Unravelling the roles of innate lymphoid cells in cerebral malaria pathogenesis.

Cerebral malaria (CM) is one complication of Plasmodium parasite infection that can lead to strong inflammatory immune responses in the central nervous system (CNS), accompanied by lung inflammation and anaemia. Here, we focus on the role of the innate immune response in experimental cerebral malaria (ECM) caused by blood-stage murine Plasmodium berghei ANKA infection. While T cells are important for ECM pathogenesis, the role of innate lymphoid cells (ILCs) is only emerging. The role of ILCs and non-lymphoid cells, such as neutrophils and platelets, contributing to the host immune response and leading to ECM and human cerebral malaria (HCM) is reviewed.

The stimulatory effects of interleukin (IL)-12 on hematopoiesis are antagonized by IL-12-induced interferon gamma in vivo.

Interleukin (IL)-12 synergizes with other cytokines to stimulate the proliferation and differentiation of early hematopoietic progenitors in vitro. However, in vivo administration of IL-12 decreases peripheral blood counts and bone marrow hematopoiesis. Here, we used interferon (IFN) gamma receptor-deficient (IFN gamma R-/-) mice to investigate whether the in vivo inhibition of hematopoiesis by IL-12 is indirectly mediated by IL-12-induced IFN-gamma. IL-12 administered for 4 d (1 microgram/mouse per day) resulted in lower peripheral blood counts and a 2-fold decrease in bone marrow cellularity in wild-type mice, but not in IFN gamma R-/- mice. Bone marrow hematopoietic progenitors were decreased after IL-12 treatment in wild-type mice, but rather increased in IFN gamma R-/- mice. Splenic cellularity was 2.3-fold higher after IL-12 administration in wild-type mice, largely due to natural killer (NK) cell and macrophage infiltration together with some extramedullary hematopoiesis. In IFN gamma R-/- mice, spleen cellularity was less increased, there were fewer infiltrating NK cells, but a strong extramedullary hematopoiesis. Thus, alterations mediated by IL-12-induced IFN-gamma include reduction in bone marrow cellularity and hematopoietic progenitors, as well as pronounced splenomegaly, largely caused by NK cell infiltration. In the absence of IFN-gamma signaling, IL-12 promotes hematopoiesis, consistent with its in vitro activities.

Correction: Interleukin-22-deficiency and microbiota contribute to the exacerbation of Toxoplasma gondii-induced intestinal inflammation.

The original version of this Article omitted the author Dr Mathias Chamaillard from the l'Institut de Pasteur, Lille, France. This has been corrected in both the PDF and HTML versions of the Article.

Efficacy of membrane TNF mediated host resistance is dependent on mycobacterial virulence.

TNF is required for protection against virulent and non-virulent mycobacterial infections. Here we compared the effect of Tm-TNF and sTNF, two different molecular forms of TNF, in virulent and non-virulent murine challenge models. Using non-virulent Mycobacterium bovis BCG intranasal infection we established that immunity is durably compromised in Tm-TNF mice, with augmented bacilli burden, leading to chronic but non-lethal infection. Acute infection by a virulent Mycobacterium tuberculosis low-dose aerosol challenge was controlled in Tm-TNF mice with bacilli burdens equivalent to that in WT mice and pulmonary pathology characterised by the formation of well-defined, bactericidal granulomas. Protective immunity was however compromised in Tm-TNF mice during the chronic phase of M. tuberculosis infection, with increased lung bacterial growth and inflammatory cell activation, dissolution of granulomas associated with dispersed iNOS expression, increased pulmonary IFNgamma and IL-10 expression but decreased IL-12 production, followed by death. In conclusion, membrane TNF is sufficient to control non-virulent, M. bovis BCG infection, and acute but not chronic infection with virulent M. tuberculosis.

mRNA-based cancer vaccine: prevention of B16 melanoma progression and metastasis by systemic injection of MART1 mRNA histidylated lipopolyplexes.

