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Abstract: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia for which we developed a nationwide network to collect data from new cases diagnosed in France. In a retrospective, observational study of 86 patients (2000-2013), we described clinical and biological data focusing on morphologies and immunophenotype. We found expression of markers associated with plasmacytoid dendritic cell origin (HLA-DRhigh, CD303+, CD304+, and cTCL1+) plus CD4 and CD56 and frequent expression of isolated markers from the myeloid, B-, and T-lymphoid lineages, whereas specific markers (myeloperoxidase, CD14, cCD3, CD19, and cCD22) were not expressed. Fifty-one percent of cytogenetic abnormalities impact chromosomes 13, 12, 9, and 15. Myelemia was associated with an adverse prognosis. We categorized chemotherapeutic regimens into 5 groups: acute myeloid leukemia (AML)–like, acute lymphoid leukemia (ALL)–like, lymphoma (cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP])–like, high-dose methotrexate with asparaginase (Aspa-MTX) chemotherapies, and not otherwise specified (NOS) treatments. Thirty patients received allogeneic hematopoietic cell transplantation (allo-HCT), and 4 patients received autologous hematopoietic cell transplantation. There was no difference in survival between patients receiving AML-like, ALL-like, or Aspa-MTX regimens; survival was longer in patients who received AML-like, ALL-like, or Aspa-MTX regimens than in those who received CHOP-like regimens or NOS. Eleven patients are in persistent complete remission after allo-HCT with a median survival of 49 months vs 8 for other patients. Our series confirms a high response rate with a lower toxicity profile with the Aspa-MTX regimen, offering the best chance of access to hematopoietic cell transplantation and a possible cure.

Recent studies have highlighted an important role for lysophosphatidylcholine acyltransferase 3 (LPCAT3) in controlling the PUFA composition of cell membranes in the liver and intestine. In these organs, LPCAT3 critically supports cell-membrane-associated processes such as lipid absorption or lipoprotein secretion. However, the role of LPCAT3 in macrophages remains controversial. Here, we investigated LPCAT3's role in macrophages both in vitro and in vivo in mice with atherosclerosis and obesity. To accomplish this, we used the LysMCre strategy to develop a mouse model with conditional Lpcat3 deficiency in myeloid cells (Lpcat3KOMac). We observed that partial Lpcat3 deficiency (approximately 75% reduction) in macrophages alters the PUFA composition of all phospholipid (PL) subclasses, including phosphatidylinositols and phosphatidylserines. A reduced incorporation of C20 PUFAs (mainly arachidonic acid [AA]) into PLs was associated with a redistribution of these FAs toward other cellular lipids such as cholesteryl esters. Lpcat3 deficiency had no obvious impact on macrophage inflammatory response or endoplasmic reticulum (ER) stress; however, Lpcat3KOMac macrophages exhibited a reduction in cholesterol efflux in vitro. In vivo, myeloid Lpcat3 deficiency did not affect atherosclerosis development in LDL receptor deficient mouse (Ldlr−/−) mice. Lpcat3KOMac mice on a high-fat diet displayed a mild increase in hepatic steatosis associated with alterations in several liver metabolic pathways and in liver eicosanoid composition. We conclude that alterations in AA metabolism along with myeloid Lpcat3 deficiency may secondarily affect AA homeostasis in the whole liver, leading to metabolic disorders and triglyceride accumulation.

Summary: Low-grade inflammation is constitutive of atherosclerosis, and anti-inflammatory therapy inhibiting interleukin-1β (IL-1β) reduces the rate of cardiovascular events. While cholesterol accumulation in atheroma plaque and macrophages is a major driver of the inflammatory process, the role of the LXR cholesterol sensors remains to be clarified. Murine and human macrophages were treated with LXR agonists for 48 h before Toll-like receptor (TLR) stimulation. Unexpectedly, we observe that, among other cytokines, LXR agonists selectively increase IL1B mRNA levels independently of TLR activation. This effect, restricted to human macrophages, is mediated by activation of HIF-1α through LXR. Accordingly, LXR agonists also potentiate other HIF-1α-dependent pathways, such as glycolysis. Treatment of human macrophages with carotid plaque homogenates also leads to induction of IL1B in an LXR-dependent manner. Thus, our work discloses a mechanism by which cholesterol and oxysterols trigger inflammation in atherosclerosis. This suggests perspectives to target IL-1β production in atherosclerotic patients.

