Sculpting MHC class II-restricted self and non-self peptidome by the class I Ag-processing machinery and its impact on Th-cell responses.

It is generally assumed that the MHC class I antigen (Ag)-processing (CAP) machinery - which supplies peptides for presentation by class I molecules - plays no role in class II-restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition, TAP deficiency or ERAAP deficiency led to dramatically altered T helper (Th)-cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4(+) T-cell repertoire, or both underlay the above finding. We found that TAP deficiency and ERAAP deficiency dramatically altered the quality of class II-associated self peptides suggesting that the CAP machinery impacts class II-restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4(+) T-cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of WT animals resulting in altered CD4(+) T-cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II-restricted peptidome, impacting the CD4(+) T-cell repertoire, and ultimately altering Th-cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II-restricted epitopes that would otherwise be capable of eliciting functional Th-cell responses.

Genome Sequences of Microbacteriophages Zada and Ioannes.

Microbacteriophages Zada and Ioannes were isolated from soil and characterized. Genomes were then sequenced and annotated. This was done using the host bacterium Microbacterium foliorum Zada and Ioannes are both lytic phages with a Siphoviridae morphotype.

Complete Genome Sequences of Cluster G Mycobacteriophage Darionha, Cluster A Mycobacteriophage Salz, and Cluster J Mycobacteriophage ThreeRngTarjay.

Mycobacteriophages Darionha, Salz, and ThreeRngTarjay are mycobacteriophages isolated using the host Mycobacterium smegmatis mc2155. Following isolation from soil samples, all three siphoviridae phages were characterized, and their genomes were sequenced and annotated.

Complete Genome Sequences of Cluster A Mycobacteriophages BobSwaget, Fred313, KADY, Lokk, MyraDee, Stagni, and StepMih.

Seven mycobacteriophages from distinct geographical locations were isolated, using Mycobacterium smegmatis mc2155 as the host, and then purified and sequenced. All of the genomes are related to cluster A mycobacteriophages, BobSwaget and Lokk in subcluster A2; Fred313, KADY, Stagni, and StepMih in subcluster A3; and MyraDee in subcluster A18, the first phage to be assigned to that subcluster.

Complement activation in the Parkinson's disease substantia nigra: an immunocytochemical study.

BACKGROUND: Inflammatory processes are increased in the Parkinson's disease (PD) brain. The long-term use of nonsteroidal anti-inflammatory drugs has been associated, in retrospective studies, with decreased risk for PD, suggesting that inflammation may contribute to development of this disorder. The objective of this study was to determine the extent of complement activation, a major inflammatory mechanism, in PD. METHODS: Substantia nigra specimens from young normal subjects (n = 11-13), aged normal subjects (n = 24-28), and subjects with PD (n = 19-20), Alzheimer's disease (AD; n = 12-13), and dementia with Lewy bodies (DLB; n = 9) were stained for iC3b and C9, representing early- and late-stage complement activation, respectively. Numbers of iC3b+, C9+, and total melanized neurons in each section were counted in a blinded fashion. Nonparametric analyses were used to evaluate differences between groups and to evaluate correlations between complement staining, numbers of melanized neurons, and the duration of PD. RESULTS: Lewy bodies in both PD and DLB specimens stained for iC3b and C9. Staining was also prominent on melanized neurons. The percentage of iC3b+ neurons was significantly increased in PD vs. aged normal and AD specimens, and in young normal vs. aged normal specimens. C9 immunoreactivity was significantly increased in PD vs. AD specimens, but unlike iC3b, the increased C9 staining in PD and young normal specimens did not achieve statistical significance vs. aged normal specimens. iC3b and C9 staining in PD specimens was not correlated with the numbers of remaining melanized neurons, nor with the duration of PD. CONCLUSION: Complement activation occurs on Lewy bodies and melanized neurons in the PD substantia nigra. Early complement activation (iC3b) is increased on melanized neurons in PD vs. aged normal specimens, and late-stage complement activation (C9) also tends to increase. This latter finding suggests that complement activation may contribute to loss of dopaminergic neurons in some individuals with PD. Complement activation on melanized neurons appears to decrease with normal aging, suggesting a possible neuroprotective role for this process in the normal substantia nigra.

Viral infection causes a shift in the self peptide repertoire presented by human MHC class I molecules.