Immunization with mRNA encoding tumor antigen is an emerging vaccine strategy for cancer. In this paper, we demonstrate that mice receiving systemic injections of MART1 mRNA histidylated lipopolyplexes were specifically and significantly protected against B16F10 melanoma tumor progression. The originality of this work concerns the use of a new tumor antigen mRNA formulation as vaccine, which allows an efficient protection against the growth of a highly aggressive tumor model after its delivery by intravenous route. Synthetic melanoma-associated antigen MART1 mRNA was formulated with a polyethylene glycol (PEG)ylated derivative of histidylated polylysine and L-histidine-(N,N-di-n-hexadecylamine)ethylamide liposomes (termed histidylated lipopolyplexes). Lipopolyplexes comprised mRNA/polymer complexes encapsulated by liposomes. The tumor protective effect was induced with MART1 mRNA carrying a poly(A) tail length of 100 adenosines at an optimal dose of 12.5 microg per mouse. MART1 mRNA lipopolyplexes elicited a cellular immune response characterized by the production of interferon-gamma and the induction of cytotoxic T lymphocytes. Finally, the anti-B16 response was enhanced using a formulation containing both MART1 mRNA and MART1-LAMP1 mRNA encoding the antigen targeted to the major histocompatibility complex class II compartments by the lysosomal sorting signal of LAMP1 protein. Our results provide a basis for the development of mRNA histidylated lipopolyplexes for cancer vaccine.

The Emerging Roles of STING in Bacterial Infections.

The STING (Stimulator of Interferon Genes) protein connects microorganism cytosolic sensing with effector functions of the host cell by sensing directly cyclic dinucleotides (CDNs), originating from pathogens or from the host upon DNA recognition. Although STING activation favors effective immune responses against viral infections, its role during bacterial diseases is controversial, ranging from protective to detrimental effects for the host. In this review, we summarize important features of the STING activation pathway and recent highlights about the role of STING in bacterial infections by Chlamydia, Listeria, Francisella, Brucella, Shigella, Salmonella, Streptococcus, and Neisseria genera, with a special focus on mycobacteria.

Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo.

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug.

Crystallization and preliminary X-ray investigation of a complex between a Fab fragment and its antigen, cyclosporin.

Preliminary crystallographic data are given for a complex between the cyclic undecapeptide cyclosporin and the Fab fragment of an anti-cyclosporin monoclonal antibody. Crystals of the complex are orthorhombic with space group P2(1)2(1)2(1) and diffract to 2.7 A resolution. The unit cell dimensions are a = 52.6 A, b = 70.2 A and c = 118.4 A. A native data set to 2.7 A resolution has been collected.

Fine specificity and cross-reactivity of monoclonal antibodies to cyclosporine.

More than 180 monoclonal antibodies (McAbs) to the cyclic undecapeptide cyclosporine (Cs) have been prepared. Several immunization protocols and antibody screening processes were compared. Two main groups of McAbs recognizing different "sides" of the Cs molecule could be differentiated. The antibodies belonged to the IgG and IgA classes and showed high affinity for Cs (up to 10(-10) -10(-11) mol/l). Based on their ability to discriminate Cs-derivatives modified singly at each of the 11 residues of the Cs molecule, the antigenic recognition pattern of different McAbs was studied at the level of individual residues. Closely related recognition patterns were found in each of the two main McAb groups. The apparent size of the Cs antigenic sites recognized by different McAbs varied from four to ten residues and did not correlate with antibody affinity.

Novel monoclonal antibodies to cyclosporine A: characterization and epitope mapping with cyclosporine analogs and cyclophilin.

Monoclonal antibodies to cyclosporine A (Cs), a potent immunosuppressant, were generated in BALB/c mice using a novel antigen prepared by linking Cs to a protein carrier via a photoactive cross-linking reagent, 4-benzoylbenzoic acid (BBa). Twenty-two monoclonal anti-Cs antibodies were generated, using Cs-BBa-bovine serum albumin (Cs-BBa-BSA) as the immunogen. They were characterized with respect to affinity by Scatchard analysis of a radioimmunoassay (RIA), and with respect to specificity by an ELISA in which a series of singly substituted Cs derivatives were examined as inhibitors. McAb affinities ranged from 5 x 10(-8) M to 2 x 10(-10) M. Based on ELISA inhibition data with Cs analogs, and on the binding to two Cs-BSA conjugates in which opposite sides of the Cs molecule are exposed, the antibodies fell into five epitope recognition groups. Binding to Cs was also studied by ELISA in competition with cyclophilin (CyP), a Cs-binding protein whose epitope specificity has been well characterized. Competition by CyP was found to correlate with antibody specificity, not with affinity, i.e. CyP competed best with antibodies having specificities most similar to that of CyP. Epitope mapping can, therefore, be accomplished in a system in which two different species of binding proteins compete for the same antigen. This type of characterization may be useful in identifying antibodies whose combining sites mimic those of a receptor.

