Identification of a CD4+ T cell-stimulating antigen of pathogenic bacteria by expression cloning.

Identifying the immunogenic proteins that elicit pathogen-specific T cell responses is key to rational vaccine design. While several approaches have succeeded in identifying major histocompatibility complex (MHC) class I bound peptides that stimulate CD8+ T cells, these approaches have been difficult to extend to peptides presented by MHC class II molecules that stimulate CD4+ T cells. We describe here a novel strategy for identifying CD4+ T cell-stimulating antigen genes. Using Listeria monocytogenes-specific, lacZ-inducible T cells as single-cell probes, we screened a Listeria monocytogenes genomic library as recombinant Escherichia coli that were fed to macrophages. The antigen gene was isolated from the E. coli clone that, when ingested by the macrophages, allowed generation of the appropriate peptide/MHC class II complex and T cell activation. We show that the antigenic peptide is derived from a previously unknown listeria gene product with characteristics of a membrane-bound protein

Cotransfer of the Eαd and Aβd genes into L cells results in the surface expression of a functional mixed-isotype Ia molecule

Ia molecules play a key role in antigen recognition by T lymphocytes. To analyze the structural features of the individual α and β chains relevant to the assembly of intact Ia molecules, mouse fibroblasts were contransfected with various combinations of haplotype- and isotype-mismatched Ia α/β gene pairs. Two important points emerged. First, the level of surface expression of a given haplotype-mismatched AαAβ pair appears to depend upon the α and β chain alleles involved. Second, transfection with some isotype-mismatched combinations such as EαdAβd results in a significant level of surface expression of a stable mixed-isotype dimer, which also appears to be normally expressed at a low level by an Iad-positive B lymphoma. Moreover, a T-cell hybridoma specific for human gamma globulin and restricted by the Ed molecule was found to be efficiently stimulated by the EαdAβd-positive transfectant in the presence of antigen. The stimulation was specifically inhibited by monoclonal antibodies directed to either the Ia or the L3T4 molecule. These findings suggest that the estimates of the potential number of Ia molecules available in an animal for restricting T-lymphocyte recognition of antigens must be revised

Second Class Minors: Molecular Identification of the Autosomal H46 Histocompatibility Locus as a Peptide Presented by Major Histocompatibility Complex Class II Molecules

CD4 T cells regulate immune responses that cause chronic graft rejection and graft versus host disease but their target antigens remain virtually unknown. We developed a new method to identify CD4 T cell–stimulating antigens. LacZ-inducible CD4 T cells were used as a probe to detect their cognate peptide/MHC II ligand generated in dendritic cells fed with Escherichia coli expressing a library of target cell genes. The murine H46 locus on chromosome 7 was thus found to encode the interleukin 4–induced IL4i1 gene. The IL4i1 precursor contains the HAFVEAIPELQGHV peptide which is presented by Ab major histocompatibility complex class II molecule via an endogenous pathway in professional antigen presenting cells. Both allelic peptides bind Ab and a single alanine to methionine substitution at p2 defines nonself. These results reveal novel features of H loci that regulate CD4 T cell responses as well as provide a general strategy for identifying elusive antigens that elicit CD4 T cell responses to tumors or self-tissues in autoimmunity

MHC Class I Molecules Can Direct Proteolytic Cleavage of Antigenic Precursors in the Endoplasmic Reticulum

AbstractThe large set of peptides presented by MHC (major histocompatibility complex) class I molecules are generated by proteolysis of diverse precursors in the cytoplasm and possibly in the endoplasmic reticulum (ER). To define the potential peptide trimming events in the ER, we analyzed proteolytic products generated in isolated microsomes. The residues flanking the N terminus of the final antigenic peptide were rapidly removed within the microsomes but only in the presence of appropriate MHC molecules. Remarkably, the precursor peptide was bound to the MHC molecules in a distinct conformation and required an aminopeptidase activity to generate the optimal peptide. The MHC molecules are therefore not only the final repositories of antigenic peptides, but they can also direct their excision from longer precursors

Transnuclear CD8 T cells specific for the immunodominant epitope Gra6 lower acute-phase Toxoplasma gondii burden.

