MHC class 1 cytoplasmic domain : defining a role for conserved amino acid residues in class 1 expression, trafficking, and antigen presentation

MHC class I molecules contain within their cytoplasmic domains a tyrosine and two or three serines that demonstrate a remarkable level of conservation between all mammals and extending to species as evolutionarily distant as the shark and zebrafish. In order to explore the role of these amino acids both in vitro and in vivo, cytoplasmic mutants of the murine H-2 Kb gene were constructed and expressed in murine L cell fibroblasts and transgenic mice. In L cell fibroblast transfectants, Kb mutant molecules reached higher cell surface levels than Kb wild-type (KbWT) molecules and demonstrated a higher capacity for binding exogenously-added peptides. While cytoplasmic mutations had no effect on β2- microglobulin binding nor Kb transport rates to the cell surface, they caused a delay in the ability of the molecules to present an immunodominant vesicular stomatitis virus (VSV) epitope following viral infection. In the transgenic mice, FACScan analysis of peripheral blood leukocytes and splenocytes revealed that cytoplasmic mutant Kb molecules reached significantly higher surface levels compared to wild-type Kb and interfered less with endogenous class I maturation and surface expression. Constitutive endocytosis of class I molecules from the surface of activated T cells was also shown to be significantly impaired by cytoplasmic point mutations. Functional studies on antigen presentation in the transgenic mice revealed that mice containing a point mutation to the conserved tyrosine were severely compromised in their ability to generate a Kb-restricted CTL response to VSV infection compared to KbWT-expressing and C57/BL control mice. Mice containing mutations to the conserved serines by contrast, demonstrated a more vigorous VSV CTL response compared to KbWT-expressing transgenic animals. The results demonstrate that the conserved tyrosine and serine residues are important for regulating lymphocyte and fibroblast MHC class I cell surface expression both in vitro and in vivo, and provide evidence of an inducible tyrosine-based endosomal targeting motif in the cytoplasmic tail of class I molecules. The functional studies suggest that the tyrosine motif in particular may be important for the generation of class I-restricted CTL responses in vivo.Science, Faculty ofZoology, Department ofGraduat

Dynamic changes during the treatment of pancreatic cancer

This manuscript follows a single patient with pancreatic adenocarcinoma for a five year period, detailing the clinical record, pathology, the dynamic evolution of molecular and cellular alterations as well as the responses to treatments with chemotherapies, targeted therapies and immunotherapies. DNA and RNA samples from biopsies and blood identified a dynamic set of changes in allelic imbalances and copy number variations in response to therapies. Organoid cultures established from biopsies over time were employed for extensive drug testing to determine if this approach was feasible for treatments. When an unusual drug response was detected, an extensive RNA sequencing analysis was employed to establish novel mechanisms of action of this drug. Organoid cell cultures were employed to identify possible antigens associated with the tumor and the patient’s T-cells were expanded against one of these antigens. Similar and identical T-cell receptor sequences were observed in the initial biopsy and the expanded T-cell population. Immunotherapy treatment failed to shrink the tumor, which had undergone an epithelial to mesenchymal transition prior to therapy. A warm autopsy of the metastatic lung tumor permitted an extensive analysis of tumor heterogeneity over five years of treatment and surgery. This detailed analysis of the clinical descriptions, imaging, pathology, molecular and cellular evolution of the tumors, treatments, and responses to chemotherapy, targeted therapies, and immunotherapies, as well as attempts at the development of personalized medical treatments for a single patient should provide a valuable guide to future directions in cancer treatment