Monoclonal antibodies to cyclosporin are representative of the major antibody populations present in antisera of immunized mice.

Two series of mouse antisera raised against cyclosporin (Cs)-carrier conjugates exposing opposite sides of the Cs molecule and more than sixty monoclonal antibodies (mAbs) derived from the same animals were compared in terms of isotype and fine specificity for Cs. The predominant isotypes of the mAbs reflected the in situ distribution of the circulating anti Cs antibodies. The fine specificity of the antibodies was studied by determining their cross-reactivity for a series of Cs-derivatives and Cs-metabolites in competitive ELISA. The antisera raised by different immunizations showed very different cross-reactivity patterns for the Cs-derivatives. However, the in situ anti Cs antibody populations and the majority of mAbs derived from the corresponding animals showed a striking similarity in fine specificity for restricted clusters of residues on the Cs molecule. These results indicate that the mAbs produced against Cs are representative of the major antibody population present in the sera of the mice used for the fusion. By determining the characteristics of antibodies found in the serum of immunized mice it may thus be possible to select animals that are likely to give rise to mAbs of a certain isotype and specificity.

Analysis of the structural diversity of monoclonal antibodies to cyclosporine.

The immunosuppressive cyclic undecapeptide cyclosporine (Cs) represents a useful model for studying the molecular basis of antibody-antigen interactions. The three-dimensional structure of the Cs molecule is known and a large panel of monoclonal antibodies (mAbs) to Cs has been well characterized by cross-reactivity studies with numerous Cs analogs. In the present study, the sequences of the variable regions of seven mAbs to Cs were determined and a striking relationship was found between the expressed variable region genes and the Cs recognition pattern. An analysis of the length and hydrophobic content of the hypervariable regions and sequence similarities suggested that the heavy chain plays a major role in Cs recognition. Different fine specificities were observed for mAbs exhibiting identical light chains, while two antibodies differed by only a single amino acid located in the heavy chain. The presence of a duplication of 12 nucleotides within the heavy chain third hypervariable region of two antibodies suggests the existence of an additional mechanism for creating antibody diversity.

Anti-cyclosporine monoclonal antibodies and their anti-idiotopic counterpart: structure and biological activity.

In order to study the structural and functional mimicry of an antigen by anti-idiotypic antibodies, we generated anti-idiotopic monoclonal antibodies (anti-Id mAbs) against a mAb (R45-45-11) with specificity for the immunosuppressive cyclic undecapeptide cyclosporine (Cs; Sandimmune). Three out of five anti-Id mAbs inhibited the binding of Cs to the anti-Cs mAb R45-45-11. All anti-Id mAbs cross-reacted only with one (anti-Cs mAb V45-271-10) out of 19 anti-Cs mAbs. The anti-Cs mAb V45-271-10 recognizes an epitope on the Cs molecule which is very similar to that recognized by R45-45-11. R45-45-11 and V45-271-10 differ only by one amino acid in the variable region. The anti-Id mAbs which recognize combining site-associated idiotopes (Ids) reverse the blocking effect of the anti-Cs mAb R45-45-11 on Cs immunosuppression in vitro. The sequences of the variable regions of heavy and light chain of one anti-Id mAb were determined. X-ray analysis of the corresponding Fab fragment, either alone or complexed with the Fab fragment of the Id, is currently in progress.

Molecular characteristics of cyclophilin-cyclosporine interaction.

