Iris Motta

In vitro induction of specific cytotoxic T lymphocytes using recombinant single-chain MHC class I/peptide complexes.

We have previously described the production and purification of a murine single-chain, soluble recombinant major histocompatibility complex (MHC) class I molecule (SC-Kd). A similar strategy was devised to produce a recombinant HLA-A2.1 (SC-A2) molecule. The latter was composed of the first three domains of the HLA-A2.1 heavy chain connected to human beta 2-microglobulin through a spacer of 15 amino acids. Immunoaffinity-purified SC-A2 molecules-were correctly folded and biologically functional. They specifically bound HLA-A2-restricted peptides and induced a peptide-specific cytotoxic T lymphocyte (CTL) clone to proliferate and secrete interleukin-2. The ability of murine and human SC-MHC molecules to elicit primary CTLs in vitro was next investigated. When coated in high density onto beads, complexes of antigenic peptide and SC-Kd or SC-A2 molecules efficiently induced a specific primary CTL response in vitro. Furthermore, the structural features of these CTLs were characterized by T cell receptor-beta chain analysis, which revealed rearrangements very similar, if not identical, to those found in CTLs generated by in vivo immunization. Such single-chain, soluble recombinant MHC class I molecules should provide a useful tool in particular for peptide binding assays and for in vitro primary CTL induction to identify immunogenic peptides such as those derived from known tumor-associated antigens.

In vitro induction of naive cytotoxic T lymphocytes with complexes of peptide and recombinant MHC class I molecules coated onto beads: role of TCR/ligand density.

We previously reported that complexes of peptide with soluble single‐chain recombinant MHC (SC‐MHC) class I molecules are able to induce cytotoxic T lymphocytes (CTL) in vitro in a murine system with an efficiency comparable to that observed with peptide‐pulsed dendritic cells as antigen‐presenting cells. In this report, we have assessed the capacity of preformed peptide/SC‐Kd complexes in monomeric or dimeric form as well as of peptide/SC‐Kd‐loaded beads to generate in vitro specific CTL responses from naive DBA/2 spleen cells. Peptide/SC‐Kd‐coated beads were consistently more efficient. We evaluated the role of co‐stimulatory molecules, using monoclonal antibodies anti‐CD80 or anti‐CD86. In addition, the capacity of peptide/SC‐Kd‐coated beads to generate a CTL response from purified naive CD8+ T cells was ascertained. Taken together, the results indicate that, under our conditions, CTL priming does not require the participation of co‐stimulatory molecules and is the consequence of a direct interaction between the cognate TCR on peptide‐specific CTL precursors and the peptide/SC‐Kd‐loaded beads. Titration of the amount of preformed complexes of SC‐Kd and peptide 170 –179 of HLA‐CW3 that need to be coated onto the beads to prime CTL precursors shows an activation threshold which can be calculated to be between 25 000 and 50 000 complexes. In effect, in cultures stimulated with specific peptide CW3/SC‐Kd complexes representing less than 50 % occupancy of the total (105 ) complexes on the beads, no peptide‐specific cytolytic activity was observed. These results suggest that the efficiency of the primary CTL induction depends on the density of specific peptide/SC‐Kd complexes present on the beads.

TNP-LPS induces an IgG anti-TNP immune response in mice.

Abstract The trinitrophenylated derivatives of lipopolysaccharide (TNP-LPS) elicit a specific anti-TNP, thymus-independent immune response in mice. After a single injection of antigen, anti-TNP antibodies of IgM and IgG isotypes are detected at the cellular and at the humoral levels, in athymic nude mice as well as in conventional (C57B1/6 × DBA/2)F 1 mice. The immune sera were resolved into IgM and IgG molecules by gel filtration; both fractions showed an anti-TNP activity, thus confirming the data obtained by the cellular analysis.

