Endogenous antigen processing drives the primary CD4+ T cell response to influenza.

By convention, CD4+ T lymphocytes recognize foreign and self peptides derived from internalized antigens in combination with major histocompatibility complex class II molecules. Alternative pathways of epitope production have been identified, but their contributions to host defense have not been established. We show here in a mouse infection model that the CD4+ T cell response to influenza, critical for durable protection from the virus, is driven principally by unconventional processing of antigen synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from infected cells. Investigation of the cellular components involved, including the H2-M molecular chaperone, the proteasome and γ-interferon-inducible lysosomal thiol reductase revealed considerable heterogeneity in the generation of individual epitopes, an arrangement that ensures peptide diversity and broad CD4+ T cell engagement. These results could fundamentally revise strategies for rational vaccine design and may lead to key insights into the induction of autoimmune and anti-tumor responses

Gauging the contribution of endogenous MHCII pathways to influenza-specific CD4+ T cell responses (106.32)

Abstract The current paradigm for MHC Class II peptide generation holds that exogenous antigen enters the endosomal/lysosomal pathway, where it is processed and presented on the cell surface for presentation to CD4+ T cells. Increasing evidence from our lab and others has elucidated pathways that process endogenous proteins for presentation on MHC Class II and operate exclusively during live infection. Despite this, the components of these pathways, the prevalence of such epitopes, and their general contribution to the immune response remain poorly characterized. Using an IFN-γ-ELISpot assay, we screened a peptide library of the flu proteome to identify potential endogenous CD4+ epitopes generated following live or UV-inactivated virus priming. The differences in specificities were striking, with “live only” hits compromising a major fraction of the response and mapping to both structural and non-structural proteins. Experiments are ongoing to identify those epitopes that can be strictly categorized as endogenous, and to identify cellular components (proteasome, TAP, H-2M,…) that contribute to their generation. A comprehensive understanding of endogenous MHCII epitopes and their corresponding presentation pathways could contribute substantially to improved vaccine design and efficacy.</jats:p

The Adaptor Protein SLP-76 Is Not a Direct Substrate for BCR-ABL in the Chronic Myeloid Leukemia Cell Line K562.

Abstract SLP-76(SH2 domain-containing leukocyte protein of 76 kD) is a hematopoietic adapter protein that is expressed in myeloid and T cells. SLP-76 is a substrate for tyrosine kinase in the src and syk family activation pathway required for T-cell receptor-mediated signaling. Cross-linking of the human FcgammaRIIa1 (CD32) in myeloid cells which contains an immune receptor tyrosine-based activation motif (ITAM) causes phosphorylation of SLP-76. Mice deficient in SLP76 develop fetal hemorrhage along with failure of T cell development and perinatal mortality. We have found that K562 cells express SLP-76. We hypothesized that SLP-76 or associated proteins may be substrates of Bcr-Abl in the K562 cell line and thus promote survival signals. Materials and Methods: The K-562 cell line was obtained from the American collection of Cell Cultures. SLP deficient (−/−) KO mice were obtained from Dr. James Clements (Roswell Park Cancer Institute). Antibodies used include Sheep polyclonal IgG Anti-Human SLP 76, Peroxidase-conjugated Affinipure Donkey Anti-Sheep IgG at 0.8mg/ml, Mouse monoclonal IgG Anti-Phosphotyrosine, and Polyclonal goat Anti-mouse. Immunoprecipitation and Immunoblot: Cells were either untreated or stimulated with purvanidate (phosphatase inhibitors). Purvanidate treated cells were used as a positive control. Spleenocytes isolated from a SLP-76 deficient mouse (KO) were used as negative control. Lysates were then subject to standard immunoprecipitation for SLP-76 followed by immunoblotting with a SLP-76 specific antibody. The denatured samples were then resolved by SDS-PAGE. Results: SLP-76 is expressed in untreated and treated K562 with purvanidate and not detectable from lysates derived from SLP-76 KO spleenocytes. To assess the phosphorylation status of SLP-76 and any co-associated proteins in K562 cells, the SLP-76 blot was stripped and then immunoblotted for total phosphotyrosine content. SLP-76 does not appear to be constitutively phosphorylated in the K562 cells. However, significant tyrosine phosphorylation of SLP-76 was readily detectable in the purvanidate treated K562 cells. Conclusion: The current studies reveal that although SLP-76 is indeed found in the K562 cell line expressing the Bcr-Abl oncogene. Somewhat surprisingly, despite the constitutive activation of the Bcr-Abl tyrosine kinase in K562 cells, we detected no obvious phosphoproteins co-precipitating with SLP-76 in the absence of purvanidate stimulation. Together, the lack of SLP-76 tyrosine phosphorylation and the lack of co-associated proteins in K562 cells suggest that SLP-76 is not a major player in the signal transduction pathways emanating from Bcr-Abl. However, a more stringent confirmation of this conclusion would require inhibiting SLP-76 expression in K562 cells and than assessing the growth and survival characteristics. Figure Figure</jats:p

Rho-family GTPase Cdc42 controls migration of Langerhans cells in vivo

Abstract Epidermal Langerhans cells (LCs) of the skin represent the prototype migratory dendritic cell (DC) subtype. In the skin, they take up Ag, migrate to the draining lymph nodes, and contribute to Ag transport and immunity. Different depletion models for LCs have revealed contrasting roles and contributions of this cell type. To target the migratory properties of DCs, we generated mice lacking the Rho-family GTPase Cdc42 specifically in DCs. In these animals, the initial seeding of the epidermis with LCs is functional, resulting in slightly reduced Langerhans cell numbers. However, Cdc42-deficient LCs fail to leave the skin in steady state as well as upon stimulation, as they do not enter the skin-draining afferent lymph vessels. Similarly, also other Cdc42-deficient migratory DC subsets fail to home properly to the corresponding draining lymph nodes. We used this novel mouse model, in which LCs are locked out, to demonstrate that these cells contribute substantially to priming of Ag-specific CD4 and CD8 T cell responses upon epicutaneous immunization, but could not detect a role in the induction of contact hypersensitivity to various doses of hapten.</jats:p