The ability of cyclophilin to react with derivatives of cyclosporine (CsA) was studied. Cyclophilin was found to interact preferentially with CsA-residues 1, 2, 10 and 11, which, together with residue 3, are the residues known to contribute to the immunosuppressive activity of CsA. The recognition of different CsA-derivatives by cyclophilin was correlated with their immunosuppressive activity in vitro. All CsA-derivatives showing a significant activity did bind to cyclophilin, although some of the CsA-derivatives able to bind cyclophilin exhibited only low activities. The results suggest that binding to cyclophilin might be one requirement for immunosuppressive activity of CsA derivatives. When tested with CsA-derivatives showing various conformational changes, the binding of cyclophilin was strongly specific for the peptide-ring conformation of CsA. No binding of calmodulin to CsA could be detected in several formats of solid-phase enzyme- or radioimmunoassay, suggesting that, in contrast to cyclophilin, calmodulin does not possess sufficient affinity for CsA to bind to it when immobilized on the solid phase.

Covalent binding of cyclosporine inhibits irreversibly T-lymphocyte activation.

A diazirine derivative of cyclosporine (PL-CS) was used to photolabel recombinant human cyclophilin (rhCyp), the cytosolic receptor for the immunosuppressant cyclosporine. The affinity of PL-CS for rhCyp and the immunosuppressive activity were 10-fold reduced as compared to cyclosporine A. Whereas cyclosporine immunosuppression was fully reversible, UV cross-linking of PL-CS resulted in permanent inhibition of lymphocyte activation as shown by proliferation of anti-CD3 stimulated human peripheral lymphocyte, interleukin (IL)-2 gene transcription and IL-2 synthesis in the human T-leukemia cell line Jurkat. In vivo photolabeling of viable Jurkat cells revealed that a 21-kDa complex was the major radiolabeled product which was identified as a cyclophilin-cyclosporine complex. In addition, cyclophilin B (25 kDa) and proteins of an unidentified nature at 40, 46 and 60 kDa were observed in Jurkat cells. The cyclosporine-resistant human fibroblast cell line MRC5 displayed a different labeling pattern: cyclophilin B (25 kDa) and a 65-kDa protein were the major labeled products, while the 46- and 60-kDa components were not detectable and cyclophilin was only faintly labeled. In summary, covalent cyclosporine binding caused irreversible lymphocyte inactivation and revealed in addition to cyclophilin other specifically labeled proteins in lymphoid cells. The role and identity of these proteins is presently unknown.

Interleukin-12-induced interferon-gamma inhibits hematopoiesis in vivo in mice.

In vivo administration of interleukin-12 decreases peripheral blood counts and bone marrow hematopoiesis, although in vitro IL-12 was shown to synergize with other cytokines to stimulate the proliferation and differentiation of early hematopoietic progenitors. We investigated whether the in vivo inhibition of hematopoiesis by IL-12 is indirectly mediated by IL-12-induced IFN-gamma. IL-12 was administered to wild-type or IFN-gamma receptor deficient (IFN gamma R-/-) mice. IL-12 treatment resulted in lower peripheral blood counts and a twofold decrease in bone marrow cellularity and hematopoietic progenitors in wild-type mice, but not in IFN gamma R-/- mice. Splenic weight and cellularity were dramatically increased after IL-12 administration in wild-type mice and somewhat less in IFN gamma R-/- mice. The increase was predominantly due to NK cell and macrophage infiltration in wild-type mice, and strong extramedullary hematopoiesis in IFN gamma R-/- mice. Thus, the reduction in total bone marrow cells and hematopoietic progenitor numbers following IL-12 treatment are largely indirect, mediated by IL-12-induced IFN gamma. In the absence of IFN-gamma signaling, IL-12 promotes both bone marrow and splenic hematopoiesis, consistent with its in vitro activities.

The novel immunosuppressant FTY720 induces peripheral lymphodepletion of both T- and B-cells in cynomolgus monkeys when given alone, with Cyclosporine Neoral or with RAD.