Relapses of psoriasis involve T cells that stem and survive in the skin. Inherited from previous flares, the tissue-resident memory T cells are epidermal IL-17-producing CD8 + and IL-22-producing CD4 + T cells. Because the capacity of resident memory T cells to take in fatty acids is essential for their residence and function, the surface composition of fatty acids may affect underlying T-cell populations. In patients treated with biologics, we used gas chromatography/mass spectrometry to decipher the fatty acid composition in both resolved and nonlesional sites. Skin T cells were activated by OKT-3 in explants from the same body sites to perform bulk transcriptomic analysis (Nanostring). The fatty acid composition differed between skin from healthy donors and normal-looking skin of patients with psoriasis but not further between nonlesional and resolved skin. Patients in whom the resolved skin was rich in oleic acid had lower T-cell-driven IL-17 epidermal transcriptomic signature upon activation of T cells in skin explants. The skin lipid composition is linked with the functions of the underlying epidermal T cells. Testing the modulating effect of custom fatty acids on skin resident T cells could help with coming closer to disease oblivion in inflammatory skin diseases., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute graft-versus-host disease (aGVHD) is a major limitation of the therapeutic potential of allogeneic hematopoietic cell transplantation. Lipopolysaccharides (LPS) derived from intestinal gram-negative bacteria are well-known aGVHD triggers and amplifiers. Here, we explored the LPS metabolism in aGVHD mouse models using an innovative quantification method. We demonstrated that systemic LPS accumulation after transplantation was due, at least partly, to a defect in its clearance through lipoprotein-mediated transport to the liver (i.e., the so-called reverse LPS transport). After transplantation, reduced circulating HDL concentration impaired LPS neutralization and elimination through biliary flux. Accordingly, HDL-deficient (Apoa1 tm1Unc ) recipient mice developed exacerbated aGVHD. Repeated administration of HDL isolated from human plasma significantly decreased the mortality and the severity of aGVHD. While the potential role of HDL in scavenging circulating LPS was examined in this study, it appears that HDL plays a more direct immunomodulatory role by limiting or controlling aGVHD. Notably, HDL infusion mitigated liver aGVHD by diminishing immune infiltration (e.g., interferon-γ-secreting CD8 + T cells and non-resident macrophages), systemic and local inflammation (notably cholangitis). Hence, our results revealed the interest of HDL-based therapies in the prevention of aGVHD., (© 2022 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Kidney transplant candidates (KTCs) who are HLA highly sensitized (calculated panel-reactive alloantibodies >95%) have poor access to deceased kidney transplantation. In this single-center prospective study, 13 highly sensitized desensitization-naïve KTCs received IV tocilizumab (8 mg/kg) every 4 weeks. We evaluated tolerability as well as immune responses, that is, T cell, B cell, T follicular helper (Tfh) subsets, blood cytokines (IL-6, soluble IL-6 receptor-sIL-6R-, IL-21), blood chemokines (CXCL10, CXCL13), and anti-HLA alloantibodies. Tocilizumab treatment was well-tolerated except in one patient who presented spondylodiscitis, raising a note of caution. Regarding immune parameters, there were no significant changes of percentages of lymphocyte subsets, that is, CD3 + , CD3 + /CD4 + , CD3 + /CD8 + T cells, and NK cells. This was also the case for Tfh cell subsets, B cells, mature B cells, plasma cells, pre-germinal center (GC) B cells, and post-GC B cells, whereas we observed a significant increase in naïve B cells (p = .02) and a significant decrease in plasmablasts (p = .046) over the tocilizumab treatment course. CXCL10, CXCL13, IL-21, total IgG, IgA, and IgM levels did not significantly change during tocilizumab therapy; conversely, there was a significant increase in IL-6 levels (p = .03) and a huge increase in sIL-6R (p = .00004). There was a marginal effect on anti-HLA alloantibodies (class I and class II). To conclude in highly sensitized KTCs, tocilizumab as a monotherapy limited B cell maturation; however, it had almost no effect on anti-HLA alloantibodies., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Recent studies have highlighted an important role for lysophosphatidylcholine acyltransferase 3 (LPCAT3) in controlling the PUFA composition of cell membranes in the liver and intestine. In these organs, LPCAT3 critically supports cell-membrane-associated processes such as lipid absorption or lipoprotein secretion. However, the role of LPCAT3 in macrophages remains controversial. Here, we investigated LPCAT3's role in macrophages both in vitro and in vivo in mice with atherosclerosis and obesity. To accomplish this, we used the LysMCre strategy to develop a mouse model with conditional Lpcat3 deficiency in myeloid cells (Lpcat3KO Mac ). We observed that partial Lpcat3 deficiency (approximately 75% reduction) in macrophages alters the PUFA composition of all phospholipid (PL) subclasses, including phosphatidylinositols and phosphatidylserines. A reduced incorporation of C20 PUFAs (mainly arachidonic acid [AA]) into PLs was associated with a redistribution of these FAs toward other cellular lipids such as cholesteryl esters. Lpcat3 deficiency had no obvious impact on macrophage inflammatory response or endoplasmic reticulum (ER) stress; however, Lpcat3KO Mac macrophages exhibited a reduction in cholesterol efflux in vitro. In vivo, myeloid Lpcat3 deficiency did not affect atherosclerosis development in LDL receptor deficient mouse (Ldlr -/- ) mice. Lpcat3KO Mac mice on a high-fat diet displayed a mild increase in hepatic steatosis associated with alterations in several liver metabolic pathways and in liver eicosanoid composition. We conclude that alterations in AA metabolism along with myeloid Lpcat3 deficiency may secondarily affect AA homeostasis in the whole liver, leading to metabolic disorders and triglyceride accumulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The ingestion of apoptotic corpses by macrophages, a process called efferocytosis, is a crucial step in inflammation resolution, since it alters macrophage phenotype toward a pro-resolving profile to foil inflammation and to favor tissue repair. Up to now, the resolving macrophages remain poorly characterized, especially in humans. Global investigations, like RNA sequencing, would be very helpful to unravel some features of these elusive cells. Nonetheless, these inquiries may be challenging in a single-species model, since the fate of ingested mRNA remains unknown and may hinder any subsequent mRNA investigations in the phagocyte. A full human model consisting of primary human neutrophil and primary human monocyte-derived macrophage co-culture was set up several decades ago to mimic in vitro the efferocytosis process. However, to our knowledge, this model has not been characterized as a suitable model to perform global mRNA investigations. Indeed, the extent of ingested neutrophil mRNA contamination has not been assessed in resolving macrophages. This work answers to this crucial question. Indeed, based on the protocols presented in this article, we demonstrate that neutrophil mRNA is severely degraded and is not able to cross-contaminate resolving macrophage mRNA, contrary to apoptotic human peripheral blood derived mononuclear cell (PBMC) or apoptotic leukemic Jurkat cell mRNA. Moreover, this allogenic co-culture system does not favor neither neutrophil activation nor macrophage pro-inflammatory cytokine release. Collectively, we highlight that this model of primary human neutrophil and primary human monocyte-derived macrophage co-culture is the best model for mRNA investigations in human resolving macrophages to help improving our knowledge on these crucial cells., Competing Interests: Declaration of Competing Interest The data presented in this manuscript are original, and have not been published or submitted elsewhere. All listed authors have approved the manuscript and agreed with the submission. In addition, we declare no financial interest related to this work., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Broad T cell depletion by polyclonal anti-thymocyte globulins (ATG) has been used for many years as a part of immunosuppressive treatment in transplantation. Currently, two different ATG are used in clinical practice, Thymoglobulin and Grafalon. Due to differences in the immunization source, these products contain different specificities and quantity of antibodies. These differences may have clinical consequences. We conducted a nested study in a large prospective multicentric cohort of kidney transplant to determine whether Grafalon-treated and Thymoglobulin-treated patients experience different lymphocyte reconstitution and clinical outcomes. 182 patients matched for age, gender, CMV status, CMV prophylaxis, number of previous transplantation, and maintenance immunosuppressive treatment were included (Thymoglobulin, [n=91]; Grafalon®, [n=91]). One-year post-transplant, recent thymic emigrants were significantly decreased (12±10% vs 21±12%; p<0.001) in Grafalon-treated patients. By contrast, T cell activation (CD38+DR+Ki67+) and senescence (CD8+CD57+CD28-) was increased in Thymoglobulin-treated patients. Compared to Grafalon, Thymoglobulin was not associated with a significantly different rate of acute rejection. CMV disease (p=0.013) was more frequent in Thymoglobulin-treated patients. Grafalon and Thymoglobulin seem to be equivalent to prevent acute rejection. CMV disease is more frequent in Thymoglobulin-treated patients. One year post-transplant immune profile profoundly differs according to the type of ATG., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Blastic plasmacytoid dendritic cell (PDC) neoplasm (BPDCN) is an aggressive hematological malignancy with a poor prognosis that derives from PDCs. No consensus for optimal treatment modalities is available today and the full characterization of this leukemia is still emerging. We identified here a BPDCN-specific transcriptomic profile when compared with those of acute myeloid leukemia and T-acute lymphoblastic leukemia, as well as the transcriptomic signature of primary PDCs. This BPDCN gene signature identified a dysregulation of genes involved in cholesterol homeostasis, some of them being liver X receptor (LXR) target genes. LXR agonist treatment of primary BPDCN cells and BPDCN cell lines restored LXR target gene expression and increased cholesterol efflux via the upregulation of adenosine triphosphate-binding cassette (ABC) transporters, ABCA1 and ABCG1. LXR agonist treatment was responsible for limiting BPDCN cell proliferation and inducing intrinsic apoptotic cell death. LXR activation in BPDCN cells was shown to interfere with 3 signaling pathways associated with leukemic cell survival, namely: NF-κB activation, as well as Akt and STAT5 phosphorylation in response to the BPDCN growth/survival factor interleukin-3. These effects were increased by the stimulation of cholesterol efflux through a lipid acceptor, the apolipoprotein A1. In vivo experiments using a mouse model of BPDCN cell xenograft revealed a decrease of leukemic cell infiltration and BPDCN-induced cytopenia associated with increased survival after LXR agonist treatment. This demonstrates that cholesterol homeostasis is modified in BPDCN and can be normalized by treatment with LXR agonists which can be proposed as a new therapeutic approach., (© 2016 by The American Society of Hematology.)

