Immunomagnetic selective donor-derived CD4+CCR7+ T cell depletion procedure for peripheral blood stem cells graft.

PURPOSE OF THE STUDY: While acute graft-versus-host-disease (GVHD) is a T cell-mediated disease caused by alloreactive donor T cells, we and others have highlighted that patients who received higher proportion of donor CD4+ naïve and central memory T cells expressing the chemokine receptor 7 (CCR7) more often developed acute GVHD than those who did not. Consequently, we then investigated in vitro the impact of selective CD4+ CCR7+ T cell depletion on immune reactions and showed that such a depletion reduced alloreactivity without altering acquired anti-infectious reactions. In order to translate these findings to clinic, we now developed a compliant procedure for a selective reduction of the CD4+ naïve and central memory T cell subset relevant to peripheral blood stem cell (PBSC) allografts. PATIENTS AND METHODS: We performed a two-step immunomagnetic depletion of CD4+ CCR7+ T cells from ten G-CSF-mobilized PBSC apheresis samples. RESULTS: A median of 89% (82-94%) of CD4+ CCR7+ T cells could be depleted. This allowed a marked reduction of the alloreactive immune response against allogenic dendritic cells compared with unmanipulated cells. The preservation of CD34+ cell number and the hematopoietic progenitor function were controlled. Functional tests showed that the selection procedure did not interfere with the capacity of pathogen-specific T cells to produce interferon-gamma in response to certain viral pathogens. CONCLUSION: Our results pave the way to a feasible procedure that can be used in patients undergoing allo-hematopoietic cell transplantation and particularly for improving haploidentical transplant results by controlling GVHD, the main immune complication.

An avoidable cause of thymoglobulin anaphylaxis.

BACKGROUND: Thymoglobulin® (anti-thymocyte globulin [rabbit]) is a purified pasteurised, gamma immune globulin obtained by immunisation of rabbits with human thymocytes. Anaphylactic allergic reactions to a first injection of thymoglobulin are rare. CASE PRESENTATION: We report a case of serious anaphylactic reaction occurring after a first intraoperative injection of thymoglobulin during renal transplantation in a patient with undiagnosed respiratory allergy to rabbit allergens. CONCLUSIONS: This case report reinforces the importance of identifying rabbit allergy by a simple combination of clinical interview followed by confirmatory skin testing or blood tests of all patients prior to injection of thymoglobulin, which is formally contraindicated in patients with a history of hypersensitivity to rabbit proteins.

Diagnosis of primary antibody and complement deficiencies in young adults after a first invasive bacterial infection.

OBJECTIVES: Screening for primary immunodeficiencies (PIDs) in adults is recommended after two severe bacterial infections. We aimed to evaluate if screening should be performed after the first invasive infection in young adults. METHODS: Eligible patients were retrospectively identified using hospital discharge and bacteriology databases in three centres during a 3-year period. Eighteen to 40-year-old patients were included if they had experienced an invasive infection with encapsulated bacteria commonly encountered in PIDs (Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), Neisseria gonorrhoeae (NG), Haemophilus influenzae (HI), or group A Streptococcus (GAS)). They were excluded in case of general or local predisposing factors. Immunological explorations and PIDs diagnoses were retrieved from medical records. Serum complement and IgG/A/M testings were systematically proposed at the time of study to patients with previously incomplete PID screening. RESULTS: The study population comprised 38 patients. Thirty-six had experienced a first invasive episode and a PID was diagnosed in seven (19%): two cases of common variable immunodeficiency revealed by SP bacteraemia, one case of idiopathic primary hypogammaglobulinaemia, and two cases of complement (C6 and C7) deficiency revealed by NM meningitis, one case of IgG2/IgG4 subclasses deficiency revealed by GAS bacteraemia, and one case of specific polysaccharide antibody deficiency revealed by HI meningitis. Two patients had previously experienced an invasive infection before the study period: in both cases, a complement deficiency was diagnosed after a second NM meningitis and a second NG bacteraemia, respectively. CONCLUSION: PID screening should be considered after a first unexplained invasive encapsulated-bacterial infection in young adults.