We generated a CD8 T-cell receptor (TCR) transnuclear (TN) mouse specific to the Ld -restricted immunodominant epitope of GRA6 from Toxoplasma gondii as a source of cells to facilitate further investigation into the CD8 T-cell-mediated response against this pathogen. The TN T cells bound Ld -Gra6 tetramer and proliferated upon unspecific and peptide-specific stimulation. The TCR beta sequence of the Gra6-specific TN CD8 T cells is identical in its V- and J-region to the TCR-β harboured by a hybridoma line generated in response to Gra6 peptide. Adoptively transferred Gra6 TN CD8 T cells proliferated upon Toxoplasma infection in vivo and exhibited an activated phenotype similar to host CD8 T cells specific to Gra6. The brain of Toxoplasma-infected mice carried Gra6 TN cells already at day 8 post-infection. Both Gra6 TN mice as well as adoptively transferred Gra6 TN cells were able to significantly reduce the parasite burden in the acute phase of Toxoplasma infection. Overall, the Gra6 TN mouse represents a functional tool to study the protective and immunodominant specific CD8 T-cell response to Toxoplasma in both the acute and the chronic phases of infection

The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion

Background: Cytotoxic T cells detect intracellular pathogens by surveying peptide loaded MHC class I molecules (pMHC I) on the cell surface. Effective immune surveillance also requires infected cells to present pMHC I promptly before viral progeny can escape. Rapid pMHC I presentation apparently occurs because infected cells can synthesize and present peptides from antigenic precursors called defective ribosomal products (DRiPs). The molecular characteristics of DRiPs are not known. Methodology/Principal Findings: Here, using a novel method for detecting antigenic precursors and proteolytic intermediates, we tracked the synthesis and processing of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1). We find that ribosomes initiated translation appropriately, but rapidly produced DRiPs representing,120 amino acid truncated EBNA1 polypeptides by premature termination. Moreover, specific sequences in EBNA1 mRNA strongly inhibited the generation of truncated DRiPs and pMHC I presentation. Significance: Our results reveal the first characterization of virus DRiPs as truncated translation products. Furthermore

Unanticipated Antigens: Translation Initiation at CUG with Leucine

Major histocompatibility class I molecules display tens of thousands of peptides on the cell surface for immune surveillance by T cells. The peptide repertoire represents virtually all cellular translation products, and can thus reveal a foreign presence inside the cell. These peptides are derived from not only conventional but also cryptic translational reading frames, including some without conventional AUG codons. To define the mechanism that generates these cryptic peptides, we used T cells as probes to analyze the peptides generated in transfected cells. We found that when CUG acts as an alternate initiation codon, it can be decoded as leucine rather than the expected methionine residue. The leucine start does not depend on an internal ribosome entry site–like mRNA structure, and its efficiency is enhanced by the Kozak nucleotide context. Furthermore, ribosomes scan 5′ to 3′ specifically for the CUG initiation codon in a eukaryotic translation initiation factor 2–independent manner. Because eukaryotic translation initiation factor 2 is frequently targeted to inhibit protein synthesis, this novel translation mechanism allows stressed cells to display antigenic peptides. This initiation mechanism could also be used at non-AUG initiation codons often found in viral transcripts as well as in a growing list of cellular genes

Fam49b dampens TCR signal strength to regulate survival of positively selected thymocytes and peripheral T cells.

The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ+ and CD8αα+TCRαβ+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival

The ER Aminopeptidases, ERAP1 and ERAP2, synergize to self-modulate their respective activities

IntroductionCritical steps in Major Histocompatibility Complex Class I (MHC-I) antigen presentation occur in the endoplasmic reticulum (ER). In general, peptides that enter the ER are longer than the optimal length for MHC-I binding. The final trimming of MHC-I epitopes is performed by two related aminopeptidases, ERAP1 and ERAP2 in humans that possess unique and complementary substrate trimming specificities. While ERAP1 efficiently trims peptides longer than 9 residues, ERAP2 preferentially trims peptides shorter than 9 residues.Materials and MethodsUsing a combination of biochemical and proteomic studies followed by biological verification.ResultsWe demonstrate that the optimal ligands for either enzyme act as inhibitors of the other enzyme. Specifically, the presence of octamers reduced the trimming of long peptides by ERAP1, while peptides longer than nonomers inhibit ERAP2 activity.DiscussionWe propose a mechanism for how ERAP1 and ERAP2 synergize to modulate their respective activities and shape the MHC-I peptidome by generating optimal peptides for presentation