Cutting edge: an essential role for Notch-1 in the development of both thymus-independent and -dependent T cells in the gut.

Whereas most T cells arise in the thymus, a distinct lineage of extrathymically derived T cells is present in the gut mucosa. The developmental origin of extrathymic T cells is poorly understood. We show here that Notch-1, a transmembrane receptor involved in T cell fate specification of bipotential T/B precursors in the thymus, is absolutely required for the development of extrathymic (as well as thymus-derived) mature T cells in the intestinal epithelium. In the absence of Notch-1, CD117(+) T cell precursors are relatively more abundant in the gut than the thymus, whereas immature B cells accumulate in the thymus but not the gut. Collectively, these data demonstrate that Notch-1 is essential for both thymic and extrathymic T cell fate specification and further suggest that bipotential T/B precursors that do not receive a Notch-1 signal adopt a B cell fate in the thymus but become developmentally arrested in the gut.

Identification of proteoglycans as the APRIL-specific binding partners.

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

Plasmodium berghei ANKA infection induces thymocyte apoptosis and thymocyte depletion in CBA mice

Immune responses to malaria infections are characterized by strong T and B cell activation, which, in addition of potentially causing immunopathology, are of poor efficacy against the infection. It is possible that the thymus is involved in the origin of immunopathological reactions and a target during malaria infections. This work was developed in an attempt to further clarify these points. We studied the sequential changes in the thymus of CBA mice infected with Plasmodium berghei ANKA, a model in which 60-90% of the infected animals develop cerebral malaria. During the acute phase of infection, different degrees of thymocyte apoptosis were recorded: (1) starry-sky pattern of diffuse apoptosis with maintenance of cortical-medullary structure; (2) intense apoptosis with cortical atrophy, with absence of large cells; (3) severe cortical thymocyte depletion, resulting in cortical-medullary inversion. In the latter, only residual clusters of small thymocytes were observed within the framework of epithelial cells. The intensity of thymus alterations could not be associated with the degree of parasitemia, the expression of clinical signs of cerebral malaria or intensity of brain lesions. The implications of these events for malaria immunity and pathology are discussed.

CD30 in PTCLS: correlation between RNA and protein levels in a large european series from the LYSA

Introduction: CD22 is expressed on most B-non-Hodgkin lymphomas (NHL); inotuzumab ozogamicin (INO) is an anti-CD22 antibody conjugated to calicheamicin. This study evaluated the safety and tolerability of INO plus R-CVP in patients (pts) with relapsed/refractory CD22+ B-NHL. Efficacy data were also collected. Methods: Part 1 of this open-label study identified a maximum tolerated dose (MTD) of INO 0.8mg/m,2 on day 2 plus R-CVP (rituximab 375mg/m,2 cyclophosphamide 750mg/m,2 and vincristine 1.4mg/m,2 on day 1; prednisone 40mg/m,2 on days 1-5) every 21 days. Subsequently, pts were enrolled in the MTD confirmation cohort (part 2, n = 10), which required a dose-limiting toxicity rate of <33% in cycle 1 and <4 pts discontinuing prior to cycle 3 due to an adverse event (AE) in the MTD expansion cohort (part 3, n = 22), which explored preliminary activity. Results: Parts 2 and 3 enrolled 32 pts: 16 pts with diffuse large B-cell lymphoma, 15 with follicular lymphoma and one with mantle cell lymphoma. Median age was 64.5 years (range 44-81 years); 34% of pts had 1 prior regimen, 34% had 2, 28% had ≥3 and 3% had none (median 2; range 0-6).Median treatment duration was five cycles (range 1-6). Part 2 confirmed the MTD as standard dose R-CVP plus INO 0.8mg/m,2; 2/10 pts had a dose-limiting toxicity (grade 3 increased ALT/AST, grade 4 neutropenia requiring G-CSF). One pt discontinued because of an AE prior to cycle 3. Common treatment-related AEs were thrombocytopenia (78%), neutropenia (66%), fatigue (50%), leukopenia (50%), nausea (41%) and lymphopenia (38%); common grade 3/4 AEs were neutropenia (63%), thrombocytopenia (53%), leukopenia (38%) and lymphopenia (31%). There was one case of treatment-related fatal pneumonia with grade 4 neutropenia. Ten pts discontinued treatment due to AEs; thrombocytopenia/delayed platelet recovery was the leading cause (grade 1/2, n = 6; grade 3/4, n = 3). Objective response rate (ORR) was 77% (n = 24/31 evaluable pts), including 26% (n=8/31) with complete response (CR); three pts had stable disease. Of the pts with follicular lymphoma, ORR was 100% (n = 15/15), including seven pts with CR. Of the pts with diffuse large B-cell lymphoma, ORR was 60% (n = 9/16), including one pt with CR. Conclusions: Results suggest that INOplus R-CVP has acceptable toxicity and promising activity in relapsed/refractory CD22+ B-NHL. The most common grade 3/4 AEs were hematologic. Follow-up for progression-free and overall survival is ongoing.