Lack of clear identification of patients at high risk of acute rejection hampers the ability to individualize immunosuppressive therapy. Here we studied whether thymic function may predict acute rejection in antithymocyte globulin (ATG)-treated renal transplant recipients in 482 patients prospectively studied during the first year post-transplant of which 86 patients experienced acute rejection. Only CD45RA(+)CD31(+)CD4(+) T cell (recent thymic emigrant [RTE]) frequency (RTE%) was marginally associated with acute rejection in the whole population. This T-cell subset accounts for 26% of CD4(+) T cells. Pretransplant RTE% was significantly associated with acute rejection in ATG-treated patients (hazard ratio, 1.04; 95% confidence interval, 1.01-1.08) for each increased percent in RTE/CD4(+) T cells), but not in anti-CD25 monoclonal (αCD25 mAb)-treated patients. Acute rejection was significantly more frequent in ATG-treated patients with high pretransplant RTE% (31.2% vs. 16.4%) or absolute number of RTE/mm(3) (31.7 vs. 16.1). This difference was not found in αCD25 monclonal antibody-treated patients. Highest values of both RTE% (>31%, hazard ratio, 2.50; 95% confidence interval, 1.09-5.74) and RTE/mm(3) (>200/mm(3), hazard ratio, 3.71; 95% confidence interval, 1.59-8.70) were predictive of acute rejection in ATG-treated patients but not in patients having received αCD25 monoclonal antibody). Results were confirmed in a retrospective cohort using T-cell receptor excision circle levels as a marker of thymic function. Thus, pretransplant thymic function predicts acute rejection in ATG-treated patients., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

We studied the impact of a set of immune cells contained within granulocyte colony-stimulating factor-mobilized peripheral blood stem cell grafts (naïve and memory T-cell subsets, B cells, regulatory T cells, invariant natural killer T cells [iNKTs], NK cells, and dendritic cell subsets) in patients (n = 80) undergoing allogeneic stem cell transplantation (SCT), using the composite end point of graft-versus-host disease (GVHD)-free and progression-free survival (GPFS) as the primary end point. We observed that GPFS incidences in patients receiving iNKT doses above and below the median were 49% vs 22%, respectively (P= .007). In multivariate analysis, the iNKT dose was the only parameter with a significant impact on GPFS (hazard ratio = 0.48; 95% confidence interval, 0.27-0.85;P= .01). The incidences of severe grade III to IV acute GVHD and National Institutes of Health grade 2 to 3 chronic GVHD (12% and 16%, respectively) were low and associated with the use of antithymocyte globulin in 91% of patients. No difference in GVHD incidence was reported according to the iNKT dose. In conclusion, a higher dose of iNKTs within the graft is associated with an improved GPFS. These data may pave the way for prospective and active interventions aiming to manipulate the graft content to improve allo-SCT outcome., (© 2016 by The American Society of Hematology.)