Role of B cells in the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is an orphan disease characterized by progressive fibrosis of the skin and internal organs. Aside from vasculopathy and fibrotic processes, its pathogenesis involves an aberrant activation of immune cells, among which B cells seem to play a significant role. Indeed, B cell homeostasis is disturbed during SSc: the memory subset is activated and displays an increased susceptibility to apoptosis, which is responsible for their decreased number. This chronic loss of B cells enhances bone marrow production of the naïve subset that accounts for their increased number in peripheral blood. This permanent activation state can be explained mainly by two mechanisms: a dysregulation of B cell receptor (BCR) signaling, and an overproduction of B cell survival signals, B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). These disturbances of B cell homeostasis induce several functional anomalies that participate in the inflammatory and fibrotic events observed during SSc: autoantibody production (some being directly pathogenic); secretion of pro-inflammatory and pro-fibrotic cytokines (interleukin-6); direct cooperation with other SSc-involved cells [fibroblasts, through transforming growth factor-ß (TGF-ß) signaling, and T cells]. These data justify the evaluation of anti-B cell strategies as therapeutic options for SSc, such as B cell depletion or blockage of B cell survival signaling.

Risk factors and outcome of graft failure after HLA matched and mismatched unrelated donor hematopoietic stem cell transplantation: a study on behalf of SFGM-TC and SFHI.

Graft failure remains a severe complication of hematopoietic stem cell transplantation (HSCT). Several risk factors have already been published. In this study, we re-evaluated them in a large cohort who had the benefit of the recent experience in HSCT (2006-2012). Data from 4684 unrelated donor HSCT from 2006 to 2012 were retrospectively collected from centers belonging to the French Society for Stem Cell Transplantation. Among the 2716 patients for whom HLA typing was available, 103 did not engraft leading to a low rate of no engraftment at 3.8%. In univariate analysis, only type of disease and status of disease at transplant for malignant diseases remained significant risk factors (P=0.04 and P<0.0001, respectively). In multivariate analysis, only status of disease was a significant risk factor (P<0.0001). Among the 61 patients who did not engraft and who were mismatched for 1 HLA class I and/or HLA-DP, 5 donor-specific antibodies (DSAs) were detected but only 1 was clearly involved in graft failure, for the others their role was more questionable. Second HSCT exhibited a protective although not statistically significant effect on OS (hazard ratio=0.57 [0.32-1.02]). In conclusion, only one parameter (disease status before graft) remains risk factor for graft failure in this recent cohort.

Suggestive evidence of a role of HLA-DRB4 mismatches in the outcome of allogeneic hematopoietic stem cell transplantation with HLA-10/10-matched unrelated donors: a French-Swiss retrospective study.

We have conducted a retrospective study on 251 patients from three centers in France and Switzerland between 2004 and 2010 with the goal to evaluate the impact of HLA-DRB3/B4/B5 allele mismatching after HLA-10/10-matched unrelated allogeneic hematopoietic stem cell transplantation (HSCT). Fourteen (5.5%) patients receiving HSCT from an HLA-10/10-matched unrelated donor had a mismatched DRB4 donor, 23 (9.5%) patients had a mismatched DRB3 donor and 214 (85%) had a fully matched unrelated donor (HLA-10/10) without DRB3- or DRB4-mismatched donor. We compared the outcomes of 37 patients with a DRB3 or DRB4 mismatch with the rest of the population. The median survival for a patient without DRB3/4 mismatch was 18 months (95% confidence interval (CI), 13-29), for DRB3-mismatched patients 32 months (95% CI, 13-NR) and for DRB4-mismatched patients 7 months (95% CI, 3-NR). The multivariate analysis showed a significant impact of DRB4 mismatching on survival (Hazards ratio (HR)=2.1 (95% CI, 1.01-4.67), P=0.045), acute GvHD (HR=2.66 (95% CI, 0.99-7.09) P=0.05) and on transplant-related mortality (HR=2.8; (95% CI, 1.7-4.4) P=0.024). In the view of an impact of DRB4 locus mismatch on clinical outcome, it would be important to confirm this observation in a prospective study as it may be worth considering DRB4 in the unrelated donor selection.

The novel HLA-B*39:93 allele was identified by sequence-based typing in a French family.

A novel HLA-B allele, B*39:93, was identified in a French family.

Impact of rabbit ATG-containing myeloablative conditioning regimens on the outcome of patients undergoing unrelated single-unit cord blood transplantation for hematological malignancies.