Induction and differentiation of B memory cells by a thymus-independent antigen, trinitrophenylated lipopolysaccharide

Abstract The capacity of a thymus-independent antigen, i.e., trinitrophenylated lipopolysaccharide (TNP-LPS), to induce and stimulate memory cells was investigated. C57BL/6 mice were immunized with an optimal immunogenic dose of TNP-LPS and the parameters of the antihapten response obtained from a later challenge with the same antigen were defined, at the antibody-secreting cell level, both in vivo and in vitro . Compared to the primary response, the secondary in vivo anti-TNP response is characterized by a shift in kinetics and the appearance of anti-TNP antibodies of the IgG isotype. The generation, by TNP-LPS, of a TNP-specific B-cell memory as expressed in IgG antibody synthesis does not require the presence of functional T cells; it can be obtained in irradiated hosts reconstituted with spleen cells depleted of Thy-1-positive cells. In vitro , TNP-LPS-primed spleen cells give rise to a T-independent anti-TNP response which is up to 15-fold higher than the primary response. This enhanced anti-hapten IgM response requires cellular division. The priming effect by TNP-LPS is specific for the TNP moiety of the TNP-LPS molecule and persists for at least 11 weeks. These results indicate that, in C57BL/6 mice, stimulation with TNP-LPS leads to the differentiation of B cells not only into antibody-forming cells but also into memory cells which can subsequently be triggered by the same antigen. Both the generation and the activation of TNP-specific B-cell memory by TNP-LPS implicate T-cell-independent events.

Absence of weight loss during Cryptosporidium infection in susceptible mice deficient in Fas-mediated apoptosis.

Apoptosis plays a major role in the development of pathogenesis due to a number of microbial infections. Epithelial cells have been previously shown to die through apoptosis during in vitro infection by the Apicomplexan parasite Cryptosporidium parvum. We now test the possibility that Fas (APO-1/CD95)-dependent apoptosis of uninfected cells, due to enhanced expression of the Fas ligand (FasL) on infected cells, may contribute to the pathology of cryptosporidiosis. Expression of the FasL increased by a large amount on the surface of intestinal epithelial cells infected with C. parvum, and the increase was limited exclusively to infected cells. In addition, a significant increase in FasL expression was observed in epithelial cells from the small intestine of mice infected with C. parvum. Finally, whereas wild-type mice depleted of CD4(+) lymphocytes lost weight during C. parvum infection, CD4(+) cell-depleted lpr mice (deficient in Fas function) infected with C. parvum gained weight at the same rate as undepleted wild-type or lpr mice. These results suggest that bystander Fas-dependent apoptosis of uninfected epithelial cells may exacerbate the weight loss associated with cryptosporidiosis.

Effects of Suramin on the immune responses to sheep red blood cells in mice: II. In vitro studies

The effect of Suramin on the secondary in vitro response to sheep erythrocytes (SRBC) was studied. Spleen cells from mice which were treated with Suramin immediately prior to sensitization with SRBC failed to respond to an in vitro SRBC challenge. This Suramin-induced immunosuppression is not related to a defect in macrophage or B-cell function(s). Suramin does not interfere with the induction by SRBC of radioresistant and radiosensitive helper-T-cell subpopulations. Cell separation studies, using wheat germ agglutinin, showed radiosensitive helper-T-cell function in the nonagglutinated fraction while the radioresistant helper activities are carried out by the agglutinated subpopulation. Evidence is presented that Suramin administration results in a suppressive T-cell activity which can be demonstrated in the subpopulation agglutinated by wheat germ agglutinin. The role of such suppressive T cells in the inhibitory effect exerted by Suramin on the cell-mediated delayed-type hypersensitivity response to SRBC is discussed.

