSCs markers in both conditions, but a remarkable variation of cell phenotype expression was distinguished after primary culture comparing to directly isolated cells by enzymatic digestion. We also studied the expression of CD271, which showed to be weakly expressed in situ on fresh fragment of WJ.

Quantitative and Qualitative CD4 T Cell Immune Responses Related to Adenovirus DNAemia in Hematopoietic Stem Cell Transplantation

The nature of adenovirus (AdV)-specific T cells that could best predict the capacity of immunocompromised host to fight AdV is unclear. To this aim, 47 pediatric patients were enrolled for at least 3 months either at allogeneic bone marrow transplantation (BMT) (23 genoidentical, 18 unrelated of which 9 were 10/10 and 9 were 9/10 HLA-matched) or at unrelated cord blood transplantation (n = 6). Enumeration of AdV-specific CD4 T cells secreting cytokines (flow cytometry) and proliferative responses to AdV ((3)HT-incorporation) were compared to AdV-DNAemia. A total of 44/47 patients did not evidence AdV-DNAemia. Thirty-two of 44 (73%) developed CD4-mediated interferon-gamma (IFN-γ) responses to AdV (median 0.36 CD4/μL of blood) since the first month post-HSCT (n = 11: 8 genoidentical and 3 unrelated) or the third month (n = 21 additional patients). At 3 months, both incidence and level intensities of AdV-specific CD4 appeared similar in genoidentical and unrelated BMT (70% and 80%; 0.36 and 0.21 CD4/μL, respectively) and not statistically different from age-matched controls (76%; 1.35 CD4/μL), whereas cord blood transplanted patients exhibited similar incidence but higher level intensities (67%; 1.49 CD4/μL). Polyfunctional (IL2 + IFN-γ) and proliferative responses appeared later, after the third month. Three of 4 9/10 HLA-matched unrelated HSCT that did not develop immunity to AdV presented chemotherapy-resistant AdV-DNAemia at 3 to 5 months post-hematopoietic stem cell transplantation (HSCT). Two were successfully treated with AdV-specific CTL infusion. Monitoring, since month 1 post-HSCT, of IFN-γ-secreting AdV-specific CD4 appears suitable for early detection of at-risk patients especially in 9/10 HLA-matched unrelated HSCT and preferable to monitoring of more delayed IL2- and proliferative responses.

Donor's age dependent proliferation decrease of human bone marrow mesenchymal stem cells is linked to diminished clonogenicity

While mesenchymal stem cells represent an interesting cell source for regenerative medicine, several points have to be investigated to improve their use in clinical, and in particular in the elderly population. This work studied the proliferation capacity of mesenchymal stem cells isolated from human bone marrow in function of donors age. Doubling time after in vitro culture, clonogenicity and phenotype were analyzed in 17 samples ranging from 3 to 85 years old (mean 47 ± 27). Results showed an increase in the doubling time for cell coming from old donor compared to cells coming from young ones. This was accompanied by a decrease in clonogenicity while no changes were observe in cell phenotype. In conclusion, this study showed an effect of donors age on the proliferation capacity of mesenchymal stem cells isolated from bone marrow that was correlated to a decrease in clonogenicity. The comprehension of molecular mechanism involved in this process could help to improve the clinical application of mesenchymal stem cells.

The clinical value of concomitant Epstein Barr virus (EBV)-DNA load and specific immune reconstitution monitoring after allogeneic hematopoietic stem cell transplantation.

BACKGROUND Monitoring of EBV DNAemia after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary, but not sufficient, to identify patients at risk of EBV-induced post-transplantation lymphoproliferative disorders (PTLD). Combining this with quantifying EBV-specific cellular immunity was shown to be helpful. In this study, we evaluated the value of IFNγ-Elispot assay in monitoring EBV DNAemia after HSCT. METHODS EBV-DNA load in whole blood was monitored at least weekly using real-time PCR in 40 recipients of HSCT. Quantitative and qualitative T-cell recoveries, including EBV-specific T-cell quantification by Elispot assay, were studied 60, 100, 180 and 360 days after HSCT. RESULTS Among the 35 evaluable patients, 14 (35%) presented EBV DNAemia, only 2/14 (14%) needing pre-emptive treatment with rituximab. The greatest risk factor for EBV DNAemia was the presence of anti-thymocyte globulin (ATG) (p=0.005). EBV-specific cellular immune recovery was monitored by IFNγ-Elispot assay. Using multivariate analysis, four factors were found to significantly influence IFNγ-Elispot results at defined times post-HSCT: EBV DNAemia, young age, global T-cell recovery and severe acute GVHD. In those cases where EBV DNAemia occurred and cleared spontaneously, Elispot results gave more than 1000 spot-forming cells (SFC)/10(6)PBMC. CONCLUSION Elispot assay may be usefully combined with EBV-DNA load monitoring to determine when a patient should receive pre-emptive treatment, or when the clinician should avoid Rituximab use which severely immunocompromises patients.

The WHIM syndrome shows a peculiar dysgranulopoiesis: myelokathexis

A 17-year-old boy had been born at term after an uneventful pregnancy to non-consanguineous parents. His siblings and parents were well. He had experienced numerous upper respiratory tract infections since birth, which had caused pulmonary atelectasia requiring surgical removal of the affected lobe. He had suffered recurrent herpetic peribuccal infection. Warts had appeared on his hands at 6 years of age and extended gradually to the elbows, arms, legs, knees and feet. Pancytopenia was first documented at the age of 1 month. White blood cell counts varied from 1Æ9 to 3Æ9 · 10/l with neutrophil counts of 0Æ9–2Æ8 · 10/l. There was no cyclic pattern of neutropenia. His haemoglobin concentration ranged between 10 and 12 g/dl and the platelet count was 47– 127 · 10/l. Blood smears showed abnormal neutrophils with condensed nuclei connected by long, stringy filaments. The bone marrow was hypercellular with an increased proportion of mature myeloid cells (left). Many neutrophils had bisegmented nuclei with dense pyknotic lobes connected by long filaments leading to bizarre forms, such as ‘eyeglasses’ or ‘clover leaf’ (right). Occasionally, these filaments were twisted around each other. Some eosinophils showed cytoplasmic vacuolation. No significant morphological abnormalities were observed in the other cell lineages. Folic acid and vitamin B12 levels were normal. Immunological investigation showed severe combined immunodeficiency. Both Band T-cell subpopulations were reduced, particularly CD4 T cells. T-cell responses to vaccine antigens were moderately affected. Global hypogammaglobulinaemia was observed (IgG < 6 g/l; normal levels of IgG2 and IgG3). No antibodies to leucocytes or platelets were detected and karyotype analysis was normal. Monthly treatment with gammaglobulins and filgrastim was initiated when the child was 7 years of age, producing a subsequent decrease in the rate of infections and an increased neutrophil count (>1 · 10/l). The patient presented the full spectrum of findings described in the WHIM syndrome, a rare immunodeficiency disorder characterised by warts, hypogammaglobulinaemia, infections and myelokathexis. Myelokathexis is characterised by a prolonged retention of neutrophils in the bone marrow compartment leading to neutropenia, despite hyperplasia of bone marrow myeloid cells, and degenerative changes in mature neutrophils. The observation of these morphologic features was strongly suggestive of myelokathexis and is helpful in the diagnosis of WHIM syndrome. This syndrome is caused by the mutation of a chemokine receptor gene, leading to production of the mutant CXCR4 protein that causes abnormal aptoptosis and migratory function, thought to be related to the cause of chronic leucopenia.