OBJECTIVE: FTY720 is a new immunosuppressant active in transplantation models, which modulates lymphocyte recirculation, leading to transient peripheral lymphopenia and increased lymphocytes in lymph nodes and Peyer's patches. Here, we investigated the susceptibility of cynomolgus monkeys to FTY720 given orally either alone or in combination with two other immunosuppressants, Cyclosporin Neoral or RAD, as an introductory study to transplantation protocols. METHODS: Each of the three phases of the study comprised a 3-week treatment period with FTY720 administered daily orally at 0.3, 0.1 or 0.03 mg/kg/day, respectively, followed by a 3-week recovery. FTY720 was given as single compound during the first week and in combination with Neoral at 20 mg/kg/day p.o. or RAD at 0.5 mg/kg/day p.o. during the subsequent 2 weeks. MAIN FINDINGS: These treatment regimen were well tolerated, except for some body weight loss at high FTY720 dose (0.3 mg/kg/day). FTY720 treatment resulted in a rapid decrease of white blood cell counts which reached a plateau after 3 days. A decrease in both T- and B-lymphocyte counts by up to 80-90% was seen with FTY720 doses of 0.1 and 0.3 mg/kg/day. FTY720 blood levels, both trough levels and AUC(0-24 h), showed a linear relationship with FTY720 dose. The reduction in lymphocyte counts was not directly proportional to FTY720 blood levels. The exposure to FTY720 significantly increased upon coadministration of Neoral. This pharmacokinetic interaction was not observed for coadministration of RAD. However, the peripheral lymphodepletion was slightly increased after coadministration of RAD but not of Neoral. This may be related to the intrinsic effects of RAD on hematopoietic cells. CONCLUSIONS: FTY720 given orally was effective in terms of peripheral T- and B-lymphodepletion and was well tolerated in cynomolgus monkeys even in combination with Cyclosporine Neoral or RAD, indicating that such combination protocols could be used in allo- and xenotransplantation in this species. However, the data indicate a potentiation of FTY720 exposure by CsA coadministration and additional lymphodepletion by coadministration of FTY720 and RAD which should be carefully monitored.

Interleukin 11.

IL-11 is a pleiotropic cytokine originally isolated from a bone marrow stromal cell line. It has been shown to share many activities with IL-6, namely to stimulate T cell-dependent B cell maturation, megakaryopoiesis and various stages of myeloid differentiation, but to inhibit adipogenesis. However, the activity of IL-11 on different stages of erythropoiesis in vitro clearly sets it apart form IL-6. IL-11 has little hematopoietic colony stimulatory activity of its own although it sustains terminal differentiation of the late erythroid progenitors CFU-E. In combination with IL-3, IL-11 has profound stimulatory effects on early multipotent hematopoietic progenitors (pre-CFCmulti), on multilineage colony-forming cells (CFCmulti), as well as on erythroid progenitors. The combination of IL-11 with the ligand for c-kit (KL) preferentially acts on early cells since it promotes the multiplication of pre-CFCmulti and stimulates highly proliferative erythroid progenitors that yields remarkable macroscopic erythroblast colonies in culture. The synergistic activity of IL-11 and KL, two stromal factors present in the bone marrow microenvironment, points to a pivotal role of IL-11 in early hematopoiesis. In vivo administration of recombinant human IL-11 elevates the number of circulating neutrophils and platelets and increased megakaryopoiesis in normal mice and primates.

Monoclonal antibody technology for cyclosporine monitoring.

Monitoring blood levels of Cyclosporine (CsA) has been the basis for adjusting individual dosage regimens in the clinic. Radioimmunoassays using polyclonal antisera reacted with CsA and some CsA metabolites, leading to overestimation when compared with high-performance liquid chromatographic measurements of CsA. Monoclonal antibodies (mAbs) have the potential to discriminate between closely related molecules. MAbs with high affinity for CsA have been prepared and their fine-specificity characterized by cross-reactivity studies using a large series of CsA-derivatives. According to the known sites of metabolism on the CsA molecule and to its three-dimensional structure, it was possible to predict which mAb would be suitable for recognizing native Cs specifically.

Cymbimicin A and B, two novel cyclophilin-binding structures isolated from actinomycetes.

Two novel metabolites, cymbimicins A and B, were isolated from the culture broth of a strain of Micromonospora sp. by screening for cyclophilin binding metabolites from actinomycete strains. Cymbimicin A binds to cyclophilin A with a high affinity six fold lower than to that of cyclosporin A. The binding affinity of cymbimicin B is about 100 times lower. The taxonomy of the producing strain, fermentation, isolation, physical and biological properties and structure elucidation are described.