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Vaccinia virus is the prototypic orthopoxvirus and was the vaccine used to eradicate smallpox, yet the expression profiles of many of its genes remain unknown. Using a genome tiling array approach, we simultaneously measured the expression levels of all 223 annotated vaccinia virus genes during infection and determined their kinetics. For 95% of these genes, significant transcript levels were detected. Most remarkably, classification of the genes by their expression profiles revealed 35 genes exhibiting immediate-early expression. Although a similar kinetic class has been described for other virus families, to our knowledge, this is the first demonstration of its existence in orthopoxviruses. Despite expression levels higher than for genes in the other three kinetic classes, the functions of more than half of these remain unknown. Additionally, genes within each kinetic class were spatially grouped together in the genome. This genome-wide picture of transcription alters our understanding of how orthopoxviruses regulate gene expression. gene transcription | genome tiling array | microarray | vaccinia virus

We have analyzed by ex vivo ELISPOT the anti-vaccinia cytotoxic T lymphocyte responses of peripheral blood mononuclear cells from humans vaccinated with Dryvax vaccine. More than 6,000 peptides from 258 putative vaccinia ORFs predicted to bind the common molecules of the HLA A1, A2, A3, A24, B7, and B44 supertypes were screened with peripheral blood mononuclear cells of 31 vaccinees. A total of 48 epitopes derived from 35 different vaccinia antigens were identified, some of which (B8R, D1R, D5R, C10L, C19L, C7L, F12, and O1L) were recognized by multiple donors and contain multiple epitopes recognized in the context of different HLA types. The antigens recognized tend to be >100 residues in length and are expressed predominantly in the early phases of infection, although some late antigens were also recognized. Viral genome regulation and virulence factor were recognized most frequently, whereas few structural proteins were immunogenic. Finally, most epitopes were highly conserved among vaccinia virus Western Reserve, variola major and modified vaccinia Ankara, supporting their potential use in vaccine and diagnostic applications. immunodominance | smallpox vaccination | virulence factors

Although there was no impairment in IL-2 secretion and proliferation of Fyn-deficient naive CD4 cells after stimulation with antigen and antigen-presenting cells, stimulation of these cells with anti-CD3 and anti-CD28 revealed profound defects. Crosslinking of purified wild-type naive CD4 cells with anti-CD3 activated Lck and initiated the signaling cascade downstream of Lck, including phosphorylation of ZAP-70, LAT, and PLC-[gamma]1; calcium flux; and dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT)p. All of these signaling events were diminished severely in Fyn-deficient na'ive cells activated by CD3 crosslinking. Coaggregation of CD3 and CD4 reconstituted this Lck-dependent signaling pathway in [Fyn.sup.-/-] T cells. These results suggest that when signaling of naive T cells is restricted to the T cell antigen receptor, Fyn plays an essential role by positive regulation of Lck activity.

The signaling pathways that lead to the localization of cellular protein to the area of interaction between T cell and antigen-presenting cell and the mechanism by which these molecules are further sorted to the peripheral supramolecular activation cluster or central supramolecular activation cluster regions of the immunologic synapse are poorly understood. In this study, we investigated the functional involvement of CD28 costimulation in the T cell receptor (TCR)-mediated immunologic synapse formation with respect to protein kinase C (PKC)[theta] localization. We showed that CD3 crosslinking alone was sufficient to induce PKC[theta] capping in naYve CD[4.sup.+] T cells. Studies with pharmacologic inhibitors and knockout mice showed that the TCR-derived signaling that drives PKC[theta] membrane translocation requires the Src family kinase, Lck, but not Fyn. In addition, a time course study of the persistence of T cell molecules to the immunologic synapse indicated that PKC[theta], unlike TCR, persisted in the synapse for at least 4 h, a time that is sufficient for commitment of a T cell to cell division. Finally, by using TCR-transgenic T cells from either wild-type or CD28-deficient mice, we showed that CD28 expression was required for the formation of the mature immunologic synapse, because antigen stimulation of CD[28.sup.-] T cells led to a diffuse pattern of localization of PKC[theta] and lymphocyte function-associated antigen-1 in the immunologic synapse, in contrast to the central supramolecular activation cluster localization of PKC[theta] in CD[28.sup.+] T cells.

Low levels of T-cell receptor interactions lead to negative selection of CD4+ CD8+ thymocytes. T-cell receptor peptide antagonists act upon thymocytes from transgenic mice for the AD10 T-cell receptor to delete CD4+ CD8+ thymocytes. The peptide antagonists do not induce mature T-cell activation.

Tumor-specific human cytotoxic T lymphocytes (CTL) may be induced in vitro, while ex vivo antigen-specific approaches may be applied to cancer immunological therapy. CTL identifies peptide antigens related to cell surface major histocompatibility complex molecules.

Changes in early biochemical T-cell activation events and membrane phenomena due to T-cellreceptor antagonism are discussed. A nonstimulatory analogue of the antigenic peptide influenza hemagglutinin was used to characterize synthesized peptide samples. The non-interference of antagonistic peptides in the cellular events leading to stable T-cell conjugate formations and its inhibition of early biochemical events required for T-cell proliferation were demonstrated.

Between 650 and 2000 different peptides are associated with the major histocompatibility complex class II molecule [I-A.sup.d]. Sequences for nine of these were obtained by a combination of automated Edman [...]

The Minimal Number of Class II MHC-Antigen Complexes Needed for T Cell Activation THE HEL PEPTIDE THAT CORREsponds to the [I-E.sup.d]-restricted determinant, HEL(107-116), can be isolated from naturally processed antigen [...]

T cell receptor (TCR) antagonists inhibit antigen-induced T cell activation and by themselves fail to induce phenotypic changes associated with T cell activation. However, we have recently shown that TCR antagonists are inducers of antigen-presenting cell (APC)-T cell conjugates. The signaling pathway associated with this cytoskeleton-dependent event appears to involve tyrosine phosphorylation and activation of Vav. In this study, we investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in antagonist-induced signaling pathway. Antagonist stimulation increased tyrosine phosphorylation and kinase activity of Fyn severalfold, whereas little or no increase in Lck and ZAP-7 activity was observed. Second, TCR stimulation of [Lck.sup.-], [Fyn.sup.hi] Jurkat cells induced strong tyrosine phosphorylation of Vav. In contrast, minimal increase in tyrosine phosphorylation of Vav was observed in [Lck.sup.hi], [Fyn.sup.10] Jurkat cells. Finally, study of T cells from a Fyn-deficient TCR transgenic mouse also showed that Fyn was required for tyrosine phosphorylation and activation of Vav induced by both antagonist and agonist peptides. The deficiency in Vav phosphorylation in Fyn-deficient T cells was associated with a defect in the formation of APC-T cell conjugates when T cells were stimulated with either agonist or antagonist peptide. We conclude from these results that Vav is a selective substrate for Fyn, especially under conditions of low-affinity TCR-mediated signaling, and that this signaling pathway involving Fyn, Vav, and Rac-1 is required for the cytoskeletal reorganization that leads to T cell-APC conjugates and the formation of the immunologic synapse.

The Relation Between Major Histocompatibility Complex (MHC) Restriction and the Capacity of Ia to Bind Immunogenic Peptides IN GENERAL, PROTEIN ANTIGENS ARE PHYSICALLY ALTERED(processed) by accessory cells as a prerequisite [...]

Autologous Peptides Constitutively Occupy the Antigen Binding Site on Ia IN GENERAL, T CELLS OF THE HELPER subset recognize complexes of altered--that is, denatured or fragmented--protein antigen and MHC Ia [...]