Anti-flagellin antibody responses elicited in mice orally immunized with attenuated Salmonella enterica serovar Typhimurium vaccine strains

In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.

Outcome after relapse of acute lymphoblastic leukemia in adult patients included in four consecutive risk-adapted trials by the PETHEMA Study Group

BACKGROUND About one half of adults with acute lymphoblastic leukemia are not cured of the disease and ultimately die. The objective of this study was to explore the factors influencing the outcome of adult patients with relapsed acute lymphoblastic leukemia. DESIGN AND METHODS. We analyzed the characteristics, the outcome and the prognostic factors for survival after first relapse in a series of 263 adult patients with acute lymphoblastic leukemia (excluding those with mature B-cell acute lymphoblastic leukemia) prospectively enrolled in four consecutive risk-adapted PETHEMA trials. RESULTS. The median overall survival after relapse was 4.5 months (95% CI, 4-5 months) with a 5-year overall survival of 10% (95% CI, 8%-12%); 45% of patients receiving intensive second-line treatment achieved a second complete remission and 22% (95% CI, 14%-30%) of them remained disease free at 5 years. Factors predicting a good outcome after rescue therapy were age less than 30 years (2-year overall survival of 21% versus 10% for those over 30 years old; P<0.022) and a first remission lasting more than 2 years (2-year overall survival of 36% versus 17% among those with a shorter first remission; P<0.001). Patients under 30 years old whose first complete remission lasted longer than 2 years had a 5-year overall survival of 38% (95% CI, 23%-53%) and a 5-year disease-free survival of 53% (95% CI, 34%-72%). CONCLUSIONS The prognosis of adult patients with acute lymphoblastic leukemia who relapse is poor. Those aged less than 30 years with a first complete remission lasting longer than 2 years have reasonable possibilities of becoming long-term survivors while patients over this age or those who relapse early cannot be successfully rescued using the therapies currently available.

Primary testicular lymphoma: review

Primary testicular lymphoma is a rare form of non-Hodgkin's lymphoma. It occurs more frequently in older patients, and it is a potentially lethal disease. For early stages (I and II), the management consists of orchidectomy followed by chemotherapy combined with monoclonal antibodies directed against the CD20 antigen and scrotal radiotherapy (sanctuary site) with/or without iliac and/or paraaortic lymph node radiotherapy. For advanced stages (III and IV), chemo-immunotherapy is the treatment of choice while scrotal radiotherapy can be discussed. Both in early or advanced disease, intrathecal chemotherapy is necessary to prevent central nervous system relapse. New molecular approaches and/or more aggressive treatments are to be explored in this rare disease. To cite this journal: Oncologie 13 (2011).

Extracellular tumor-related mRNA in plasma of lymphoma patients and survival implications.