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation, limiting the success of this therapy. We previously reported that interleukin-22 (IL-22) participates to aGVHD development, but the underlying mechanisms of its contribution remain poorly understood. In this study, we analyzed the mechanism of the pathological function of IL-22 in intestinal aGVHD. Ex-vivo colon culture experiments indicated that IL-22 was able to induce Th1-like inflammation via signal transducer and activator of transcription factor-1 (STAT1) and CXCL10 induction in the presence of type I interferon (IFN). To evaluate a potential synergy between IL-22 and type I IFN in aGVHD, we transplanted recipient mice, either wild-type (WT) or type I IFN receptor deficient (IFNAR(-/-)), with bone marrow cells and WT or IL-22 deficient (IL-22(-/-)) T cells. We observed a decreased GVHD severity in IFNAR(-/-) recipient of IL-22(-/-) T cells, which was associated with a lower level of STAT1 activation and reduced CXCL10 expression in the large intestine. Finally, immunohistochemistry staining of STAT1 performed on gastrointestinal biopsies of 20 transplanted patients showed exacerbated STAT1 activation in gastrointestinal tissues of patients with aGVHD as compared with those without aGVHD. Thus, interfering with both IL-22 and type I IFN signaling may provide a novel approach to limit aGVHD.

Background Aims: This study aimed to characterize the immune effectors contained in the grafts from donor mice mobilized by granulocyte colony-stimulating factor (G-CSF) and plerixafor and to evaluate their impact on the development of acute graft-versus-host-disease (aGVHD)., Methods: Mobilization was done with G-CSF alone or G-CSF plus plerixafor (G+P)., Results: In grafts collected after G+P mobilization, we observed a significantly higher proportion of c-kit(+)Sca-1(+) hematopoietic stem cells compared with G-CSF. A significant increase in the percentage of plasmacytoid dendritic cells was detected in the G+P graft compared with G-CSF graft. We also studied the ability of stem cell grafts mobilized with G+P to induce GVHD in a mouse model. We observed higher mortality (P < 0.001) associated with increased aGVHD clinical score (P < 0.0001) as well as higher pathology score in the intestine of mice receiving G+P as compared with G-CSF grafts (P < 0.001). Moreover, the exacerbated aGVHD severity was associated with upregulation of CCR6 expression on both CD4(+) and CD8(+) T cells from the G+P grafts, as well as on T cells from mice transplanted with G+P grafts., Conclusions: In conclusion, we showed that grafts mobilized with G+P exhibited functional features different from those mobilized with G-CSF alone, which increase the severity of aGVHD in the recipients., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Persistent ATG-induced CD4(+) T cell lymphopenia is associated with serious clinical complications. We tested the hypothesis that ATG induces accelerated immune senescence in renal transplant recipients (RTR). Immune senescence biomarkers were analyzed at transplant and one-year later in 97 incident RTR -62 patients receiving ATG and 35 receiving anti-CD25 mAb (α-CD25). This consisted in: (i) thymic output; (ii) bone marrow renewal of CD34(+) hematopoietic progenitor cells (CD34(+) HPC) and lymphoid (l-HPC) and myeloid (m-HPC) progenitor ratio; (iii) T cell phenotype; and (iv) measurement of T cell relative telomere length (RTL) and telomerase activity (RTA). Clinical correlates were analyzed with a 3 year follow-up. Thymic output significantly decreased one-year posttransplant in ATG-treated patients. ATG was associated with a significant decrease in l-HPC/m-HPC ratio. Late stage differentiated CD57(+) /CD28(-) T cells increased in ATG-treated patients. One-year posttransplant T cell RTL and RTA were consequently lower in ATG-treated patients. ATG is associated with accelerated immune senescence. Increased frequency of late differentiated CD4(+) T cell frequency at transplantation tended to be predictive of a higher risk of subsequent opportunistic infections and of acute rejection only in ATG-treated patients but this needs confirmation. Considering pretransplant immune profile may help to select those patients who may benefit from ATG to prevent severe infections and acute rejection., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Antiplatelet-antibody-producing B cells play a key role in immune thrombocytopenia (ITP) pathogenesis; however, little is known about T-cell dysregulations that support B-cell differentiation. During the past decade, T follicular helper cells (TFHs) have been characterized as the main T-cell subset within secondary lymphoid organs that promotes B-cell differentiation leading to antibody class-switch recombination and secretion. Herein, we characterized TFHs within the spleen of 8 controls and 13 ITP patients. We show that human splenic TFHs are the main producers of interleukin (IL)-21, express CD40 ligand (CD154), and are located within the germinal center of secondary follicles. Compared with controls, splenic TFH frequency is higher in ITP patients and correlates with germinal center and plasma cell percentages that are also increased. In vitro, IL-21 stimulation combined with an anti-CD40 agonist antibody led to the differentiation of splenic B cells into plasma cells and to the secretion of antiplatelet antibodies in ITP patients. Overall, these results point out the involvement of TFH in ITP pathophysiology and the potential interest of IL-21 and CD40 as therapeutic targets in ITP., (© 2014 by The American Society of Hematology.)

