MHC Class I Antigen Presentation is Regulated by the SUMO-Conjugating Enzyme UBC9: a Dissertation

CD8 T cells recognize complexes of MHC class I and peptide on the surface of target cells. MHC class I antigen presentation is a long pathway, in which proteins are degraded by proteasomes to generating oligopeptides, which may be further trimmed by aminopeptidases in the cytosol. Peptides are transported into the ER, where they may be further trimmed by ER lumenal aminopeptidases and bind to newly-synthesized MHC class I complexes. Proteins degraded by the proteasome are generally tagged with ubiquitin by a combination of ubiquitin-conjugating enzymes and ubiquitin ligases. UBC9 is one ubiquitin conjugating enzyme, which does not conjugate ubiquitin, but instead conjugates small ubiquitin-like molecules (SUMO) to target protein. UBC9 has been found to regulate the functions of many proteins in vivo, most importantly by modifying nuclear transportation and function. Curing [During] my thesis work, I studied the function of UBC9 in MHC class I antigen presentation. UBC9 over-expression in COS cells co-expressing ovalbumin markedly increased presentation SIINFEKL (the immunodominant epitope from ovalbumin in the context of H-2Kb), and UBC9 overexpression increased cell surface H-2Kbin general, suggesting that Ubc9 increased MHC class I antigen presentation by increasing peptide supply. UBC9 did not increase synthesis or degradation of ovalbumin. In transient transfection experiments, Ubc9 increased presentation of SIINFEKL precursors that did, and that did not, depend on proteasomes for processing, as well as SIINFEKL precursors targeted to the ER, bypassing cytosolic processing altogether. However, a C-terminal extended precursor of SIINFEKL, which requires only proteasomal processing before presentation, was the most markedly affected by UBC9 overexpression. This suggested that UBC9 was affecting the pattern of cleavages made by proteasomes in ways that enhance the generation of the C-terminus of SIINFEKL. Because presentation of SIINFEKL itself (which requires no further proteolytic processing) was also enhanced, UBC9 must also affect steps in the class I pathway that occur after the generation of the mature epitopes. UBC9 did not affect the rate of peptide degradation in cytosolic extracts or in intact cells. These findings suggested that UBC9 might have multiple effects on the MHC class I antigen presentation pathway. Immunofluorescent microscopy demonstrated that UBC9 increased the expression of the beta subunits of immunoproteasomes (LMP2, LMP7, and MECL1) as well as of TAP1 and tapasin. In contrast, UBC9 expression did not increase levels of calnexin, calreticulin, ERp57, or Protein disulfide isomerase (PDI). Similarly, levels of leucine aminopeptidase were not increased in UBC9-transfected cells. Therefore, UBC9 overexpression increases the levels of some but not all components of the class I pathway. UBC9 overexpression increased protein levels of MECL1, LMP2 or LMP7 that were under the control of viral promoters, and levels of MECL1 mRNA were similar in control vector and UBC9 transfected cells. Therefore, UBC9 did not increase the level of expression of these subunits through increased transcription. Pulse-chase experiments showed that UBC9 overexpression reduced the degradation of MECL1. Therefore, UBC9 increases the levels of at least some of these components of the MHC class I antigen presentation pathway by increasing their stability. To know the biological significance of UBC9 in MHC class I antigen presentation, I used small interfering RNA (siRNA) to knock down UBC9. Though UBC9 can be successfully knocked down by siRNA, the UBC9-negative cells became very sick, and were not suitable for the study of MHC class I antigen presentation. There are three forms of SUMO molecules in mammalian cells: SUMO-1, SUMO-2 and SUMO-3. My study suggested that SUMO-2 may be involved in UBC9\u27s regulation of MHC class I antigen presentation, since mutant SUMO-2 blocked UBC9\u27s ability to increase H-2Kb-SIINFEKL levels on the cell surface after the cells were loaded with ovalbumin. To further study the function of UBC9, I mutated the active amino acid Cys 93 of UBC9 to Ser (UBC9OH). Unexpectedly, this mutant form (UBC9OH) has very similar effects as wild-type UBC9, increasing Kb-SIINFEKL levels at the cells surface. This suggested that UBC9 protein regulates MHC class I antigen presentation pathway proteins by direct or indirect protein interaction, rather than (or as well as) by SUMO conjugation. Taking account of SUMO-2 results, I propose that wild-type UBC9 (either transfected or endogenous) conjugates SUMO-2 to its substrates, and then UBC9 (wild-type or mutant) interacts with its sumoylated targets, thus affecting protein functions. I also studied heat shock protein Hsp27, which is known to be a substrate for UBC9 in vivo. Hsp27 is expressed in a variety of tissues in the absence of stress, and may regulate actin dynamics. Hsp27 overexpression decreased generation of H-2Kb-SIINFEKL complexes from SIINFEKL precursors that did, and did not, require proteasomes for processing, or that were targeted to the ER. Hsp27 over-expression did not affect protein synthesis, and globally decreased cell surface H2-Kb and H2-Dblevels, but did not affect HLA-A0302 level. Hsp27 overexpression inhibits the presentation of ER-localized SIINFEKL. Taken together, my data suggested that HSP27 may inhibit MHC class I antigen presentation by affecting MHC class I molecules itself rather than peptide supply. After Hsp27 was eliminated with siRNA, the effects were very similar to those seen with Hsp27 overexpression. Levels of H-2Kb-SIINFEKL decreased, and overall cell surface H-2Kb and H-2Db levels decreased. It is possible that when Hsp27 is over-expressed, it acts as a dominant negative form, conferring a similar phenotype to Hsp27 knockdown. These observations suggest that Hsp27 plays an important role in MHC class I antigen presentation

Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma

Interference with inhibitory immunological checkpoints controlling T cell activation provides new opportunities to augment cancer immunotherapies. Whereas cytotoxic T lymphocyte–associated antigen-4 blockade has shown promising preclinical and clinical results, therapeutic CD4+CD25+ T reg cell depletion has failed to consistently enhance immune-based therapies. Using B16/BL6, a transplantable murine melanoma model, we show a dichotomy between the effects of T reg cell depletion on tumor rejection dependent on whether depletion occurs before (prophylactic) or after (therapeutic) tumor engraftment. Failure to promote rejection with therapeutic depletion is not related to lack of T reg cell depletion, to elimination of CD25+ effector T cells, or to a failure to enhance systemic antitumor T cell responses, but correlates with failure of effector cells to infiltrate the tumor and increase the intratumor ratio of effector T cell/T reg cell. Finally, systemic antitumor responses generated upon therapeutic T reg cell depletion are significantly stronger than those generated in the presence of T reg cells, and are capable of eliciting rejection of established tumors after transfer into immunoablated recipients receiving combination immunotherapy. The data demonstrate a dissociation between measurable systemic responses and tumor rejection during CD25-directed T reg cell depletion, and suggest an alternative, clinically applicable strategy for the treatment of established tumors

Impact of Petrophysical Properties on Hydraulic Fracturing and Development in Tight Volcanic Gas Reservoirs

The volcanic reservoir is an important kind of unconventional reservoir. The aqueous phase trapping (APT) appears because of fracturing fluids filtration. However, APT can be autoremoved for some wells after certain shut-in time. But there is significant distinction for different reservoirs. Experiments were performed to study the petrophysical properties of a volcanic reservoir and the spontaneous imbibition is monitored by nuclear magnetic resonance (NMR) and pulse-decay permeability. Results showed that natural cracks appear in the samples as well as high irreducible water saturation. There is a quick decrease of rock permeability once the rock contacts water. The pores filled during spontaneous imbibition are mainly the nanopores from NMR spectra. Full understanding of the mineralogical effect and sample heterogeneity benefits the selection of segments to fracturing. The fast flow-back scheme is applicable in this reservoir to minimize the damage. Because lots of water imbibed into the nanopores, the main flow channels become larger, which are beneficial to the permeability recovery after flow-back of hydraulic fracturing. This is helpful in understanding the APT autoremoval after certain shut-in time. Also, Keeping the appropriate production differential pressure is very important in achieving the long term efficient development of volcanic gas reservoirs

The cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent of MHC class I antigen presentation

Most antigenic peptides presented on MHC class I molecules are generated by proteasomes during protein breakdown. It is unknown whether these peptides are protected from destruction by cytosolic peptidases. In cytosolic extracts, most antigenic peptides are degraded by the metalloendopeptidase, thimet oligopeptidase (TOP). We therefore examined whether TOP destroys antigenic peptides in vivo. When TOP was overexpressed in cells, class I presentation of antigenic peptides was reduced. In contrast, TOP overexpression didn\u27t reduce presentation of peptides generated in the endoplasmic reticulum or endosomes. Conversely, preventing TOP expression with siRNA enhanced presentation of antigenic peptides. TOP therefore plays an important role in vivo in degrading peptides released by proteasomes and is a significant factor limiting the extent of antigen presentation

In Situ Passivation of PbBr₆^4– Octahedra toward Blue Luminescent CsPbBr₃ Nanoplatelets with Near 100% Absolute Quantum Yield

Recently, the pursuit of high photoluminescence quantum yields (PLQYs) for blue emission in perovskite nanocrystals (NCs) has attracted increased attention because the QY of blue NCs lags behind those of green and red ones severely, which is fatal for three-primary-color displays. Here, we propose an in situ PbBr₆^4– octahedra passivation strategy to achieve a 96% absolute QY for the ultrapure (line width = 12 nm) blue emission from CsPbBr₃ nanoplatelets (NPLs), and both values rank first among perovskite NCs with blue emission. From the aspect of constructing intact PbBr₆^4– octahedra, additional Br^– was introduced to drive the ionic equilibrium to form intact Pb–Br octahedra. The reduced Br vacancy and inhibited nonradiative recombination processes are well proved by reduced Urbach energy, increased Pb–Br bonds, and slower transient absorption delay. Blue light-emitting diodes (LEDs) using NPLs were fabricated, and a high external quantum efficiency (EQE) of 0.124% with an emission line width of ∼12 nm was realized. This work will provide good references to break the “blue-wall” in perovskite NCs