An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies.

Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this "antigen." Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small ( approximately 180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.

Transgenic Leishmania and the immune response to infection.

Genetic manipulation of single-celled organisms such as the Leishmania parasite enables in depth analysis of the consequences of genotypic change on biological function. In probing the immune responses to infection, use of transgenic Leishmania has the potential to unravel both the contribution of the parasite to the infection process and the cellular interactions and mechanisms that characterize the innate and adaptive immune responses of the host. Here, we briefly review recent technical advances in parasite genetics and explore how these methods are being used to investigate parasite virulence factors, elucidate immune regulatory mechanisms and contribute to the development of novel therapeutics for the leishmaniases. Recent developments in imaging technology, such as bioluminescence and intravital imaging, combined with parasite transfection with fluorescent or enzyme-encoding marker genes, provides a rich opportunity for novel assessment of intimate, real-time host-parasite interactions at a previously unexplored level. Further advances in transgenic technology, such as the introduction of robust inducible gene cassettes for expression in intracellular parasite stages or the development of RNA interference methods for down-regulation of parasite gene expression in the host, will further advance our ability to probe host-parasite interactions and unravel disease-promoting mechanisms in the leishmaniases.

A modified colorimetric assay of macrophage activation for intracellular cytotoxicity against Leishmania parasites.

An in vitro method is described which colorimetrically assesses the activation of macrophages for intracellular cytotoxicity against the obligate intracellular parasite Leishmania donovani. The assay system uses a highly purified macrophage population derived from 10-day murine bone marrow cultures. These were infected in vitro as a suspension culture with viable L. donovani amastigotes and then exposed to activating agents. After 48 h the intracellular parasites were released by SDS lysis of the macrophages. Surviving Leishmania organisms were quantitated by their conversion of the chromophore MTT. The sensitivity of this method was comparable with the established method of [3H]dThd incorporation. This assay system has been used to show that there is a dual signal requirement (recombinant interferon-gamma and bacterial endotoxin (LPS] for effective activation of macrophages for leishmanicidal activity.

Interleukin-13 in Iranian patients with visceral leishmaniasis: relationship to other Th2 and Th1 cytokines.

The role of interleukin (IL)-13, a Th2 cytokine sharing many of the features of IL-4, has not previously been examined in patients with visceral leishmaniasis (VL). We examined sera from Iranian patients with VL caused by Leishmania infantum. Serum IL-13 was detected in 50% (22/44) of patients with active primary disease. In comparison, IL-10 was detected in 79.5% (35/44), interferon gamma (IFN gamma) in 38.5% (17/44), and IL-4 in only 5% (2/44) of these patients. With few exceptions all 3 cytokines were undetectable after clinical recovery following antimony therapy. Five of 7 patients (71%) who failed antimony therapy and had relapsing disease had similar levels of IL-10 to patients with active primary disease. However, with only 1 exception, IL-13, IFN gamma and IL-4 were not detected in such patients. These data suggest that relapsing disease may result from defective cellular immunity, unrelated to immunosuppression mediated by IL-10.

The effect of parasitization by Leishmania mexicana mexicana on macrophage function in vitro.

Macrophages infected with amastigotes of Leishmania mexicana mexicana as compared to normal macrophages show decreased migration both randomly and through a 5 microns pore in response to a known chemotaxin, an increased ability to pinocytose and an increased bactericidal ability. Unless very heavily parasitized their ability to phagocytose is unaltered. Parasitized macrophages are unaltered in their ability to secrete extracellularly lysosomal enzymes, prostaglandins and lysozyme in response to known stimuli, or to kill target cells in an antibody dependent cell mediated cytotoxicity assay.

IgG subclass responses in a longitudinal study of canine visceral leishmaniasis.

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an important disease of domestic dogs. Here, we present data on the IgG subclass antibody response to crude L. infantum antigen in a cohort of naturally infected Brazilian dogs. Specific IgG1-IgG4 responses could be detected in 98, 58, 70 and 82%, respectively of 57 dogs that were seropositive for specific IgG. Levels of all IgG subclasses were strongly inter-correlated. Levels of all IgG subclasses increased at the time of seroconversion, before reaching a plateau after several months. Levels of all IgG subclasses were higher in sick dogs than healthy dogs, and levels of all except IgG2 were higher in parasite-positive (by PCR) than parasite-negative dogs. However, levels of IgG2 relative to IgG1 were lower in sick or parasite-positive dogs compared to healthy or parasite-negative infected dogs. In contrast to previous studies, the results suggest that canine VL is associated with upregulation of specific antibody of all IgG subclasses, particularly IgG1, IgG3 and IgG4.

Visceral leishmaniasis: immunology and prospects for a vaccine.

Human visceral leishmaniasis (HVL) is the most severe clinical form of a spectrum of neglected tropical diseases caused by protozoan parasites of the genus Leishmania. Caused mainly by L. donovani and L. infantum/chagasi, HVL accounts for more than 50 000 deaths every year. Drug therapy is available but costly, and resistance against several drug classes has evolved. Here, we review our current understanding of the immunology of HVL and approaches to and the status of vaccine development against this disease.

