Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule.

Introduction: Microbial systems, such as Escherichia coli, as host recombinant expression is the most versatile and the cheapest system for protein production, however, several obstacles still remain, such as recovery of soluble and functional proteins from inclusion bodies, elimination of lipopolysaccharides (LPS) contamination, incomplete synthesis, degradation by proteases, and the lack of post-translational modifications, which becomes even more complex when comes to membrane proteins, because they are difficult not only to produce but also to keep in solution in its active state. T-cell Immunoglobulin and Mucin domain 3 (TIM-3) is a type I transmembrane protein that is predominantly expressed on the surface of T lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages, playing a role as a negative immune checkpoint receptor. TIM-3 comprises a single ectodomain for interaction with immune system soluble and cellular components, a transmembrane domain, and a cytoplasmic tail, responsible for the binding of signaling and scaffolding molecules. TIM-3 pathway holds potential as a therapeutic target for immunotherapy against tumors, autoimmunity, chronic virus infections, and various malignancies, however, many aspects of the biology of this receptor are still incompletely understood, especially regarding its ligands. Methods: Here we overcome, for the first time, the challenge of the production of active immune checkpoint protein recovered from bacterial cytoplasmic inclusion bodies, being able to obtain an active, and non-glycosylated TIM-3 ectodomain (TIM-3-ECD), which can be used as a tool to better understand the interactions and roles of this immune checkpoint. The TIM-3 refolding was obtained by the association of high pressure and alkaline pH. Results: The purified TIM-3-ECD showed the correct secondary structure and was recognized from anti-TIM-3 structural-dependent antibodies likewise commercial TIM-3-ECD was produced by a mammal cells system. Furthermore, immunofluorescence showed the ability of TIM-3-ECD to bind to the surface of lung cancer A549 cells and to provide an additional boost for the expression of the lymphocyte activation marker CD69 in anti-CD3/CD28 activated human PBMC. Discussion: Taken together these results validated a methodology able to obtain active checkpoint proteins from bacterial inclusion bodies, which will be helpful to further investigate the interactions of this and others not yet explored immune checkpoints.

Effects of long-term pretreatment with isoproterenol on bromocriptine-induced tachycardia in conscious rats.

It has been shown that bromocriptine-induced tachycardia, which persisted after adrenalectomy, is (i) mediated by central dopamine D2 receptor activation and (ii) reduced by 5-day isoproterenol pretreatment, supporting therefore the hypothesis that this effect is dependent on sympathetic outflow to the heart. This study was conducted to examine whether prolonged pretreatment with isoproterenol could abolish bromocriptine-induced tachycardia in conscious rats. Isoproterenol pretreatment for 15 days caused cardiac hypertrophy without affecting baseline blood pressure and heart rate. In control rats, intravenous bromocriptine (150 microg/kg) induced significant hypotension and tachycardia. Bromocriptine-induced hypotension was unaffected by isoproterenol pretreatment, while tachycardia was reversed to significant bradycardia, an effect that was partly reduced by i.v. domperidone (0.5 mg/kg). Neither cardiac vagal nor sympathetic tone was altered by isoproterenol pretreatment. In isolated perfused heart preparations from isoproterenol-pretreated rats, the isoproterenol-induced maximal increase in left ventricular systolic pressure was significantly reduced, compared with saline-pretreated rats (the EC50 of the isoproterenol-induced increase in left ventricular systolic pressure was enhanced approximately 22-fold). These results show that 15-day isoproterenol pretreatment not only abolished but reversed bromocriptine-induced tachycardia to bradycardia, an effect that is mainly related to further cardiac beta-adrenoceptor desensitization rather than to impairment of autonomic regulation of the heart. They suggest that, in normal conscious rats, the central tachycardia of bromocriptine appears to predominate and to mask the bradycardia of this agonist at peripheral dopamine D2 receptors.

T-cell compartment involvement in two high antibody responder lines of mice (HI and HII Biozzi mice) respectively susceptible and resistant to collagen-induced arthritis.

The T-cell compartment was investigated in two high antibody responder lines of mice respectively susceptible (HI) and resistant (HII) to chicken collagen (CII)-induced arthritis (CIA). Previous data had shown that both lines were high anti-CII Ab producers, without any TCR V-beta gene defect or membrane expression impairment. The present studies demonstrate that anti-CII proliferation is much lower in HII than in HI. These results are confirmed by the limiting dilution analysis of anti-CII T-precursor frequencies (1/991 in HI and 1/12175 in HII). The percentage of CD8+ T cells is constitutively higher in HII mice, this difference increasing after CII immunization. This finding suggests a suppressive effect accounting for resistance to CIA. However, no restoration of specific response was achieved by in-vivo or in-vitro depletion of CD8+ T cells. T clones specific for Chicken CII could be obtained only from primed HI mice. Four of five clones with CD8+ phenotype proliferated in vitro to native and denatured CII and showed cytotoxic function in an anti-CD3 redirected assay. The CD4+ clone was shown to proliferate on both HI and HII-pulsed APC, which rules out a major CII processing/presentation defect in HII.