PURPOSE: MHC class I presentation of peptides allows T cells to survey the cytoplasmic protein milieu of host cells. During infection, presentation of self peptides is, in part, replaced by presentation of microbial peptides. However, little is known about the self peptides presented during infection, despite the fact that microbial infections alter host cell gene expression patterns and protein metabolism. EXPERIMENTAL DESIGN: The self peptide repertoire presented by HLA-A*01;01, HLA-A*02;01, HLA-B*07;02, HLA-B*35;01, and HLA-B*45;01 (where HLA is human leukocyte antigen) was determined by tandem MS before and after vaccinia virus infection. RESULTS: We observed a profound alteration in the self peptide repertoire with hundreds of self peptides uniquely presented after infection for which we have coined the term "self peptidome shift." The fraction of novel self peptides presented following infection varied for different HLA class I molecules. A large part (approximately 40%) of the self peptidome shift arose from peptides derived from type I interferon-inducible genes, consistent with cellular responses to viral infection. Interestingly, approximately 12% of self peptides presented after infection showed allelic variation when searched against approximately 300 human genomes. CONCLUSION AND CLINICAL RELEVANCE: Self peptidome shift in a clinical transplant setting could result in alloreactivity by presenting new self peptides in the context of infection-induced inflammation.

MHC class II peptide flanking residues of exogenous antigens influence recognition by autoreactive T cells.

Molecular mimicry between exogenous microbial antigens and self-epitopes has been proposed as a triggering mechanism for autoimmune diseases for many years. We reported that a peptide from a protein specific to Chlamydia pneumoniae (Cpn0483) which shares a motif with the dominant encephalitogenic epitope of the self-antigen, rat myelin basic protein (rat68-86), elicits experimental autoimmune encephalomyelitis (EAE) in Lewis rats. We recently observed that rat68-86 utilizes aspartic acid (D) and arginine (R) in the common motif as primary and secondary TCR contacts, respectively. In contrast, the encephalitogenic activity of Cpn0483 is dependent on R and the C-terminal asparagine (N), which flanks the MHC class II-P9 anchor residue. Thus, rat68-86 and Cpn0483 share a common motif, are encephalitogenic and are both restricted by MHC class II RT1.B(l). T cells from rats immunized with the encephalitogenic Cpn0483 peptide proliferated to the priming peptide as well as to the non-encephalitogenic CpnN>A analog. However, CpnN>A-primed T cells did not respond to the native Cpn0483 peptide. We conclude that the MHC-flanking C-terminal asparagine residue markedly influences T cell recognition by the chlamydial peptide.

Discovering naturally processed antigenic determinants that confer protective T cell immunity.

CD8+ T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infection - information that is critical for the rational design of TCD8-targeted vaccines. We employed a proteomics-based approach for large-scale discovery of naturally processed determinants derived from a complex pathogen, vaccinia virus (VACV), that are presented by the most frequent representatives of four major HLA class I supertypes. Immunologic characterization revealed that many previously unidentified VACV determinants were recognized by smallpox-vaccinated human peripheral blood cells in a variegated manner. Many such determinants were recognized by HLA class I-transgenic mouse immune TCD8 too and elicited protective TCD8 immunity against lethal intranasal VACV infection. Notably, efficient processing and stable presentation of immune determinants as well as the availability of naive TCD8 precursors were sufficient to drive a multifunctional, protective TCD8 response. Our approach uses fundamental insights into T cell epitope processing and presentation to define targets of protective TCD8 immunity within human pathogens that have complex proteomes, suggesting that this approach has general applicability in vaccine sciences.

Autoreactive T cells persist in rats protected against experimental autoimmune encephalomyelitis and can be activated through stimulation of innate immunity.

Lewis rats can be rendered unresponsive to experimental autoimmune encephalomyelitis by immunization with myelin basic protein (MBP), or MBP68-86, the dominant encephalitogenic MBP epitope for this strain, administered in IFA. However, protected rats harbor potentially encephalitogenic T cells, which are maintained in an inactive state. We investigated whether these quiescent effector cells could be activated in vitro. Although these T cells respond poorly to MBP68-86, they proliferate vigorously whether cocultured with MBP68-86 and either IL-2 or IL-12, suggesting that the T cells are in a state of anergy. Moreover, we could activate these anergic T cells with peptide and cytosine-guanine dinucleotide (CpG) oligonucleotide, but not control oligonucleotide, suggesting that products of the innate immune response are capable of activating anergic autoreactive T cells. The activated T cells produced the proinflammatory cytokine, IFN-gamma in response to IL-12, and IL-6 was secreted in response to CpG oligonucleotide. IL-6 has been reported to play a role in T cell activation by blocking T regulatory/suppressor (Treg) cell-mediated suppression through a Toll-like receptor-dependent pathway. However, anti-IL-6 mAb did not block CpG activation of the anergized cells. In contrast, anti-TGF-beta(1) Ab released the unresponsive T cells from the anergic state in the presence of MBP68-86, whereas TGF-beta(1) inhibited proliferation of MBP68-86- plus CpG-activated T cells. Because TGF-beta(1) has previously been implicated in Treg activity, this finding is consistent with a role for Treg cells in maintaining autoreactive T cells in the anergic state.