David Michonneau

CD24 hi CD27 + and plasmablast-like regulatory B cells in human chronic graft-versus-host disease

Interleukin 10 (IL-10)-producing B cells (regulatory B cells [Bregs]) regulate autoimmunity in mice and humans, and a regulatory role of IL-10-producing plasma cells has been described in mice. Dysfunction of B cells that maintain homeostasis may play a role in the pathogenesis of chronic graft-versus-host disease (cGVHD) after allogeneic stem cell transplantation. Here, we found a relation between decreased Breg frequencies and cGVHD severity. An impaired ability of B cells to produce IL-10, possibly linked to poor signal transducer and activator of transcription 3 and extracellular signal-regulated kinase phosphorylation, was found in patients with active cGVHD. IL-10 production was not confined to a single B-cell subset, but enriched in both the CD24(hi)CD27(+) and CD27(hi)CD38(hi) plasmablast B-cell compartments. In vitro plasmablast differentiation increased the frequency of IL-10-producing B cells. We confirmed that allogeneic transplant recipients had an impaired reconstitution of the memory B-cell pool. cGVHD patients had less CD24(hi)CD27(+) B cells and IL-10-producing CD24(hi)CD27(+) B cells. Patients with cGVHD had increased plasmablast frequencies but decreased IL-10-producing plasmablasts. These results suggest a role of CD24(hi)CD27(+) B-cell and plasmablast-derived IL-10 in the regulation of human cGVHD.

In vivo imaging of inflammasome activation reveals a subcapsular macrophage burst response that mobilizes innate and adaptive immunity

The inflammasome is activated in response to a variety of pathogens and has an important role in shaping adaptive immunity, yet the spatiotemporal orchestration of inflammasome activation in vivo and the mechanisms by which it promotes an effective immune response are not fully understood. Using an in vivo reporter to visualize inflammasome assembly, we establish the distribution, kinetics and propagation of the inflammasome response to a local viral infection. We show that modified vaccinia Ankara virus induces inflammasome activation in subcapsular sinus (SCS) macrophages, which is immediately followed by cell death and release of extracellular ASC specks. This transient inflammasome signaling in the lymph node generates a robust influx of inflammatory cells and mobilizes T cells from the circulation to increase the magnitude of T cell responses. We propose that after infection, SCS macrophages deliver a burst response of inflammasome activity and cell death that translates into the broadening of T cell responses, identifying an important aspect of inflammasome-driven vaccination strategies.

The PD-1 Axis Enforces an Anatomical Segregation of CTL Activity that Creates Tumor Niches after Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a curative treatment for hematologic malignancies, relies on donor cytotoxic T lymphocyte (CTL)-mediated graft-versus-leukemia (GVL) effect. Major complications of HSCT are graft-versus-host disease (GVHD) that targets specific tissues and tumor relapses. However, the mechanisms dictating the anatomical features of GVHD and GVL remain unclear. Here, we show that after HSCT, CTLs exhibited different killing activity in distinct tissues, being highest in the liver and lowest in lymph nodes. Differences were imposed by the microenvironment, partly through differential PD-1 ligand expression, which was strongly elevated in lymph nodes. Two-photon imaging revealed that PD-1 blockade restored CTL sensitivity to antigen and killing in lymph nodes. Weak CTL activity in lymph nodes promoted local tumor escape but could be reversed by anti-PD-1 treatment. Our results uncover a mechanism generating an anatomical segregation of CTL activity that might dictate sites of GVHD and create niches for tumor escape.

Maintaining calcineurin inhibition after the diagnosis of post-transplant lymphoproliferative disorder improves renal graft survival

Post-transplant lymphoproliferative disorder (PTLD) is an uncontrolled proliferation of transformed lymphocytes fostered by immunosuppression. In addition to chemotherapy, treatment of PTLD includes a reduction of maintenance immunosuppression. Patients with PTLD have an increased risk of graft loss, suggesting that reduced immunosuppression strategy needs to be optimized with regard to graft outcome. Here we retrospectively reviewed 101 cases involving PTLD to identify the risks associated with graft loss. During a median follow-up of 70 months, 39 patients died and 21 lost their graft. Multivariate analysis found that an eGFR under 30 ml/min per 1.73 m(2) at PTLD diagnosis, a biopsy-proven acute rejection episode following reduction of immunosuppression, and the absence of calcineurin inhibition in maintenance immunosuppression are independent risk factors for allograft loss. Neither the type of PTLD nor the chemotherapy regimen was predictive of allograft failure. Histological analysis of graft biopsies showed that maintaining calcineurin inhibition after the diagnosis of PTLD reduced the risk of developing de novo anti-HLA antibodies and humoral rejection. Remarkably, calcineurin inhibitor maintenance was neither associated with higher mortality nor with worse progression-free survival. Thus, maintaining calcineurin inhibition at a reduced dose after the diagnosis of PTLD seems safe and may improve renal graft outcome, possibly through better control of the recipients humoral immune response.