Generation of immune memory by haptenated derivatives of thymus-independent antigens in C57BL/6 mice: 1. The differentiation of memory B lymphocytes into antibody-secreting cells depends on the nature of the thymus-independent carrier used for memory induction and/or revelation

It has been previously reported that trinitrophenylated lipopolysaccharide (TNP-LPS), a thymus-independent (TI)-1 antigen, elicits an anamnestic response to TNP in C57BL/6 mice. The ability of these mice to mount a secondary response to TI-2 antigens was analyzed. Priming with DNP-Ficoll or DNP-Dextran, both TI-2 antigens, resulted in an increased frequency of TNP-binding B lymphocytes. Evidence is presented that memory cell-induction by DNP-Ficoll does not require functional T cells. The differentiation into antibody-forming cells (AFC) of memory cells generated by DNP-Dextran or DNP-Ficoll cannot be obtained by a challenge with either antigen. There was no indication that the lack of a secondary response to TI-2 antigens was related to suppressive T cells interfering with memory expression. Memory cells induced by DNP-Dextran or DNP-Ficoll can nevertheless be activated by TNP-LPS. In contrast to the restricted sensitivity of TNP-memory cells generated by TI-2 antigens, TNP-LPS-induced memory cells are indifferently susceptible to TI-1 or TI-2 antigenic stimulation. These results are discussed in terms of memory B-cell subpopulations.

Effect(s) of lipopolysaccharide on lectin-induced T-cell activation

The present study focuses on the effect of lipopolysaccharide (LPS) on the cellular events leading to T-cell activation by concanavalin A (Con A). Interleukin 2 (Il-2) production is much reduced in Con A-stimulated cultures of spleen cells derived from LPS-treated mice. This depressed Il-2 synthesis is not related to the eventual activity of LPS-activated suppressive B cells. Rather, it reflects an ineffective collaboration between adherent cells and T lymphocytes. The low level of Il-2 produced by LPS-sensitized spleen cells is sufficient for lectin-induced T-cell proliferation. Moreover, acquisition of responsiveness to Il-2 is unaltered by LPS. No strict correlation was found between the deficiency in Il-2 production and the inability of LPS-sensitized spleen cells to generate a thymus-dependent response. Less time (5 hr) is needed for LPS to exert its inhibitory effect on an anti-sheep red blood cell response than on Il-2 synthesis (at least 24 hr). Results are discussed in terms of cellular interactions implicated in a polyclonal T-cell response and with regard to the contribution of Il-2 to the LPS-induced immune unresponsiveness.

Heterogeneity of mouse thy 1.2 antigen expression revealed by monoclonal antibodies

Abstract A different sensitivity of T cells from C57B1/6 and DBA/2 mice to treatment with the monoclonal anti-Thy 1.2 F7D5 serum as compared with a conventional alloantiserum is reported. Depletion of T helper cells, Con A-, PHA-, MLC-, and GVH-reactive cells from a DBA/2 or C57B1/6 spleen cell population was readily achieved with the conventional alloserum. In contrast, the F7D5 antiserum abolished all T functions studied in C57B1/6 spleen cells whereas it was totally or partially ineffective on DBA/2 spleen cells when T helper, MLC, or GVH reactivity were assayed. It did however eliminate the capacity of DBA/2 spleen cells to respond to stimulation with Con A or PHA. Analysis in an Ortho-Cytofluorograf of thymocytes and sIg − lymphocytes labeled with either GAMB-F or F7D5 + RAM Ig-F showed no difference at the level of the thymocytes: Thy 1.2 antigen as revealed by either GAMB or F7D5 is similarly expressed in the two mouse strains. The fluorescence profiles of splenic T lymphocytes indicated a reduced representation per unit cell basis of the Thy 1.2 antigenic determinant recognized by F7D5 in DBA/2 mice. Moreover, this same determinant is expressed in only 70% of all Thy 1.2-positive cells detected in DBA/2 sIg − population. This implies that, in DBA/2 mice, maturation of T cells is accompanied by a complete or partial loss of the F7D5 Thy 1.2 determinant and that T helper functions and MLC and GVH reactivity are mediated by T cells which express little or none of this F7D5 Thy 1.2 determinant.

Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line in vitro.

Stem cells, having the property of self renewal, offer the promise of lifelong repair of damaged tissue. However, somatic tissue‐committed primary stem cells are rare and difficult to expand in vitro. Genetically modified stem‐like cells with the ability to expand conditionally provide a valuable tool with which to study stem cell biology, especially the cellular events of proliferation and differentiation. In addition, stem cells may be appropriate candidates for therapeutic applications.