Jean-Hervé Colle

Identification and Biochemical Characterization of Human Plasma Soluble IL-7R: Lower Concentrations in HIV-1-Infected Patients

The IL-7R α-chain and the common γ-chain (γc) are both components of IL-7R. Human plasma harbors soluble forms of IL-7R (sIL-7Rα and sγc) that are detected and assayed by Western blotting, showing that the levels of sIL-7Rα are higher than the levels of sγc (47.5 ng/ml and 1.5 ng/ml, respectively). Gel electrophoresis and tandem mass spectrometry used to analyze deglycosylated, affinity-purified protein showed that sIL-7Rα is generated through differentially spliced mRNA, not by membrane receptor shedding. Plasma sIL-7Rα and sγc are present as heterocomplexes and sγc was found to be mainly associated with sIL-7Rα. The affinities of two IL-7 binding sites (Kd = 35 ± 8 pM and Kd = 3 ± 1 nM) were similar to that of the membrane receptor, suggesting that the sIL-7Rα/sγc complex retains high affinity for IL-7. sIL-7Rα mRNA is constitutively present among peripheral T lymphocytes and is down-modulated in vitro by IL-7. Chronically HIV-1-infected patients (n = 20) showed no significant (p > 0.714) variation in sγc levels and a significant (p < 0.0014) 2-fold decrease in plasma sIL-7Rα levels compared with those in control healthy individuals. Plasma IL-7 and sIL-7Rα levels did not show any obvious relationship.

Leishmania major reaches distant cutaneous sites where it persists transiently while persisting durably in the primary dermal site and its draining lymph node: a study with laboratory mice.

ABSTRACT So far, studies of Leishmania persistence in mice have used injections of parasites administered either intravenously in the tail vein or subcutaneously in the footpad. These routes poorly reflect the natural conditions when the sandfly delivers metacyclic promastigotes intradermally. In this study B10D2 and BALB/c mice were inoculated within the ear dermis with 104Leishmania major metacyclic promastigotes. The parasite load was monitored by quantitative PCR in different tissues from the dermal inoculation site to distant tissues. The two sites of multiplication and persistence of parasites were the site of L. majorinoculation and the draining lymph node (DLN), with a different pattern in the two mouse inbred lines. These two organs were the only sites harboring parasites 12 months postinoculation, with the DLN of BALB/c mice harboring around 107 parasites, a stable load from months 3 to 12. In these two sites, 8 and 12 months after inoculation, interleukin 4 (IL-4), gamma interferon, and inducible nitric oxide synthase transcripts parallel the parasite load while IL-10 transcript levels remain high. In addition, at early time points until month 3, parasite DNA was also detected in distant tissues such as the contralateral noninoculated ear or the tail skin, indicating that blood was at least transiently disseminating the parasites. In contrast,L. major DNA in liver, spleen, and femoral bone marrow remained sporadic in mice of both lines. This study is discussed within the framework of Leishmania transmission from the vertebrate host to the sandfly vector, a complex process still poorly understood.

Listeria monocytogenes as a Short-Lived Delivery System for the Induction of Type 1 Cell-Mediated Immunity against the p36/LACK Antigen of Leishmania major

ABSTRACT Listeria monocytogenes has been used as an experimental live vector for the induction of CD8-mediated immune responses in various viral and tumoral experimental models. Susceptibility of BALB/c mice to Leishmania major infection has been correlated to the preferential development of Th2 CD4 T cells through an early production of interleukin 4 (IL-4) by a restricted population of CD4 T cells which react to a single parasite antigen, LACK (stands forLeishmania homologue of receptors for activated C kinase). Experimental vaccination with LACK can redirect the differentiation of CD4+ T cells towards the Th1 pathway if LACK is coadministrated with IL-12. As IL-12 is known to be induced by L. monocytogenes, we have tested the ability of a recombinant attenuated actA mutant L. monocytogenes strain expressing LACK to induce the development of LACK-specific Th1 cells in both B10.D2 and BALB/c mice, which are resistant and susceptible toL. major, respectively. After a single injection of LACK-expressing L. monocytogenes, IL-12/p40 transcripts showed a rapid burst, and peaks of gamma interferon (IFN-γ)-secreting LACK-specific Th1 cells were detected around day 5 in the spleens and livers of mice of both strains. These primed IFN-γ-secreting LACK-reactive T cells were not detected ex vivo after day 7 of immunization but could be recruited and detected 15 days later in the draining lymph node after an L. major footpad challenge. Although immunization of BALB/c mice with LACK-expressing L. monocytogenes did not change the course of the infection withL. major, immunized B10.D2 mice exhibited significantly smaller lesions than nonimmunized controls. Thus, our results demonstrate that, in addition of its recognized use for the induction of effector CD8 T cells, L. monocytogenes can also be used as a live recombinant vector to favor the development of potentially protective IFN-γ-secreting Th1 CD4 T lymphocytes.