Objective: Plasmacytoid dendritic cells (PDC) represent a rare subset of dendritic cells specialized in the production of type I IFN in response to microbial pathogens. Recent data suggested that histone deacetylase (HDAC) inhibitors possess potent immunomodulatory properties both in vitro and in vivo. In this study, we assayed the ability of the HDAC inhibitor, valproic acid (VPA), to influence the phenotype and functional properties of human PDC isolated from peripheral blood., Methods and Results: We showed that VPA inhibited the production of IFN-α and the proinflammatory cytokines TNF-α and IL-6 by CpG-activated PDC. VPA also affected the phenotype of PDC by reducing the expression of costimulatory molecules induced by CpG activation. Moreover, VPA reduced the capacity of CpG-stimulated PDC to promote CD4(+) T cell proliferation and IFN-γ production, while enhancing the proportion of IL-10 positive T cells., Conclusion: These results suggest that HDAC inhibition by VPA alters essential human PDC functions, highlighting the need for monitoring immune functions in cancer patients receiving HDAC inhibitors, but also making these drugs attractive therapies in inflammatory, and autoimmune diseases implicating PDC., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Hematopoietic cell transplantation (HCT) is a curative treatment for hematological malignancies. This therapeutic approach is associated with a profound immune deficiency and an increased rate of opportunistic infections. Nocardiosis is a rare bacterial infection occurring mainly in patients with deficient cell-mediated immunity, such as AIDS patients or transplant recipients. Diagnosis of nocardiosis can be challenging, as signs and symptoms are non-specific. Routine prophylaxis with trimethoprin/sulfamethoxazole (TMP/SMZ) does not prevent the risk of infection. Between May 2001 and December 2009, five cases of nocardiosis were diagnosed from the 366 allogeneic HCT recipients in our centre. Four patients developed a disseminated nocardiosis within the first year after HCT. The fifth patient presented a localized cutaneous nocardiosis. In disseminated cases, median total CD4+ T-cells were below 100 cells/μL. Naive CD4+ CD45RA+/RO- T-cells were almost undetectable. CD8(+) T-cells and NK cells were below the normal range and CD19+ B-cell reconstitution was completely deficient. In a localized case, we observed a lack of naive thymic emigrants CD4+ CD45RA+/RO- T-cells., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Microparticles (MPs) are known to be increased in various malignancies and are involved in tumor invasion, angiogenesis, coagulation, and metastasis. We investigated the plasma levels of annexin-V MPs (AV(+)MPs), platelet-derived MPs (PMPs), and endothelial-derived MPs (EMPs) in patients with melanoma (n=129) and in healthy controls (n=49). A functional coagulation test STA Procoag-PPL measuring the clotting time was performed on samples containing MPs to evaluate their procoagulant potential. The plasma levels of PMPs, EMPs, and AV(+)MPs were significantly higher, and the clotting time-PPL was significantly lower in melanoma patients than in healthy controls. The plasma levels of PMPs, EMPs, and AV(+)MPs were higher in stage IV than in the other stages of melanoma, but with no significant difference. In addition, we observed an inverse correlation between PMPs, AV(+)MPs, and clotting times. Our data suggest that MPs are involved in the progression of melanoma and may be associated to melanoma-associated thrombogenesis.

The pathogenic role of B cells in immune thrombocytopenia (ITP) has justified the therapeutic use of anti-CD20 antibodies such as rituximab (RTX). However, 60% of ITP patients do not respond to RTX. To decipher the mechanisms implicated in the failure of RTX, and because the spleen plays a well-recognized role in ITP pathogenesis, 12 spleens from ITP patients who had been nonresponders to RTX therapy were compared with 11 spleens from RTX-untreated ITP patients and 9 controls. We here demonstrate that in RTX-nonresponder ITP patients, preferential Th1 and Tc1 T lymphocyte polarizations occur, associated with an increase in splenic effector memory CD8(+) T-cell frequency. Moreover, in the RTX- nonresponder patient group, the CD8(+) T-cell repertoire displays a restricted pattern. In the blood, the phenotype of CD8(+) T cells before and after RTX treatment is not modified in responders or nonresponders. Altogether, these results demonstrate for the first time an activation of splenic CD8(+) T cells in ITP patients who did not respond to RTX and suggest their involvement in platelet destruction in these patients.

Background Aims: This study aimed to characterize the immune effectors contained in apheresis samples obtained from patients with grafts mobilized with plerixafor and granulocyte colony-stimulating factor (G-CSF) (P+G) compared with grafts mobilized with G-CSF alone (G)., Methods: Aliquots of apheresis samples were obtained from 36 patients with malignant diseases after mobilization with G (n = 18) or P+G (n = 18). The phenotype and cytokine secretion profile of T cell and dendritic cell subsets were characterized by multicolor cytometry including intracellular cytokine staining., Results: In grafts collected after mobilization with P+G, there was a significantly higher percentage of CD3(+) T cells compared with samples collected after mobilization with G alone. On a functional level, a significant increase of interferon-γ and tumor necrosis factor-α secreting CD8(+) T cells was observed in the P+G group compared with the G group. CD4(+)Foxp3(+) regulatory T cells were similar in both groups but exhibited a lower expression of inducible costimulatory molecule and a significantly higher expression of CD127 in the P+G group. Myeloid dendritic cells (MDCs) and BDCA3(+) dendritic cells were similar in both groups. In contrast, plasmacytoid dendritic cells (PDCs) (CD123(+)BDCA2(+)HLA-DR(+)) were significantly increased in the P+G grafts, leading to a higher PDC-to-MDC ratio. PDCs mobilized by P+G displayed different functional markers--a higher percentage of ILT7(+) PDCs and decreased expression of CD86--suggesting a potential regulatory capacity of PDCs mobilized by P+G., Conclusions: Grafts mobilized with P+G exhibited major different functional features compared with grafts mobilized with G alone, suggesting that such grafts may have an impact on patient outcome after autologous stem cell transplantation., (Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The natural history and clinical significance of posttransplant Epstein-Barr virus (EBV) infection remain largely unknown. The aims of this study are to describe the incidence, risk factors and consequences of EBV infection after kidney transplantation. A total of 383 consecutive patients having received a kidney transplant between January 2002 and December 2010 were included. EBV polymerase chain reaction (PCR) was performed every 2 weeks for 3 months, and every 4 weeks for the next 9 months. A total of 155 of the 383 patients (40%) had at least one positive viremia during the first year posttransplant. The median time to viremia was day 31 posttransplant (14-329). A total of 73 (47%) had EBV viremia > 10(3) log and 23 (15%) had positive viremia for more than 6 months. EBV D+/R- patients (12/18 (67%) versus 143/365 (39%), p = 0.02) and those having received antithymocyte globulins (ATG) (54% vs. 35%; p<0.001) were more likely to develop EBV infection. EBV infection (hazard ratio [HR], 3.03; 95% confidence interval [CI], 1.72-8.29; p = 0.01) was associated with the occurrence of opportunistic infections. A positive EBV PCR during the first 6 months posttransplant was associated with graft loss (HR, 3.04; 95% CI, 1.36-6.79; p = 0.014). EBV reactivation is frequent after transplantation and reflects overimmunosuppression. Prospective studies should examine the association between EBV and graft loss., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Prolonged CD4 T cell lymphopenia after polyclonal antithymocyte globulins (ATG) is associated with an increased rate of cancers. Here, we examined whether pre-transplant thymic function estimated by TREC levels is predictive of cancer occurrence following ATG treatment., Patients and Methods: The impact of TREC on cancer occurrence was analyzed in 115 consecutive incident renal transplant recipients having received ATG., Results: Mean follow-up was 7.5±2.6years. After ATG induction, patients with the lowest pre-transplant TREC values had lower post-transplant CD4(+) and CD4(+) CD45RA(+) CD45RO(-) T cell counts, and a higher frequency of T cells with a regulatory phenotype (CD127(+)CD4(+)CD25(+)Foxp3(+)). Log-transformed pre-transplant TREC values were significantly lower in patients who developed cancer after transplantation (p<0.0001). The cumulative incidence of cancer was higher in patients having the lowest pre-transplant TREC values (T1 [low]: 47.4%, T2 [medium]: 12.5%, and T3 [high]: 2.7%; p<0.0001). In multivariate analysis, pre-transplant TREC value was the only predictive factor of cancer (HR, 0.39; 95% CI, 0.16 to 0.97, for one log (TREC/10(6) PBMC); p=0.046)., Conclusions: Pre-transplant thymic function is associated with an increased rate of post-transplant cancer in patients having received ATG. Omitting ATG in recipients with low pre-transplant TREC values should be considered., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A G-->C polymorphism has been identified in the human cyclooxygenase-2 (COX-2) gene promoter at position -765 with C allele leading to a decreased promoter activity with low prostaglandin E2 (PGE2) production. PGE2 has strong immunomodulatory properties that could influence graft survival. We studied the association between this polymorphism and allograft failure in two independent cohorts of renal transplant recipients (RTRs) including a total of 603 patients. The functional effect of COX-2 gene promoter polymorphism was analyzed by measuring serum levels of PGE2. Median follow-up was 8.7 and 7.9 years for the first and second cohort, respectively. Analysis of 603 patients identified 20 CC (3.3%), 179 GC (29.7%) and 404 GG (67%) carriers. Patients with the GG genotype had significantly higher serum PGE2 concentrations than patients with the C allele. Carriers with a C allele have an independent increased risk of graft loss (hazard ratio (HR) 2.43 [95% CI 1.19-4.97], p = 0.015 for cohort 1; HR 1.72 [95% CI 0.99-3.77], p = 0.051 for cohort 2) compared to GG patients. COX-2 gene promoter polymorphism at position -765 (G-->C) is associated with a higher rate of graft loss in RTRs. Such findings may be used to influence immunosuppressive strategies and optimize patient management.

Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radioactivity- or effector-domain delivery. There is now a growing interest in using anti-idiotypic antibodies or other antigen mimics to induce potent immune responses against antigen structures in question. We have earlier reported on the functional rescue of antibodies that are active when fused to the phage, but inactive as soluble protein [Jensen, K.B., Larsen, M., Pedersen, J.S., Christensen, P.A., Alvarez-Vallina, L., Goletz, S., Clark, B.F. and Kristensen, P. (2002) Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochem. Biophys. Res. Commun. 298, 566-73.]. The rescue was accomplished by maintaining the fusion between the antibody fragment and portions of the filamentous bacteriophage coat protein 3, as present in the original antibody-displaying phage. In the present study, we have applied this system in an attempt to improve immunogenicity of anti-idiotypic antibodies isolated by phage display. Here we demonstrate that by preserving linkage between phage antibody and the N-terminal domain of phage coat protein 3, we induce multimerization of the antibody fragments, and improve their immunogenicity. This immunization approach allows induction of anti-idiotypic antibodies in mice, and facilitates the use of antibodies that are non-functional as non-fused soluble protein.

Background With the development of cord blood banking, solutions have to be found to solve the storage space problem, by reducing the volume of cord blood units (CBU). Methods We compared total nucleated cell (TNC) and CD34(+) cell counts before and after processing with three different CBU volume reduction methods used consecutively in our bank: a manual method based on hydroxyethyl starch sedimentation (HES) (n=447), a top-and-bottom (TB) semi-automated method (n=181) using Optipress II, and the Sepax automated method (n=213). Statistical analysis was done using t-tests, linear regression and Spearman correlation coefficients. Adjusted variables included TNC, CD34(+) cell counts, CD34(+) cell percentage and CB volume before processing. Results TNC recovery was higher with Sepax (80.3+/-7.7%) than with HES (76.8+/-9.1%) and TB (60.7+/-13.5%) (P<0.0001, both). It was higher with HES than with TB (P<0.0001). CD34(+) cell recovery was higher with Sepax (86+/-11.6%) than with HES (81.5+/-12.5%) and TB (82.0+/-17.7%) (P<0.008 and <0.0001, respectively) and results with HES and TB were not significantly different (P=0.7). Interestingly, with Sepax, TNC and CD34(+) cell recoveries were not correlated with pre-processing values (P=0.8 and 0.4, respectively). Discussion In conclusion, the Sepax volume reduction method allows higher TNC and CD34(+) cell recoveries.

Background: Polymorphisms in Toll-like receptor-4 (TLR4) have been reported to be associated with a blunted immune response to microbial pathogens, as well as a decreased risk of atherosclerosis in the general population. We assessed the impact of the two TLR4 variants on the risk of severe infection, the incidence of acute rejection, and the occurrence of atherosclerotic complications in renal transplant recipients (RTR)., Methods: TLR-4 polymorphisms were assessed in a cohort of 238 RTR. Post-transplant atherosclerotic events, acute rejection, severe bacterial infection, cytomegalovirus (CMV) disease, and opportunistic infections were evaluated as outcomes., Results: The patients were followed for a mean duration of 95 +/- 29 months after transplant. TLR4 polymorphism was observed in 27 (11.3%) RTR. Subjects with TLR4 polymorphisms were less likely to experience post-transplant atherosclerotic events (RR 0.44; 95% CI 0.21 to 0.93; P= 0.02) and acute rejection (RR 0.41; 95% CI 0.30 to 0.83; P= 0.01), but presented severe bacterial infections (RR 1.33; 95% CI 1.12 to 1.67; P= 0.01) and opportunistic infections (RR 3.03; 95% CI 1.72 to 8.29; P= 0.008) more frequently. TLR4 polymorphism was marginally associated with CMV disease (RR 1.47; 95% CI 0.95 to 2.64; P= 0.08)., Conclusion: RTR with TLR4 polymorphism present a lower risk of post-transplant atherosclerotic events and acute allograft rejection, but experience severe infectious episodes more frequently. This subset of RTR may benefit from a less potent immunosuppression regimen, along with increased preventive measures against infectious agents.