BACKGROUND We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL) and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC) and favorable outcome (LMO2, BCL6, FN1) in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma. METHODOLOGY/PRINCIPAL FINDINGS mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA. CONCLUSIONS/SIGNIFICANCE Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.

Identification of Schistosoma mansoni candidate antigens for diagnosis of schistosomiasis

The development of a more sensitive diagnostic test for schistosomiasis is needed to overcome the limitations of the use of stool examination in low endemic areas. Using parasite antigens in enzyme linked immunosorbent assay is a promising strategy, however a more rational selection of parasite antigens is necessary. In this study we performed in silico analysis of the Schistosoma mansoni genome, using SchistoDB database and bioinformatic tools for screening immunogenic antigens. Based on evidence of expression in all parasite life stage within the definitive host, extracellular or plasmatic membrane localization, low similarity to human and other helminthic proteins and presence of predicted B cell epitopes, six candidates were selected: a glycosylphosphatidylinositol-anchored 200 kDa protein, two putative cytochrome oxidase subunits, two expressed proteins and one hypothetical protein. The recognition in unidimensional and bidimensional Western blot of protein with similar molecular weight and isoelectric point to the selected antigens by sera from S. mansoni infected mice indicate a good correlation between these two approaches in selecting immunogenic proteins.

Primary renal lymphoma: report of three new cases and literature review.

OBJECTIVES We report the cases of three patients with primary renal lymphoma. Diagnosis and subsequent treatment are discussed. METHODS The literature on the origin, epidemiology, clinical presentation, diagnosis, treatment and prognosis of primary renal lymphoma was reviewed. RESULTS The first patient was diagnosed after radical nephrectomy and subsequently was given six cycles of CVP (cyclophosphamide, vincristine, prednisone). The diagnosis of the second patient was established by renal biopsy, and the patient received six cycles of CHOP (cyclophosphamide, adriamycin, vincristine and predisone). The last patient had a lymphoma, secondary to immunosuppression, in a transplanted kidney. In this case transplant nephrectomy sufficed to cure the patient's lymphoma. All patients had B-cell non-Hodgkin lymphoma (an extrarenal origin was ruled out by bone marrow biopsy), and were disease-free 15 months, 7 months, and 6.5 years after diagnosis, respectively. CONCLUSIONS Primary renal lymphoma is rare. Diagnosis is established by renal biopsy, although it often presents as a mass simulating renal cell cancer and diagnosis is obtained after radical nephrectomy. Treatment consists of chemotherapy (CHOP). associated with rituximab.

TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.

Lymphoblastic transformation of follicular lymphoma: a clinicopathologic and molecular analysis of 7 patients.

Approximately 30% of patients with follicular lymphoma (FL) transform to a more aggressive malignancy, most commonly diffuse large B cell lymphoma. Rarely, FL transformation results in clinical findings, histology, and immunophenotype reminiscent of B-lymphoblastic leukemia/lymphoma. We report the largest series to date with detailed analysis of 7 such patients. Lymphoblastic transformation occurred on average 2 years after initial diagnosis of FL. Five patients had prior intensive chemotherapy. Two patients developed mature high-grade lymphoma, followed by the lymphoblastic transformation. FL had BCL2 gene rearrangement in 4 of 5 cases. High-grade transformation was accompanied by MYC gene rearrangement (5 of 5). Transformation was characterized by expression of TdT, loss of Bcl6, variable loss of immunoglobulin light chain, and persistence of Pax-5, Bcl2, and CD10. Whole-exome sequencing in 1 case revealed presence of several actionable mutations (CD79B, CCND3, CDK12). FL, aggressive mature B cell lymphoma, and lymphoblastic transformation were clonally related in 6 evaluable cases. After transformation, survival ranged from 1 to 14 months. Four patients died of disease, 2 were in remission after stem cell transplant, and 1 was alive with disease.

No Evidence That Soluble TACI Induces Signalling via Membrane-Expressed BAFF and APRIL in Myeloid Cells.