Pulmonary dendritic cells and alveolar macrophages are regulated by gammadelta T cells during the resolution of S. pneumoniae-induced inflammation.

gammadelta T cells commonly associate with mucosal and epithelial sites, fulfilling a variety of immunoregulatory functions. While lung gammadelta T cells have well-characterized pro-inflammatory activity, their potential role in the resolution of lung inflammation has yet to be explored in any detail. Indeed, given the importance of minimizing inflammation, the cellular mechanisms driving the resolution of lung inflammation are poorly understood. Using a murine model of acute Streptococcus pneumoniae-mediated lung inflammation, we now show that resolution of inflammation following bacterial clearance is associated with a > 30-fold increase in gammadelta T-cell number. Although inflammation eventually resolves in TCR delta(-/-) mice, elevated numbers of alveolar macrophages and pulmonary dendritic cells, and the appearance of well-formed granulomas in lungs of TCR delta(-/-) mice, together indicated a role for gammadelta T cells in regulating mononuclear phagocyte number. Ex vivo, both alveolar macrophages and pulmonary dendritic cells were susceptible to lung gammadelta T cell-mediated cytotoxicity, the first demonstration of such activity against a dendritic cell population. These findings support a model whereby expansion of gammadelta T cells helps restore mononuclear phagocyte numbers to homeostatic levels, protecting the lung from the consequences of inappropriate inflammation.

Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis.

The protective capabilities of three Leishmania recombinant proteins - histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide, and the polyprotein MML immunized with MPL-SE adjuvant - were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide or the combination of H1+HASPB1 with Montanidetrade mark, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions.

Antigen presentation and the response to parasitic infection.

Antigen presentation to T-cells is central to the induction and maintenance of the cell-mediated immune response. Many approaches have been used to define a complex accessory cell compartment, which performs the variety of functional roles encompassed by this term. Resulting from this, antigen presentation has become the cornerstone behind many theories of disease susceptibility and is an important consideration for vaccine design. In this article, Paul Kaye reviews aspects of antigen presentation by accessory cells with particular emphasis on how parasites and their antigens interact with this heterogeneous group of cells.

Costimulation and the regulation of antimicrobial immunity.

The regulated expression of costimulatory molecules is a major factor limiting T-cell responses to self-antigens. However, the development of effective antimicrobial immunity requires that these molecules be induced on a variety of tissues, but most notably on macrophages. Here, Paul Kaye discusses the regulation of costimulatory molecules on macrophages and suggests that microbial interference in this process has important implications for immune regulation.

Inflammatory cells in murine visceral leishmaniasis express a dendritic cell marker.

Immunohistological studies of the murine liver following Leishmania donovani infection have been performed. We describe here the identification of cells expressing a recently defined dendritic cell marker, as detected by monoclonal antibody NLDC 145. Such cells are numerous in the developing inflammatory foci but are not detected individually at any other site in the infected liver. This finding suggests that mature tissue DC are not recruited during infection and that expression of this antigen is under precise microenvironmental control.

Acquisition of cell-mediated immunity to Leishmania. I. Primary T-cell activation detected by IL-2 receptor expression.

In order to analyse the early stages of the T-cell response to Leishmania, bioassays for detecting low levels of IL-2 receptor expression both in bulk culture and under limiting dilution conditions have been used. Infection of C57BL/10 mice with Leishmania donovani amastigotes leads to the appearance of antigen-specific T lymphocytes bearing high-affinity IL-2 receptors 24-72 hr later. Phenotypic analysis by complement-mediated cytotoxicity indicates that these activated T cells comprise both L3T4+, Lyt2- and L3T4-, Lyt2+ populations. The data also suggest the existence of activated cells bearing both these markers. By both assay techniques, the appearance of receptor-positive populations appears transitory, with few such cells detectable at 7 days post-infection. The implications of these data for further studies of murine leishmaniasis are discussed.

Regulation of macrophage accessory cell activity by mycobacteria. I. Ia expression in normal and irradiated mice infected with Mycobacterium microti.

CBA/Ca mice were infected by either the intravenous or intraperitoneal route with Mycobacterium microti and the subsequent changes in local macrophage populations examined. Following infection, the number of macrophages increased and they showed greater expression of both MHC Class II molecules. This response was not dependent on viability of the mycobacteria, in contrast to reports with other microorganisms such as Listeria. Studies in sublethally irradiated mice indicated that persistent antigen could give rise to a response after a period of host recovery which was radiation dose dependent. This procedure also highlighted differences in the regulation of different murine class II antigens in vivo, as seen by delayed re-expression of I-E antigens. Macrophage accessory cell function, as assessed by an in vitro T cell proliferation assay, correlated with Ia expression after fixation, but not after indomethacin treatment; this highlights the diverse nature of regulatory molecules produced by these cells.

Leishmania donovani infection in scid mice: lack of tissue response and in vivo macrophage activation correlates with failure to trigger natural killer cell-derived gamma interferon production in vitro.