This study aimed to assess the impact of antithymocyte globulin (ATG) on patient outcome in a retrospective series of 91 patients (median age: 12 years) who underwent unrelated single-unit cord blood transplantation (allo-CBT) following a myeloablative conditioning regimen. Cord blood units were HLA-matched (6/6, n=18; 21%), one-Ag mismatched (n=30, 35%) or two-Ag mismatched (n=38; 44%). In this series, the OS, nonrelapse mortality (NRM) and cumulative incidence of relapse were 47±6%, 23±4% and 48±5%, respectively. Among 46 patients who received ATG as part of the conditioning regimen, the incidence of acute and chronic GVHD was lower than that in the group of 45 patients who did not receive ATG (20% vs 43%; P=0.03). However, multivariate statistical analysis revealed that the ATG use was associated with decreased OS and EFS rates and a high incidence of NRM (hazard ratio (HR)=1.99, 95% confidence interval (CI): 1.11-3.59, P=0.02), (HR=1.83, 95% CI: 1.08-3.10, P=0.02) and (HR=2.54, 95% CI: 1.03-6.26, P=0.04), respectively. Therefore, our results do not support the use of ATG as part of a myeloablative-conditioning regimen before single-unit allo-CBT in younger patients with hematological malignancies.

[The impact of donor naive and memory T cell subsets on patient outcome following allogeneic stem cell transplantation: relationship between infused donor CD4+/CCR7+ T cell subsets and acute graft-versus-host disease]. / Impact de la composition du greffon sur le devenir des patients après une allogreffe de cellules souches hématopoïétiques: corrélation entre proportion des lymphocytes T CD4+ du greffon exprimant le CCR7 et la survenue d'une GVH aiguë

In a previous prospective study on 62 patients who underwent an HLA-matched allogeneic stem cell transplantation, we have observed that proportion of donor-derived CCR7(+)/CD4(+) T cells in the graft provided a predictive indicator of acute GVHD without interfering on chronic GVHD and relapse rate. Here we present our results on a confirmatory cohort of 137 consecutive patients. Indeed patients who received more than 76% of CCR7(+)/CD4(+) T cells in the graft developed more often acute GVHD be it of low or high grade than those who did not. Determination of the CCR7(+)/CCR7(neg) ratio of CD4(+) T cells in the graft provides a predictive indicator of acute GVHD and could help to define strategies of partial selective T cell depleted transplantation.

Donor-derived CD4(+)/CCR7(+) T-cell partial selective depletion does not alter acquired anti-infective immunity.

In previous studies, we observed that a high proportion of donor-derived CD4(+) T cells expressing the chemokine receptor 7 (CCR7) was a major determinant of acute GVHD, without interfering with the incidence of other post-transplant outcomes, especially relapse and nonrelapse mortality rates. Here, we investigated in vitro the impact of partially selective CD4(+)/CCR7(+) T lymphocytes on acquired anti-infective immune response in 10 donors who underwent G-CSF-primed PBSC collection. Similar quantitative and functional proliferative reactions were observed in lymphocyte cultures in the presence of adenovirus and pp65 Ags with unmanipulated and partially depleted donor samples. No responses were observed in the presence of human T-cell lymphotropic virus type 1 used as a negative control. These results complete the proof of concept needed to build a clinical trial investigating partially selective CD4(+)/CCR7(+) T cell-depleted allo-SCT.

Plasma levels of IL-7 and IL-15 after reduced intensity conditioned allo-SCT and relationship to acute GVHD.

To assess the impact of homeostatic expansion on the occurrence of acute GVHD after reduced intensity conditioning (RIC) transplantation, systemic levels of IL-7 and IL-15 and expression of their specific receptor chains were prospectively investigated in 45 fully HLA-matched allograft recipients. IL-7 and IL-15 levels peaked at four- to fivefold over pre-conditioning values. IL-7 levels were inversely correlated to absolute T-cell counts. Peak IL-15 levels positively correlated to concurrent CRP levels, but normalized earlier than IL-7. These results indicate that the kinetic course of IL-7 depends mainly on initiation of T-cell recovery, while IL-15 depends more on peri-transplant inflammation after RIC. Longer duration of the rise in IL-7 levels was associated with preservation of a normal CD4/CD8 ratio. In all, 16 (35%) patients developed grade 2-4 acute GVHD at a median of 42 days post graft, preceded by higher IL-7 levels and more downregulation of IL-7 receptor α chain on CD4(+) T cells than in patients without acute GVHD, suggesting enhanced homeostatic expansion. In multivariate analysis, IL-7 level measured on day +30 was the foremost predictive factor for grade 2-4 acute GVHD (P=0.002). Measurement of IL-7 level after RIC transplantation might help predict risk of subsequent acute GvHD.

Plasma levels of IL-7 and IL-15 in the first month after myeloablative BMT are predictive biomarkers of both acute GVHD and relapse.