IL-4 mRNA transcription is induced in mouse bone marrow-derived mast cells through an MHC class II-dependent signaling pathway

We have previously shown that mouse bone marrow‐derived mast cells (BMMC) can process and present immunogenic peptides to CD4 T cells. Here, we report on a T cell‐dependent MHC class II‐mediated mast cell activation resulting in IL‐4 transcription and protein release. Presentation of optimal doses of ovalbumin peptide 323 – 339 resulted in IL‐2 production by a specific T cell hybridoma and increase in IL‐4 mRNA transcription in mast cells. IL‐4 mRNA transcription increased by 200‐fold in mast cells treated in IL‐3/IL‐4/granulocyte‐macrophage colony‐stimulating factor (high presenters) whereas only a tenfold increase or no increase were obtained with IL‐3/IL‐4/IFN‐γ‐ or IL‐3‐treated mast cells (low presenters), respectively. Induction of IL‐4 mRNA transcription in purified mast cells by direct ligation of MHC class II molecules, using anti‐I‐A and anti‐I‐E‐coated beads, indicates that MHC class II molecules are critical in this signaling pathway. However, when compared to T cells, anti‐MHC class II‐coated beads were less efficient, indicating a potential role of accessory molecules in this mast cell activation process. IgE‐independent IL‐4 production by mast cells as a result of cognate interaction with CD4 T cells could be critical for the development of type 2 responses. This novel mechanism may contribute to the induction and/or amplification of specific IgE‐mediated allergic responses.

PP2A targeting by viral proteins: a widespread biological strategy from DNA/RNA tumor viruses to HIV-1.

Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. Although initially viewed as constitutive housekeeping enzymes, it is now well established that PP2A proteins represent a family of highly and sophistically regulated phosphatases. The past decade, multiple complementary studies have improved our knowledge about structural and functional regulation of PP2A holoenzymes. In this regard, after summarizing major cellular regulation, this review will mainly focus on discussing a particulate biological strategy, used by various viruses, which is based on the targeting of PP2A enzymes by viral proteins in order to specifically deregulate, for their own benefit, cellular pathways of their hosts. The impact of such PP2A targeting for research in human diseases, and in further therapeutic developments, is also discussed.

PP2A1 Binding, Cell Transducing and Apoptotic Properties of Vpr77–92: A New Functional Domain of HIV-1 Vpr Proteins

Background The hallmark of HIV-1 pathogenesis is the progressive CD4+ T cell depletion and high propensity of CD4+ T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr71-82 mitochondriotoxic domain containing the conserved sequence 71-HFRIGCRHSRIG-82 with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A1 is involved in the induction of G2 arrest by HIV-1 Vpr. Principal Findings Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A1 binding sequence from Vpr77–92 is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR83–85 and T89A substitutions in the Vpr77–92 sequence prevent PP2A1 binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr71–82 domain, has no effect on the biological properties of the Vpr77–92 domain. Conclusion Together our data provide evidence for the first time that the Vpr77–92 sequence delineates a biological active domain of Vpr with PP2A1 binding and pro-apopototic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr77–92 domain in full length Vpr protein and also in entire HIV provirus.

Effect of intragastric and intraperitoneal immunisation with attenuated and wild-type LACK-expressing Listeria monocytogenes on control of murine Leishmania major infection

Stable chromosomal constructs of attenuated DeltaactA and wild-type Listeria monocytogenes expressing the Leishmania major protein LACK were tested as live vaccine vectors in the Th2-orientated chronic L. major murine infection model. These vectors, either by intraperitoneal (i.p.) or intragastric (i.g.) route, were able to induce a strong CD4 Th1 immune response that was correlated with slower parasite growth in the infected footpad. Significant protection against L. major infection was observed in BALB/c mice, ranging from delay in the lesion onset to full protection in 80% of the challenged animals, depending on the size of the parasite inoculum challenge. The i.g. route gave clinically higher protection level than the i.p. route. Both bacterial vectors were as efficient, suggesting that the extent of in vivo bacterial dissemination and multiplication did not seem to be a key parameter for induction of an efficient protective immune response.

