The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte–antigen-presenting cell interface

During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav−/− mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav+/− mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav−/− mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.

Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells.

Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused into H-2b or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating F1sc-->H-2b and F1sc-->H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gp120 glycoproteins elicited antiviral CTL activity. F1sc-->H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b restricted gB epitope, whereas F1sc-->H-2b chimeras responded to the H-2b but not the H-2d restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved in initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.

Peptide analogs to pathogenic epitopes of the human acetylcholine receptor alpha subunit as potential modulators of myasthenia gravis.

Myasthenia gravis is an autoimmune disease in which T cells specific to epitopes of the autoantigen, the human acetylcholine receptor, play a role. We identified two peptides, p195-212 and p259-271, from the alpha subunit of the receptor, which bound to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) from peripheral blood lymphocytes of myasthenia gravis patients and stimulated lymphocytes of >80% of the patients. We have prepared analogs of these myasthenogenic peptides and tested their ability to bind to MHC class II determinants and to interfere specifically with T-cell stimulation. We first determined relative binding efficiency of the myasthenogenic peptides and their analogs to APCs of patients. We found that single substituted analogs of p195-212 (Ala-207) and p259-271 (Lys-262) could bind to human MHC molecules on APCs as efficiently as the original peptides. Moreover, dual analogs containing the two single substituted analogs in one stretch (either sequentially, Ala-207/Lys-262, or reciprocally, Lys-262/Ala-207) could also bind to APCs of patients, including those that failed to bind one of the single substituted analogs. The single substituted analogs significantly inhibited T-cell stimulation induced by their respective myasthenogenic peptides in >95% of the patients. The dual analogs were capable of inhibiting stimulation induced by either of the peptides: They inhibited the response to p195-212 and p259-271 in >95% and >90% of the patients, respectively. Thus, the dual analogs are good candidates for inhibition of T-cell responses of myasthenia gravis patients and might have therapeutic potential.

Interleukin 3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex "re-presentation" of exogenous antigen by tumor-infiltrating antigen-presenting cells.

We show that interleukin 3 (IL-3) enhances the generation of tumor-specific cytotoxic T lymphocytes (CTLs) through the stimulation of host antigen-presenting cells (APCs). The BALB/c (H-2d) spontaneous lung carcinoma line 1 was modified by gene transfection to express ovalbumin as a nominal "tumor antigen" and to secrete IL-3, a cytokine enhancing myeloid development. IL-3-transfected tumor cells are less tumorigenic than the parental cell line, and tumor-infiltrating lymphocytes isolated from these tumors contain increased numbers of tumor-specific CTLs. By using B3Z86/90.14 (B3Z), a unique T-cell hybridoma system restricted to ovalbumin/H-2b and implanting the tumors in (BALB/c x C57BL/6)F1 (H-2d/b) mice, we demonstrate that the IL-3-transfected tumors contain an increased number of a rare population of host cells that can process and "re-present" tumor antigen to CTLs. Electron microscopy allowed direct visualization of these host APCs, and these studies, along with surface marker phenotyping, indicate that these APCs are macrophage-like. The identification of these cells and their enhancement by IL-3 offers a new opportunity for tumor immunotherapy.

Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells.

Human melanoma cells can process the MAGE-1 gene product and present the processed nonapeptide EADPTGHSY on their major histocompatibility complex class I molecules, HLA-A1, as a determinant for cytolytic T lymphocytes (CTLs). Considering that autologous antigen presenting cells (APCs) pulsed with the synthetic nonapeptide might, therefore, be immunogenic, melanoma patients whose tumor cells express the MAGE-1 gene and who are HLA-A1+ were immunized with a vaccine made of cultured autologous APCs pulsed with the synthetic nonapeptide. Analyses of the nature of the in vivo host immune response to the vaccine revealed that the peptide-pulsed APCs are capable of inducing autologous melanoma-reactive and the nonapeptide-specific CTLs in situ at the immunization site and at distant metastatic disease sites.