Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity.

T cell receptor (TCR)-T cell immunotherapy, in which T cells are engineered to express a TCR specific for a tumor epitope, is a form of adoptive cell therapy (ACT) that has demonstrated promise against various tumor types. Mutants of oncoprotein KRAS, particularly at glycine-12 (G12), are frequent drivers of tumorigenicity, but also attractive targets for TCR-T cell therapy. However, MHC class I-restricted TCRs specifically targeting G12-mutant KRAS epitopes in the context of tumors expressing HLA-A2, the most common human HLA-A allele, have remained elusive despite evidence that an epitope encompassing the mutation can bind HLA-A2 and induce T cell responses. We report that post-translational modifications of the protein on this epitope may allow tumor cells to evade immunologic pressure from TCR-T cells. A lysine side chain-methylated KRAS G12V peptide, rather than unmodified epitope, may be presented in HLA-A2 by tumor cells and impact recognition by TCRs. Using a novel computationally guided approach to design TCRs, we developed by mutagenesis TCRs that recognize this methylated peptide, enhancing tumor recognition and destruction. Additionally, we identified TCRs with similar functional activity in normal repertoires from rare primary T cells by stimulation with modified peptide, clonal expansion, and selection. Mechanistically, a gene knockout screen to identify mechanism(s) by which tumor cells methylate or demethylate this epitope unveiled SPT6 as a demethylating protein that could be targeted to improve effectiveness of these TCRs. These findings highlight the role of post-translational modifications in immune evasion and suggest that identifying and targeting such modifications should facilitate development of more effective TCR-T cell therapies.

Internal Standard Triggered-Parallel Reaction Monitoring Mass Spectrometry Enables Multiplexed Quantification of Candidate Biomarkers in Plasma.

Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Previously, mass spectrometry-based approaches have been used to fill this gap but feature poor quantitative performance and were generally limited to hundreds of proteins. Here, we demonstrate the capability of an internal standard triggered-parallel reaction monitoring (IS-PRM) assay to greatly expand the numbers of candidates that can be tested with improved quantitative performance. The assay couples immunodepletion and fractionation with IS-PRM and was developed and implemented in human plasma to quantify 5176 peptides representing 1314 breast cancer biomarker candidates. Characterization of the IS-PRM assay demonstrated the precision (median % CV of 7.7%), linearity (median R2 > 0.999 over 4 orders of magnitude), and sensitivity (median LLOQ < 1 fmol, approximately) to enable rank-ordering of candidate biomarkers for validation studies. Using three plasma pools from breast cancer patients and three control pools, 893 proteins were quantified, of which 162 candidate biomarkers were verified in at least one of the cancer pools and 22 were verified in all three cancer pools. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.

ARTEMIS: A Novel Mass-Spec Platform for HLA-Restricted Self and Disease-Associated Peptide Discovery.

Conventional immunoprecipitation/mass spectroscopy identification of HLA-restricted peptides remains the purview of specializing laboratories, due to the complexity of the methodology, and requires computational post-analysis to assign peptides to individual alleles when using pan-HLA antibodies. We have addressed these limitations with ARTEMIS: a simple, robust, and flexible platform for peptide discovery across ligandomes, optionally including specific proteins-of-interest, that combines novel, secreted HLA-I discovery reagents spanning multiple alleles, optimized lentiviral transduction, and streamlined affinity-tag purification to improve upon conventional methods. This platform fills a middle ground between existing techniques: sensitive and adaptable, but easy and affordable enough to be widely employed by general laboratories. We used ARTEMIS to catalog allele-specific ligandomes from HEK293 cells for seven classical HLA alleles and compared results across replicates, against computational predictions, and against high-quality conventional datasets. We also applied ARTEMIS to identify potentially useful, novel HLA-restricted peptide targets from oncovirus oncoproteins and tumor-associated antigens.

The Helicobacter pylori cell shape promoting protein Csd5 interacts with the cell wall, MurF, and the bacterial cytoskeleton.