A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory, cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression, we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies, we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry, reverse transcriptase-polymerase chain reaction, and immunoblot analysis. Furthermore, CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion, thus confirming the presence of a functional CD33 on these leukemic cells. Moreover, we found that circulating pDCs in healthy individuals also weakly express CD33. Overall, our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies.

The monitoring of immune reconstitution in murine models of HC transplantation, using accurate and automated methods, is necessary in view of the recent developments of hematopoietic cell (HC) transplantation (including reduced intensity conditioning regimens) as well as emerging immunological concepts (such as the involvement of dendritic cells or regulatory T cells). Here, we describe the use of a single-platform approach based on flow cytometry and tubes that contain a defined number of microbeads to evaluate absolute blood cell counts in mice. This method, previously used in humans to quantify CD34+ stem cells or CD4+ T cells in HIV infected patients, was adapted for mouse blood samples. A CD45 gating strategy in this "lyse no wash" protocol makes it possible to discriminate erythroblasts or red blood cell debris from CD45+ leukocytes, thus avoiding cell loss. Tubes contain a lyophilized brightly fluorescent microbead pellet permitting the acquisition of absolute counts of leukocytes after flow cytometric analysis. We compared this method to determine absolute counts of circulating cells with another method combining Unopette reservoir diluted blood samples, hemocytometer, microscopic examination and flow cytometry. The sensitivity of this single-platform approach was evaluated in different situations encountered in allogeneic HC transplantation, including immune cell depletion after different conditioning regimens, activation status of circulating cells after transplantation, evaluation of in vivo cell depletion and hematopoietic progenitor mobilization in the periphery. This single-platform flow cytometric assay can also be proposed to standardize murine (or other mammalian species) leukocyte count determination for physiological, pharmacological/toxicological and diagnostic applications in veterinary practice.

Intravenous infusion of apoptotic donor or third-party leukocytes simultaneously with an allogeneic donor bone marrow (BM) graft favors engraftment across major histocompatibility barriers. While verifying that such apoptotic cell infusion might not also be associated with antibody (Ab)-mediated allo-immune responses, we found, rather strikingly, that apoptotic cell infusion could in fact successfully prevent a humoral allo-immunization against a BM graft in mice. Indeed, among recipients having rejected their BM graft, prior apoptotic cell infusion was associated with a near absence of Ab-mediated allo-responses, while such an immunization was frequently observed in the absence of apoptotic cell infusion. This was also observed when infusing host apoptotic cells, thus showing that the prevention of immunization was linked to the apoptotic state of the cells rather than mediated by residual anti-recipient activity. In vivo anti-transforming growth factor-beta (TGF-beta) treatment resulted in the loss of this apoptotic cell infusion-associated protective effect on humoral allo-responses. Further studies will determine whether apoptotic cell infusion, in addition to hematopoietic graft facilitation might also contribute to preventing deleterious Ab-mediated allo-responses in various transplantation settings.

Donor allorecognition of the recipient after hematopoietic transplantation can result in graft-versus-host disease, a potent graft-vs-leukemia effect as well as a graft facilitation effect. Danger signals, host Ag-presenting cells and minor histocompatibility Ag have recently emerged as major determinants of such an alloreactivity. A better understanding of the involved immune mechanisms, the development of novel immunomonitoring tools and cell engineering approaches should result in a significantly increased therapeutic index of allogeneic alloreactivity., (John Libbey Eurotext 2003)

Recent evidence suggests that recipient as well as donor dendritic cell (DC) subsets are implicated in hematopoietic engraftment, graft-vs.-host disease occurrence, immune reconstitution and graft-vs.-leukemia effects observed after allogeneic hematopoietic cell transplantation. Although further data are needed to better understand the precise role of different DC subsets, strategies based on their manipulation to obtain tolerogenic DC can be envisaged. Here, we propose that DC blocked in an immature stage, using immunosuppressive agents, or lymphoid DC can be adequate candidates to control the alloreactive conflict post-allograft.

The deoxyspergualin derivative LF 15-0195 has demonstrated some efficacy in animal models of autoimmune and graft-versus-host diseases and is currently tested in clinics. The molecular mechanisms of LF 15-0195 immunosuppressive activity remained unknown. We show that exposure to LF 15-0195 sensitizes Jurkat T cells to apoptosis induced by an agonistic anti-CD95 antibody (CH11 clone) and by the cytokine TNF-related apoptosis-inducing ligand. LF 15-0195 does not demonstrate any significant effect on the postmitochondrial activation of caspases, nor does it modify overall expression of CD95, Fas-associated death domain, and procaspase-8. The compound facilitates the recruitment of these molecules to the death-inducing signaling complex (DISC) and enhances caspase-8 and -10 activation, thus increasing cytochrome c and direct IAP binding with low pI (DIABLO)/Smac mitochondrial release. LF 15-0195 also sensitizes Jurkat T cells to CD3-mediated apoptosis, an in vitro model for activation-induced T-cell death (AICD). LF 15-0195-mediated sensitization to AICD was further confirmed in human peripheral T cells exposed to anti-CD3 antibodies, then cultured in the presence of interleukin-2. In these cells, LF 15-0195 increased apoptosis triggered by either anti-CD95 antibodies or CD3 restimulation, whereas no effect was observed on "passive apoptosis." Finally, in bone marrow recipient mice, LF 15-0195 enhanced allogeneic donor T-cell death, which required a functional CD95 pathway. These results suggest that LF 15-0195 sensitizes T cells to AICD by increasing caspase activation at the DISC level in response to CD95 engagement. This original mechanism, together with LF 15-0195 efficacy in various disease models, makes this compound a promising immunosuppressive drug.