Trypanosoma cruzi: maintenance of parasite-specific T cell responses in lymph nodes during the acute phase of the infection.

Mice infected with 5 x 10(3) forms of Trypanosoma cruzi showed a transient, but severe impairment of in vitro spleen cell responses to parasite antigens and to Concanavalin A (Con A). In contrast, inguinal and periaortic lymph node (LN) cells displayed high parasite-specific proliferative responses and only a partial reduction of the Con A-induced proliferation during the acute and chronic phases of infection. Lymphocytes that underwent blastic transformation in T. cruzi-stimulated cell cultures were of the L3T4+ phenotype. Suppression of spleen cell responses occurred in the acute phase whether mice were infected with high (3 x 10(5] or low (5 x 10(3] doses of T. cruzi by intraperitoneal or subcutaneous route. Suppression of the T. cruzi-specific proliferative response of LN cells was only observed in mice infected with high subcutaneous inocula. This suppression, however, was restricted to the LNs draining the site of inoculation without affecting distant LNs. Supernatants from parasite-stimulated proliferating LN cells displayed low or undetectable T cell growth factor (TCGF) activity, in contrast with the high TCGF levels found in supernatants of the same cells stimulated with Con A. Low levels of TCGF were also detected in cultures of LN cells from mice immunized with T. cruzi extracts. Neither the T. cruzi antigen used for in vitro stimulation nor the LN cell supernatants from infected mice inhibited TCGF activity. These findings indicate that (1) parasite-specific responses are present in the LN compartment throughout the acute phase of T. cruzi infection in mice and (2) the proliferative response of L3T4+ LN cells from infected mice to T. cruzi antigens is not associated with a high TCGF secretory response.

Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T cell populations capable of mediating Leishmania major-specific delayed-type hypersensitivity.

The effect of adoptive transfer of in vitro-propagated Leishmania major-specific T cell populations on the course of experimentally induced cutaneous leishmaniasis was studied in mice. The L. major-specific T cells expressed the T helper/inducer phenotype and were able in vitro to a) mount a specific proliferative response, b) provide specific helper activity for antibody responses, c) activate parasitized macrophages resulting in L. major destruction, and d) secrete macrophage-activating factors as tested in a tumoricidal assay. These T cells were also found capable of transferring parasite-specific delayed-type hypersensitivity responses to normal syngeneic mice. Results indicated that the i.v. transfer of these L. major-specific T cell populations into normal syngeneic mice exacerbated cutaneous lesions induced by infection with L. major. This effect on the disease process appeared to be dependent upon recognition of parasite antigens by the injected T cells because no exacerbation of the disease process was seen after the transfer of similar T cell populations specific for an antigen unrelated to the parasite, namely ovalbumin. However, the inclusion of ovalbumin in the L. major infecting inoculum resulted in an exacerbating effect of ovalbumin-specific T cells on cutaneous leishmaniasis. These unexpected results were supported by observations showing that immunization of mice with L. major antigens in complete Freund's adjuvant 7 days before infection with L. major led to exacerbated lesions. A similar aggravation of L. major-induced cutaneous lesions was also observed in mice previously immunized with an unrelated antigen provided that this antigen was included in the L. major infecting inoculum.

Adoptive transfer of delayed type hypersensitivity reactions specific for Leishmania major antigens to normal mice using murine T cell populations and clones generated in vitro.

Leishmania major specific murine T cell blasts and clones maintained in vitro were tested for their ability to adoptively transfer delayed type hypersensitivity (DTH) reactions to normal mice. These effector cells exhibited a Lyt 1+2- cell surface phenotype. They induced specific DTH reactions in syngeneic DBA/2 mice after local transfer in the footpad, (together with parasite antigens) or intravenous injection followed by challenge with parasite antigens in the footpad. The DTH reactions were specific for L. major antigens and required H-2 identity between the injected T cells (clones) and the adoptively transferred host. Using radioactively labelled T cell blasts for intravenous transfer, it was demonstrated that a large fraction of these functionally active cells localized in the spleen and footpad which had been challenged with parasite antigens.

Antibody suppression in mice infected with Nippostrongylus brasiliensis.

A/SN mice infected with N. Brasiliensis showed depressed anti-DNP antibody responses following immunization with DNP-Asc in alum. The immunosuppression was only observed when infection preceded immunization by between 2 and 7 days, and was not achieved when the interval was extended to 10 days. The suppression lasted at least 50 days, and affected IgE levels more than IgG1 or IgG agglutinating anti-DNP antibodies. A high dose of infective larvae (500-1000 per mouse) was necessary to induce suppression. Use of low dose irradiation indicated a parasite-induced radiosensitive component of the mouse immune system which negatively regulated the anti-DNP IgE response. These results suggested that the parasite could induce suppression in an analogous manner to sequential antigen-induced suppression (AIS).