Myeloid cells express the TNF family ligands BAFF/BLyS and APRIL, which exert their effects on B cells at different stages of differentiation via the receptors BAFFR, TACI (Transmembrane Activator and CAML-Interactor) and/or BCMA (B Cell Maturation Antigen). BAFF and APRIL are proteins expressed at the cell membrane, with both extracellular and intracellular domains. Therefore, receptor/ligand engagement may also result in signals in ligand-expressing cells via so-called "reverse signalling". In order to understand how TACI-Fc (atacicept) technically may mediate immune stimulation instead of suppression, we investigated its potential to activate reverse signalling through BAFF and APRIL. BAFFR-Fc and TACI-Fc, but not Fn14-Fc, reproducibly stimulated the ERK and other signalling pathways in bone marrow-derived mouse macrophages. However, these effects were independent of BAFF or APRIL since the same activation profile was observed with BAFF- or APRIL-deficient cells. Instead, cell activation correlated with the presence of high molecular mass forms of BAFFR-Fc and TACI-Fc and was strongly impaired in macrophages deficient for Fc receptor gamma chain. Moreover, a TACI-Fc defective for Fc receptor binding elicited no detectable signal. Although these results do not formally rule out the existence of BAFF or APRIL reverse signalling (via pathways not tested in this study), they provide no evidence in support of reverse signalling and point to the importance of using appropriate specificity controls when working with Fc receptor-expressing myeloid cells.

Virological and Immunological Characterization of Novel NYVAC-Based HIV/AIDS Vaccine Candidates Expressing Clade C Trimeric Soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as Virus-Like Particles.

The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen. In this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors. IMPORTANCE: We have generated two novel NYVAC-based HIV vaccine candidates expressing HIV-1 clade C trimeric soluble gp140 (ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs. These vectors are stable and express high levels of both HIV-1 antigens. Gag-induced VLPs do not interfere with NYVAC morphogenesis, are highly attenuated in immunocompromised and newborn mice after intracranial inoculation, trigger specific innate immune responses in human cells, and activate T (Env-specific CD4 and Gag-specific CD8) and B cell immune responses to the HIV antigens, leading to high antibody titers against gp140. For these reasons, these vectors can be considered vaccine candidates against HIV/AIDS and currently are being tested in macaques and humans.

Electrophoretic characteristics of monoclonal immunoglobulin G of different subclasses.

Monoclonal IgG are commonly observed in various B cell disorders, of which multiple myeloma is the most clinically relevant. In a series of serum samples, we identified by immunofixation 73 monoclonal IgG, including 63 IgG(1), 4 IgG(2), 5 IgG(3), and 1 IgG(4). The light chains were of kappa type in 45 cases, and of lambda type in 28 cases. These monoclonal IgG were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) in various isoelectric focusing conditions, as well as by 3-DE (2-DE of the proteins extracted from agarose after serum protein agarose electrophoresis). After 2-DE, 38 out of 73 monoclonal gamma chains (52%) were visualized using immobilized pH 3-10 gradients for isoelectric focusing. In 6 cases (8%), gamma chains were only detected using alkaline immobilized pH 6-11 gradients. In 3 cases (4%), 3-DE revealed monoclonal gamma chains hidden by polyclonal gamma chains. Finally, in 26 cases (36%), no monoclonal gamma chains were clearly visualized. Sixty-one monoclonal light chains (84%) were detected using immobilized pH 3-10 gradients, whereas 12 (16%) were not. Monoclonal gamma chains and light chains were highly heterogeneous in terms of pI and M(r). However, a statistically significant correlation (P<0.05) was observed between the position of the monoclonal IgG in agarose gel and the pI of their heavy and light chains (R=0.733, multiple linear regression). Because of the extreme diversity of their heavy and light chains, it appears that a classification of monoclonal IgG based only on their electrophoretic properties is not possible.