T-cell reconstitution after allo-SCT initially depends on homeostatic peripheral expansion of donor T cells, the level of which may promote the differentiation of alloreactive and tumor-reactive effectors. IL-7 and IL-15 exert their effect as key homeostatic cytokines. We prospectively investigated plasma levels of IL-7 and IL-15 in a homogeneous group of 40 patients in CR of their hematologic malignancy undergoing myeloablative, fully (10/10) HLA-matched BMT. IL-7 and IL-15 proceeded along similar kinetic courses, peaking at wide ranges (3.8-30.2 and 14.3-66 pg/ml, respectively) on day +14 when all patients were profoundly lymphopenic. Occurrence and grade of subsequent acute GVHD were significantly associated with heightened day +14 IL-7 and IL-15 levels. Association of peak IL-7 level to grade 2-4 acute GVHD was confirmed by Cox multivariate analysis (hazard ratio (HR)=5.38; P=0.022). Malignancy relapse was significantly associated with reduced day +14 levels of IL-15 (Cox multivariate analysis: HR=0.93; P=0.035). Plasma IL-7 and IL-15 levels in the early post transplantation period are therefore biomarkers that can help predict subsequent development of acute GVHD and malignancy relapse.

Long-term kinetics of a T-lymphocytes subset in kidney transplant recipients: relationship with posttransplant malignancies.

UNLABELLED: Immunosuppression is associated with a high incidence of malignancies among renal transplant patients. In this study, we investigated the relationship between CD4 lymphopenia and the development of posttransplant malignancy (PTM) after induction therapies in renal transplant recipients (RTR). PATIENTS AND METHODS: This retrospective study included 966 RTR who were transplanted between 1993 and 2005 and had a mean follow-up of 83 +/- 46 months. Induction with antithymocyte globulin (ATG) was employed in 747 patients, while remaining 219 recipients received anti-CD25 antibodies. CD4 T-cell counts determined yearly were correlated with the occurrence of PTM. RESULTS: Eighty-five (8.8%) patients developed a PTM: cutaneous neoplasia (n = 33), lymphoma (n = 14), noncutaneous solid cancer (n = 36). Only age was observed to be significantly different among patients with versus without PTM (48 +/- 10 vs 41 +/- 12 years; P < .001). An early CD4 lymphopenia (<300/mm(3)) was frequent after ATG as compared with anti-CD25 induction (69.8% vs 12.1% at 3 months; P < .0001). The proportion of T CD4 lymphopenic patients progressively decreased over time remaining stable at 5- and 10-year follow-ups (12% and 10.8%, respectively). However, CD4 lymphopenia was not associated with a greater incidence of PTM. CONCLUSION: ATG induced CD4 lymphopenia, which persisted in a small proportion of patients in the long term, but did not seem to be correlated with the occurrence of PTM.

Cytokine concentrations in exhaled breath condensates in systemic sclerosis.

BACKGROUND: Pulmonary fibrosis in systemic sclerosis (SSc) involves inflammatory processes in the lower respiratory tract. Analysis of exhaled breath condensate (EBC) is a non-invasive method for studying inflammatory mediators, such as cytokines, which are of interest from both physiological and therapeutic perspectives. The aim of this study was to assess and compare cytokine concentrations in the EBC of SSc patients and controls. MATERIAL AND METHODS: EBC was collected from 19 SSc patients and 19 controls. We used a multiplex assay test kit to assay interleukin (IL)-2, -4, -6, -10, tumour necrosis factor-alpha, and interferon-gamma in samples concentrated by lyophilization. RESULTS: Cytokine concentrations in EBC were higher in SSc patients than in controls. The stepwise analyses showed that IL-4 was the biomarker which contributed most to the discrimination between controls and patients (Wilk's Lambda = 0.55, p < 0.001). We observed significant negative correlations of EBC cytokines with total lung capacity and diffusion capacity of the lung for carbon monoxide. CONCLUSIONS: These findings suggest that EBC sampling permits the non-invasive study of inflammation in SSc patients, and may be correlated with the severity of interstitial lung disease.

Peripheral blood natural killer cell count is associated with clinical outcome in patients with aaIPI 2-3 diffuse large B-cell lymphoma.