The levels and patterns of cytokines produced by CD4 T lymphocytes of BALB/c mice infected with Leishmania major by inoculation into the ear dermis depend on the infectiousness and size of the inoculum

ABSTRACT The production of cytokines by CD4 lymph node T lymphocytes derived from BALB/c mice recently infected in the ear dermis with high (106 parasites) or low (103 parasites) doses of Leishmania major metacyclic promastigotes (MP) was examined over a 3-week period following inoculation. Results were compared with those obtained when mice were injected with less infectious parasite populations, namely, stationary-phase or log-phase promastigotes (LP). Cells were purified 16 h and 3, 8, and 19 days after inoculation, and the amounts of gamma interferon (IFN-γ) and interleukin-4 (IL-4) released in response to LACK (Leishmania homolog of receptors for activated C kinase) or total L. major antigens were assessed. We found that LACK-reactive T cells from mice inoculated with a high dose of parasites first produced IFN-γ and later on IL-4; the level of IFN-γ produced early by these cells was dependent upon the stage of the promastigotes inoculated, the highest level being reached with cells recovered from mice inoculated with the least infectious parasites, LP; sequential production of IFN-γ and then of IL-4 also characterized L. major antigen-reactive CD4 T cells, suggesting that the early production of IFN-γ does not impede the subsequent rise of IL-4 and finally the expansion of the parasites; after low-dose inoculation of MP, cutaneous lesions developed with kinetics similar to that of lesions induced after inoculation of 106 LP, but in this case CD4 T lymphocytes did not release IFN-γ or IL-4 in the presence of LACK and neither cytokine was produced in response to L. major antigens before the onset of lesion signs. These results suggest the existence of a discreet phase in terms of CD4 T-cell reactivity for at least the first 8 days following inoculation, a time period during which parasites are able to grow moderately. In conclusion, the levels and profiles of cytokines produced by Leishmania-specific CD4 T lymphocytes clearly depend on both the stage of differentiation and number of parasites used for inoculation.

Quorum Sensing Contributes to Activated IgM-Secreting B Cell Homeostasis

Maintenance of plasma IgM levels is critical for immune system function and homeostasis in humans and mice. However, the mechanisms that control homeostasis of the activated IgM-secreting B cells are unknown. After adoptive transfer into immune-deficient hosts, B lymphocytes expand poorly, but fully reconstitute the pool of natural IgM-secreting cells and circulating IgM levels. By using sequential cell transfers and B cell populations from several mutant mice, we were able to identify novel mechanisms regulating the size of the IgM-secreting B cell pool. Contrary to previous mechanisms described regulating homeostasis, which involve competition for the same niche by cells having overlapping survival requirements, homeostasis of the innate IgM-secreting B cell pool is also achieved when B cell populations are able to monitor the number of activated B cells by detecting their secreted products. Notably, B cell populations are able to assess the density of activated B cells by sensing their secreted IgG. This process involves the FcγRIIB, a low-affinity IgG receptor that is expressed on B cells and acts as a negative regulator of B cell activation, and its intracellular effector the inositol phosphatase SHIP. As a result of the engagement of this inhibitory pathway, the number of activated IgM-secreting B cells is kept under control. We hypothesize that malfunction of this quorum-sensing mechanism may lead to uncontrolled B cell activation and autoimmunity.

Mouse T-lymphocyte activation by Urtica dioica agglutinin: II. — Original pattern of cell activation and cytokine production induced by UDA

Urtica dioica agglutinin (UDA) is a T-lymphocyte-specific polyclonal activator that differs from ConA, the classical mouse T-cell mitogen, by inducing a late and limited proliferation of a distinct T-cell subset recruited among both CD4+ and CD8+ lymphocytes. We investigated the possibility that the particular kinetics may originate from UDA-specific activation processes in which the known early mandatory signals were completed only after an extended delay. We report that the time of contact required between lectin and the cell membrane to acquire the capacity to proceed into cell cycle was much longer (36-40 h) for UDA than for ConA (8-10 h). Addition of phorbol ester, which artificially induces PKC translocation, or ionomycin, which provokes Ca2+ mobilization, did not accelerate the proliferative kinetics, suggesting that these early mandatory signals are not the limiting factors in the delayed proliferation. The induction of c-myc was retarded in the UDA group, and there was a good correlation between the kinetics of c-myc induction and the kinetics of cell proliferation. The comparison of the level of transcription of the genes encoding different cytokines revealed additional differences between the two mitogens: the whole wave of cytokine gene expression was delayed with UDA. In particular, IL2, IL3 and IFN gamma gene expression was retarded compared to the ConA-induced single wave. An even later transcriptional wave took place at around 72 h for IL4 and IL5. Finally, this particular kinetics corresponded to an unusually high level of IL3 and IFN gamma and a low level of IL4 and IL5 gene transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)