We have recently shown that the use of allogeneic granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood hematopoietic stem cell transplantation (PBHSCT), as compared with bone marrow transplantation (BMT), is associated with increased titers of antibodies (Abs) directed against red blood cell ABO antigens. To further evaluate the influence of a G-CSF-mobilized PBHSCT graft on alloimmune Ab responses, we examined the frequency of anti-HLA Abs after transplantation in the setting of the same randomized study, comparing PBHSCT with BMT in adults. Anti-HLA Ab presence was determined by complement-dependent cytotoxicity assay (CDC) and flow cytometry in the recipient before and 30 days after transplantation as well as in the donor before graft donation. The use of PBHSCT was significantly associated with increased detection of anti-HLA immunoglobulin G (IgG) Abs early after transplantation as evidenced by flow cytometry (11 of 24 versus 4 of 27 transplant recipients, P =.03) and, less so, by CDC (5 of 24 versus 1 of 27 transplant recipients, P =.09). The difference between PBHSCT and BMT was further heightened when analysis was restricted to anti-HLA IgG Ab-negative donor/recipient pairs. In such a setting, early anti-HLA Ab was never detected after BMT but was repeatedly detected after PBHSCT (flow cytometry, 6 of 18 versus 0 of 17 transplant recipients, P =.02; CDC, 4 of 23 versus 0 of 26 transplant recipients, P =.04). Importantly, the PBHSCT-associated increase in anti-HLA Ab detection was observed despite a reduction in the median number of platelet-transfusion episodes per patient in PBHSC transplant versus BM transplant recipients (3 platelet-transfusion episodes [range, 1-21] in PBHSCT group vs 6 platelet-transfusion episodes [range, 3-33] in the BMT group; P =.02). In conclusion, this study strongly suggests that G-CSF-mobilized PBHSCT results in an increased incidence of circulating anti-HLA Abs and further confirms that the use of such a graft alters alloimmune Ab responses.

Cross-tolerization of T lymphocytes after apoptotic cell uptake by dendritic cells may be involved in self-tolerance maintenance. Furthermore, immunosuppressive properties are attributed to apoptotic cells. This study evaluated the consequences of apoptotic leukocyte administration in a restrictive engraftment model of murine bone marrow (BM) transplantation. Sublethally irradiated recipients received a limited number of allogeneic BM, with or without irradiated apoptotic leukocytes of different origins. No graft-versus-host disease was observed. Whereas only a low proportion of mice receiving BM cells alone engrafted, addition of apoptotic irradiated leukocytes, independently of the origin (donor, recipient, third-party mice, as well as xenogeneic peripheral blood mononuclear cells), significantly enhanced engraftment. Similar results were obtained after infusion of leukocytes rendered apoptotic by UVB irradiation or by anti-Fas monoclonal antibody stimulation, thus confirming the role of apoptotic cells in engraftment facilitation. Overall, these results suggest that apoptotic leukocytes can nonspecifically facilitate allogeneic BM engraftment. Such a simple approach could be of interest in BM transplantation settings involving an important HLA donor/recipient disparity, a T-cell-depleted graft, or reduced conditioning regimen intensity.

Administration of donor T cells expressing the herpes simplex-thymidine kinase (HS-tk) with a hematopoietic stem cell (HSC) transplantation could allow, if graft-versus-host disease (GVHD) was to occur, a selective in vivo depletion of these T cells by the use of ganciclovir (GCV). The study evaluates the feasibility of such an approach. Escalating numbers of donor HS-tk-expressing CD3(+) gene-modified cells (GMCs) are infused with a T-cell-depleted bone marrow transplantation (BMT). Twelve patients with hematological malignancies received 2 x 10(5) (n = 5), 6 x 10(5) (n = 5), or 20 x 10(5) (n = 2) donor CD3(+) GMCs/kg with a BMT from a human leukocyte antigen (HLA)-identical sibling. No acute toxicity was associated with GMC administration. An early increase of circulating GMCs followed by a progressive decrease and long-lasting circulation of GMCs was documented. GCV treatment resulted in significant rapid decrease in circulating GMCs. Three patients developed acute GVHD, with a grade of at least II, while one patient developed chronic GVHD. Treatment with GCV alone was associated with a complete remission (CR) in 2 patients with acute GVHD, while the addition of glucocorticoids was necessary to achieve a CR in the last case. Long-lasting CR occurred with GCV treatment in the patient with chronic GVHD. Unfortunately, Epstein-Barr virus-lymphoproliferative disease occurred in 3 patients. Overall, the administration of low numbers of HS-tk-expressing T cells early following an HLA-identical BMT is associated with no acute toxicity, persistent circulation of the GMCs, and GCV-sensitive GVHD. Such findings open the way to the infusion of higher numbers of gene-modified donor T cells to enhance post-BMT immune competence while preserving GCV-sensitive alloreactivity.

Elimination of clonally expanded peripheral CD8 T cells was thought to involve apoptosis induction mediated principally by TNF, but recently Fas (CD95/APO-1) has been shown to play a role in certain responses. Here we study Fas expression and sensitivity to its ligation on murine CD8 cells specific for the CW3 antigen expressed by transfected P815 cells. Fas was progressively downregulated after successive in vitro restimulations of antigen-specific CD8 cells, until clones became Fas negative and totally resistant to the effects of recombinant Fas ligand. In contrast, Fas expression by in vivo restimulated antigen-specific cells did not diminish. Loss of Fas expression in vitro was not totally irreversible, since it could be reinduced by inhibition of DNA methylation. Understanding how Fas may be differentially regulated in vivo and in vitro is an important issue for the optimal manipulation of T cells for adoptive immunotherapy protocols., (Copyright 2000 Academic Press.)

Tumors of diverse origin have recently been shown to express CD95 ligand and to induce apoptosis in CD95-expressing targets in vitro; however, in vivo, enhanced tumor growth and rejection have both been observed as a consequence of either immunosuppressive or proinflammatory functions of CD95 ligand. The final in vivo outcome of CD95 ligand expression will depend upon a complex balance of interactions relevant for each tumor in its particular microenvironment.