Severe Aplastic Anemia Associated With Eosinophilic Fasciitis: Report of 4 Cases and Review of the Literature

AbstractDiffuse eosinophilic fasciitis (Shulman disease) is a rare sclerodermiform syndrome that, in most cases, resolves spontaneously or after corticosteroid therapy. It has been associated with hematologic disorders, such as aplastic anemia. The clinical features and long-term outcomes of patients with eosinophilic fasciitis and associated aplastic anemia have been poorly described. We report the cases of 4 patients with eosinophilic fasciitis and associated severe aplastic anemia. For 3 of these patients, aplastic anemia was refractory to conventional immunosuppressive therapy with antithymocyte globulin and cyclosporine. One of the patients received rituximab as a second-line therapy with significant efficacy for both the skin and hematologic symptoms. To our knowledge, this report is the first to describe rituximab used to treat eosinophilic fasciitis with associated aplastic anemia.In a literature review, we identified 19 additional cases of eosinophilic fasciitis and aplastic anemia. Compared to patients with isolated eosinophilic fasciitis, patients with eosinophilic fasciitis and associated aplastic anemia were more likely to be men (70%) and older (mean age, 56 yr; range, 18–71 yr). Corticosteroid-containing regimens improved skin symptoms in 5 (42%) of 12 cases but were ineffective in the treatment of associated aplastic anemia in all but 1 case. Aplastic anemia was profound in 13 cases (57%) and was the cause of death in 8 cases (35%). Only 5 patients (22%) achieved long-term remission (allogeneic hematopoietic stem cell transplantation: n = 2; cyclosporine-containing regimen: n = 2; high-dose corticosteroid-based regimen: n = 1).

Influence of bone marrow graft B lymphocyte subsets on outcome after HLA‐identical sibling transplants

The potential role of the infused B cell subset after Hematopoietic Stem Cell Transplantation has not been yet studied. The present study analyzed the impact of B cells on transplant outcome in 254 patients who received a bone marrow graft from a human leucocyte antigen‐identical sibling donor. The influence of B lineage‐specific hematopoietic progenitor cells (CD34+ CD19+) and B cells (immature and mature B cells, CD34− CD19+) was also analyzed. All included patients received a myeloablative regimen. The cumulative incidence function of acute graft‐versus‐host (GvHD) grade II to IV was 48% and was inversely associated with the number of CD34+ CD19+. There were no statistically significant associations between B cell subsets and chronic GvHD or survival. The CD34+ CD19+ B cell subset remained significantly associated with acute GvHD in multivariate analysis (Relative risk = 0·32, 95% confidence interval: 0·11–0·92, P = 0·035). In conclusion, a higher B lineage‐specific hematopoietic progenitor cells (CD34+ CD19+) cell dose is associated with a significant decrease incidence of acute GvHD.

Overexpression of the antiapoptotic protein A1 promotes the survival of double positive thymocytes awaiting positive selection.

As it has been shown for Mcl-1, Bcl-xl and Bcl-2, proteins of the Bcl-2 family play a crucial role during T-cell development in the thymus. We here show that the expression of the antiapoptotic gene A1 is specifically enhanced at the DN3/DN4 transition and in DP thymocytes that have been positively selected suggesting that A1 expression might be considered as a transcriptional signature of thymocytes that have received pre-TCR or TCR survival signal. Furthermore, we observed that A1-a overexpression in recombination activation gene 1-deficient mice transgenic for the major histocompatibillity complex class I-restricted F5 TCR enhances cell survival of DP thymocytes and permits accumulation of DP cells awaiting positive selection. However, A1-a overexpression has no effect on negative selection. Therefore, our results suggest that A1 plays a specialized role in allowing survival of DP thymocytes and that its role can be distinguished from that of Mcl-1, Bcl-xl and Bcl-2.

Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies.

Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs.

Late-onset post-transplantation lymphoproliferative disorders after kidney transplantation: a monocentric study over three decades

BACKGROUND Late-onset post-transplantation lymphoproliferative disorders (PTLDs) occur 1 year after transplantation and are associated with poor prognosis. Initial treatment usually involves a reduction in immunosuppressive treatment. While early-onset PTLDs have a good prognosis following RI, this approach is generally inadequate for late-onset PTLDs. We assessed the specific outcome of late-onset PTLDs after kidney transplantation during the past three decades. METHODS We reviewed the clinical and biological data of 52 kidney transplant recipients who developed late-onset PTLDs at our centre between 1980 and 2010. We compared clinical features, long-term outcome and renal prognosis of late-onset PTLDs both before and after the era of rituximab. RESULTS Before 2000, 38% of the patients underwent surgery and 76% received chemotherapy either immediately or after surgery. After 2000, rituximab was administrated to 70% of the patients either alone (23%) or in combination with chemotherapy (77%). Chemotherapy alone was administrated in 26% of the cases. Before and after 2000, complete remission was achieved in 38 and 87% of the cases, respectively (P = 0.0005). The 5-year overall survival (OS) was 33.3 and 69% (P = 0.003), and 5-year disease-free survival was 37.5 and 80%, respectively (P = 0.19). Renal function was preserved in 70% of the cases at the end of the follow-up. CONCLUSIONS This study shows an increase in OS and low graft loss for patients with late-onset PTLDs during the last decade, which may be attributed to multiple changes in clinical practice, including a more standardized treatment and the use of rituximab in combination with chemotherapy.