BACKGROUND: Lymphocytopenia is a prognostic factor in Hodgkin's disease. In diffuse large B-cell lymphoma (DLBCL), data are much less established, in spite of numerous reports on immune system-lymphoma interactions. This study addresses the prognostic value of blood lymphocyte subsets at diagnosis in DLBCL. PATIENTS AND METHODS: Absolute values of blood lymphocyte subsets and monocytes were prospectively determined by flow cytometry in 140 patients with 2 or 3 adverse age-adjusted International Prognostic Index (aaIPI) factors included in a Groupe d'Etude des Lymphomes de l'Adulte protocol (LNH98B3). Absolute cell counts at diagnosis and aaIPI were evaluated with regard to clinical outcome. RESULTS: Low median cell counts of 337, 211, and 104/mul were evidenced for the CD4+, CD8+ T, and natural killer (NK) cells, respectively. In univariate analysis, only NK cell count [odds ratio (OR) = 1.81 (1.27, 2.57), P = 0.001] and aaIPI [OR = 2.29 (0.95, 5.45), P = 0.06] were associated with induction treatment response. Low NK cell count [Hazard ratio (HR) = 1.27 (1.06, 1.52), P = 0.01] and aaIPI 3 [HR = 1.95 (1.20, 3.16), P = 0.01] were also associated with a shorter event free survival (EFS). In multivariate analysis, NK cell count was associated with response [OR = 1.77 (1.24, 2.54), P = 0.002] and EFS [HR = 1.25 (1.04, 1.50) P = 0.02] independently of aaIPI. CONCLUSIONS: This study shows an association between circulating NK cell number and clinical outcome in DLBCL, possibly important in the context of the broadening use of rituximab, a likely NK-dependent therapy.

Cytokine profile of human septic shock serum inducing cardiomyocyte contractile dysfunction.

This study was designed to measure nitrite/nitrate and cytokine levels of serum obtained from septic shock patients and to describe potential depressant effects of human septic serum on rat cardiomyocytes. Serum was prepared from 10 non-septic patients and 10 patients with documented septic shock. Adult rat ventricular myocytes were exposed to 20 % serum in the medium. Cardiomyocyte contractility was assessed by measuring shortening fraction and shortening velocity. Serum levels of nitrite/nitrate, a marker of nitric oxide final metabolites, and cytokines (tumor necrosis factor (TNF)-alpha, interleukin (IL) 1beta, 6, 10, 8 and 12p70) were measured. Compared with serum from non-septic patients, serum of septic shock patients induced rapid reduction of the extent and velocity of shortening in isolated cardiomyocytes. Nitrite/nitrate, TNF-alpha, IL-1beta and IL-12p70 concentrations of tested serum for cardiomyocyte studies were not increased in septic serum compared with controls. In contrast, septic serum that induced a depression of in vitro contractility, had increased levels of IL-6, IL-8 and IL-10. We can conclude that the depression of in vitro contractility induced by septic serum is not directly dependent on elevated levels of nitric oxide metabolites, TNF-alpha or IL-1beta. Our results support the view that other cytokines, including IL-6, IL-8 and IL-10, are potent circulating mediators of myocardial depression in cardiomyocytes.

A high proportion of donor CD4+ T cells expressing the lymph node-homing chemokine receptor CCR7 increases incidence and severity of acute graft-versus-host disease in patients undergoing allogeneic stem cell transplantation for hematological malignancy.

CC-chemokine receptor 7 (CCR7), a chemokine receptor required for transmigration into lymphoid organs, is only expressed by naive and central memory T cells. T cells with a capacity of homing into lymphoid organs can initiate acute graft-versus-host disease (GVHD) in mice and respond vigorously in vitro to alloantigens in humans, but their impact on clinical outcomes is unknown. We evaluated prospectively the distribution of naive, central memory and CCR7neg memory T-cell subsets in 39 bone marrow and 23 granulocyte colony-stimulating factor-mobilized peripheral blood stem cell allografts and investigated their impact on patient outcomes. Ranges of the relative proportions of CCR7+ cells within CD4+ and CD8+ T-cell populations were broad, but did not differ between the two sources of allografts. By multivariate analysis, high percentage of donor-derived CD4+CCR7+ T cells (>73.5%) significantly correlated with incidence, earliness of onset and severity of acute GVHD, conferring the highest adjusted hazard ratio (HR=3.9; 95% confidence interval 1.4-10.8; P=0.008) without interfering in other clinical events, especially chronic GVHD and relapse. Determination of the percentage of CD4+CCR7+ T cells in the graft provides a predictive indicator of acute GVHD. Partial depletion of this subset may reduce the risk of acute GVHD while preserving immunotherapeutic effects.