Infection of immunocompetent mice with Leishmania donovani is characterized by the development of a tissue granulomatous response, in vivo macrophage activation, and a predominantly Th1-type CD4+ T-cell response. To determine whether a recently described T-cell-independent pathway of gamma interferon (IFN-gamma) production involving the collaboration of macrophages and natural killer (NK) cells contributed to this pattern of events, we have investigated the responses of scid mice to L. donovani infection. The multiplication of parasites in the livers of scid mice progressed at a rate equivalent to that seen in BALB/c mice over the first 14 days of infection, but by day 28 scid mice had a fivefold-higher parasite burden. This infection was not, however, accompanied by any demonstrable histological response in the liver or by elevated major histocompatibility complex class II expression on splenic macrophages. In vitro, L. donovani was unable to trigger IFN-gamma production from scid spleen cell cultures under conditions which allowed efficient triggering by bacterial stimuli. Although L. donovani also failed to stimulate the release of tumor necrosis factor, an important macrophage-derived cofactor for IFN-gamma secretion by NK cells, exogenous recombinant tumor necrosis factor alpha could not restore the IFN-gamma response. Even with the potent synergistic effect of exogenous interleukin-2, L. donovani was unable to stimulate this pathway to the same extent as Listeria monocytogenes. Indeed, L. donovani inhibited the response to L. monocytogenes in a dose-dependent fashion. Experiments involving the transfer of supernatants and the use of neutralizing monoclonal antibodies have failed to find evidence that interleukin-10 is involved in this inhibition. These data suggest that NK cell-derived IFN-gamma is unlikely to participate in the early regulation of visceral leishmaniasis in the mouse.

Recombinant interleukin-1 alpha augments granuloma formation and cytokine production but not parasite clearance in mice infected with Leishmania donovani.

In vivo administration of various doses of recombinant interleukin-1 alpha to B10.D2/n mice chronically infected with Leishmania donovani resulted in enhanced formation of granulomas and in vitro production of gamma interferon. By direct microscopical enumeration, reduction in gross parasite burden in the viscera was not observed, however. These data highlight an important discordance between granuloma formation per se and parasite elimination and suggest that interleukin-1 deficiency alone cannot account for the chronicity of this disease.

Organ-specific immune responses associated with infectious disease.

The immune response to infection can vary markedly in different organs of the same animal. In some organs, the infection can resolve with subsequent immunity to re-infection, whereas in other organs, pathogens can persist. Here, Christian Engwerda and Paul Kaye highlight the importance of defining organ-specific immune mechanisms for developing strategies that deal effectively with infectious diseases and their associated pathologies.

Presentation of Leishmania donovani promastigotes occurs via a brefeldin A-sensitive pathway.

For the presentation of Leishmania promastigotes to polyclonal CD4+ T cells, a processing period within activated macrophages of 3-4 h is required. Presentation can be inhibited by both chloroquine and brefeldin A (BFA), the latter implicating a requirement for newly synthesized MHC class II molecules. This inhibition is both reversible and specific, in that BFA did not inhibit mixed lymphocyte reaction stimulation by these infected macrophages. Immunogold labeling demonstrated that class II was associated with the parasite-containing phagolysosome. The level of class II was not significantly altered in BFA-treated cells in the time period studied, suggesting that antigen may exist the phagolysosome and interact with class II in another cellular compartment.

Immunoregulatory pathways in murine leishmaniasis: different regulatory control during Leishmania mexicana mexicana and Leishmania major infections.

The effect of whole body sublethal gamma irradiation on the subsequent growth of Leishmania mexicana mexicana and Leishmania major was studied in CBA/Ca and BALB/c mice. Whereas BALB/c mice are highly susceptible to both parasites developing non healing progressively growing lesions at the site of cutaneous infection, CBA/Ca mice develop small healing cutaneous ulcers following subcutaneous infection with L. major but non healing lesions following subcutaneous infection with L.m. mexicana. Prior whole body sublethal irradiation of CBA/Ca mice, but not BALB/c mice, resulted in strong resistance against infection with L.m. mexicana: no lesions developed at the site of cutaneous infection. Irradiated BALB/c mice did, however, develop small lesions which healed when infected with L. major. The protective effects of irradiation coincided with the development of delayed type hypersensitivity. Both naive and sensitized nylon wool purified lymphocytes could restore susceptibility to L. major in irradiated BALB/c mice but only lymphocytes from long term infected donor mice adoptively transferred a non healing response to irradiated CBA/Ca mice infected with L.m. mexicana. Non-irradiated, L. major infected, CBA/Ca mice, but not similarly treated BALB/c mice, were found to be resistant to subsequent infection with L.m. mexicana. On the other hand, irradiated BALB/c mice infected with L. major were resistant to subsequent infectious challenge with L.m. mexicana. We suggest that the susceptibility of CBA/Ca mice to L.m. mexicana is under the control of an as yet unidentified gene which is not dependent on the generation of T suppressor cells and is bypassed by previous infection with L. major. Therefore, BALB/c mice immunized against L. major by prior sublethal irradiation are also resistant to L.m. mexicana.