Disseminated Scopulariopsis brevicaulis infection in an allogeneic stem cell recipient

In patients with hematological diseases complicated by neutropenia, Candida and Aspergillus infections are commonly described. However, over the last few years, opportunistic infections due to multi-resistant fungi have been increasingly reported. We describe here a fatal case of disseminated Scopulariopsis brevicaulis infection in an allogenic stem cell recipient. In January 2007, a 43-year-old woman was diagnosed with myelodysplasia. In March 2008, she developed secondary AML. In July 2008, induction chemotherapy with a classical 3þ 7 regimen failed to induce CR and a second induction course of chemotherapy induction was administered with anthracycline and high-dose cytarabine. The chemotherapy resulted in a prolonged period of profound neutropenia. The patient’s fever was addressed by empirical broadspectrum antibiotherapy. Finally, she achieved CR and fever resolved without any infection being documented. In October 2008, the patient underwent allogeneic PBSC transplantation from an unrelated donor. GVHD prophylaxis with corticosteroids and mycophenolate mofetil was used. In November 2008, computed tomography (CT) showed a lesion in the upper right lung suggestive of pulmonary aspergillosis. Treatment by voriconazole (200mg 2/day) was started. On 2 February 2009, the patient developed chronic neutropenia and treatment by vesanoid was then initiated. On 8 February 2009, she developed temperature, with chest pain, coughing and progressive dyspnea. Voriconazole was continued and large-spectrum antibiotherapy (tazocillin, gentamicin and zyvoxid) was added. Aspergillus antigenemia was negative; no bacteria or viruses were found in the blood, urine or after catheter culture. On 20 February 2009, due to the persistence of respiratory symptoms and fever, whole-body CT was performed, revealing destruction of the posterior wall of sphenoidal sinus, infiltrate in the upper right lung and multiple intra-hepatic nodules. Zygomycosis was suspected, because of the lengthy voriconazole treatment (4 months) and the CT-detected lesion of the sinus. Voriconazole was replaced by liposomal 2 amphotericin B (10mg/kg/day). Direct microscopical examination of the samples on 24, 26 and 27 February showed evidence of fungus mycelia. The sputum culture aroused suspicion of S. brevicularis on 4 March 2009. The molecular identification that allowed the formal identification was based on the ITS1 DNA sequence analysis of a fragment after PCR amplification based on the 28S ribosomal RNA sequence. In vitro study by the Etest method revealed high antifungal minimal inhibitory concentration (MIC): amphotericin B (2mg/mL), posaconazole (1mg/mL), itraconazole (32mg/mL) and voriconazole (32 mg/mL). On 5 March, terbinafine (250mg/day) was added to liposomal amphotericin B. MICs obtained by Etest were confirmed by the reference procedure of the European Committee on Antifungal Susceptibility (EUCAST) for spore-forming molds. Despite the antifungal treatment, the patient died 4 days later. The genus Scopulariopsis mainly comes from soil, food and organic materials. The majority of Scopulariopsis human infections are due to S. brevicularis. It can cause onychomycosis and soft tissue infection after traumatic injuries. In immunocompromised patients, cases are rare, but increasing. Eight cases, all of them concerning hematological patients, have been reported in medical publications. All of them were allogeneic transplanted patients with prolonged neutropenia (seven acute leukemia, one aplastic anemia). Furthermore, 5/8 had been on steroid therapy. In six cases, S. brevicularis infections were localized in the lung. The clinical presentation does not give many clues. Fever seems to have been persisting in all the cases despite large-spectrum antibiotherapy. Complaints of coughing and shortness of breath were observed in two cases. Tomography displayed pulmonary infiltrates (2/6), or pulmonary lesions evoking Aspergillus infection (3/6). It is noticeable that in three cases skin lesions appeared before the pulmonary symptoms arose. In two cases sinonasal localizations, diagnosed by biopsies, have been reported, and in one case the sinus localization was linked to a brain abscess. In the case of the patient described herein, the lung was affected. A sinusal dissemination was suspected, but no biopsy was performed because of the localization of the lesion and no definitive conclusion was possible. Furthermore, Scopulariopsis fungemia had not been detected. Concerning other non-Aspergillus fungal disseminated infections, it must be stressed that fungemia may be positive in Fusarium or Scedosporium infections. To our knowledge, Scopulariopsis-positive hemoculture has been reported in only one case. As post-mortem examination was not performed, we do not know the origin of the hepatic nodules, but in two reported cases, autopsy showed disseminated infection in the lung, brain, kidney and liver. In the present case, direct microscopical examinations and culture on repeated sputum samples led to the detection of mycelium. In medical publications, pulmonary infections are reported to be demonstrated by bronchoscopy (1/6) or lung biopsy (1/6). We did not observe any onyxis Bone Marrow Transplantation (2011) 46, 1276–1277 & 2011 Macmillan Publishers Limited All rights reserved 0268-3369/11