Chronic infection with Helicobacter pylori can lead to the development of gastric ulcers and stomach cancers. The helical cell shape of H. pylori promotes stomach colonization. Screens for loss of helical shape have identified several periplasmic peptidoglycan (PG) hydrolases and non-enzymatic putative scaffolding proteins, including Csd5. Both over and under expression of the PG hydrolases perturb helical shape, but the mechanism used to coordinate and localize their enzymatic activities is not known. Using immunoprecipitation and mass spectrometry we identified Csd5 interactions with cytosolic proteins CcmA, a bactofilin required for helical shape, and MurF, a PG precursor synthase, as well as the inner membrane spanning ATP synthase. A combination of Csd5 domain deletions, point mutations, and transmembrane domain chimeras revealed that the N-terminal transmembrane domain promotes MurF, CcmA, and ATP synthase interactions, while the C-terminal SH3 domain mediates PG binding. We conclude that Csd5 promotes helical shape as part of a membrane associated, multi-protein shape complex that includes interactions with the periplasmic cell wall, a PG precursor synthesis enzyme, the bacterial cytoskeleton, and ATP synthase.

EF-P Posttranslational Modification Has Variable Impact on Polyproline Translation in Bacillus subtilis.

Elongation factor P (EF-P) is a ubiquitous translation factor that facilitates translation of polyproline motifs. In order to perform this function, EF-P generally requires posttranslational modification (PTM) on a conserved residue. Although the position of the modification is highly conserved, the structure can vary widely between organisms. In Bacillus subtilis, EF-P is modified at Lys32 with a 5-aminopentanol moiety. Here, we use a forward genetic screen to identify genes involved in 5-aminopentanolylation. Tandem mass spectrometry analysis of the PTM mutant strains indicated that ynbB, gsaB, and ymfI are required for modification and that yaaO, yfkA, and ywlG influence the level of modification. Structural analyses also showed that EF-P can retain unique intermediate modifications, suggesting that 5-aminopentanol is likely directly assembled on EF-P through a novel modification pathway. Phenotypic characterization of these PTM mutants showed that each mutant does not strictly phenocopy the efp mutant, as has previously been observed in other organisms. Rather, each mutant displays phenotypic characteristics consistent with those of either the efp mutant or wild-type B. subtilis depending on the growth condition. In vivo polyproline reporter data indicate that the observed phenotypic differences result from variation in both the severity of polyproline translation defects and altered EF-P context dependence in each mutant. Together, these findings establish a new EF-P PTM pathway and also highlight a unique relationship between EF-P modification and polyproline context dependence.IMPORTANCE Despite the high level of conservation of EF-P, the posttranslational modification pathway that activates EF-P is highly divergent between species. Here, we have identified and characterized in B. subtilis a novel posttranslational modification pathway. This pathway not only broadens the scope of potential EF-P modification strategies, but it also indicates that EF-P modifications can be assembled directly on EF-P. Furthermore, characterization of these PTM mutants has established that an altered modification state can impact both the severity of polyproline translational defects and context dependence.

UV light inactivation of human and plant pathogens in unfiltered surface irrigation water.

Fruit and vegetable growers continually battle plant diseases and food safety concerns. Surface water is commonly used in the production of fruits and vegetables and can harbor both human- and plant-pathogenic microorganisms that can contaminate crops when used for irrigation or other agricultural purposes. Treatment methods for surface water are currently limited, and there is a need for suitable treatment options. A liquid-processing unit that uses UV light for the decontamination of turbid juices was analyzed for its efficacy in the treatment of surface waters contaminated with bacterial or oomycete pathogens, i.e., Escherichia coli, Salmonella enterica, Listeria monocytogenes, Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, and Phytophthora capsici. Five-strain cocktails of each pathogen, containing approximately 10(8) or 10(9) CFU/liter for bacteria or 10(4) or 10(5) zoospores/liter for Ph. capsici, were inoculated into aliquots of two turbid surface water irrigation sources and processed with the UV unit. Pathogens were enumerated before and after treatment. In general, as the turbidity of the water source increased, the effectiveness of the UV treatment decreased, but in all cases, 99.9% or higher inactivation was achieved. Log reductions ranged from 10.0 to 6.1 and from 5.0 to 4.2 for bacterial pathogens and Ph. capsici, respectively.

A targeted proteomics-based pipeline for verification of biomarkers in plasma.

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.

Repeatability of autorefraction and axial length measurements after laser in situ keratomileusis.

PURPOSE: To assess the repeatability and agreement of refractive error measurements and the repeatability of axial length (AL) measurements in patients after laser in situ keratomileusis (LASIK). SETTING: The Ohio State University College of Optometry, Columbus, Ohio, USA. METHODS: Subjective refraction, autorefraction measurements with the Grand Seiko and Humphrey autorefractors, and AL measurements with the IOLMaster were completed for 40 previously myopic LASIK patients under noncycloplegic and cycloplegic conditions on 2 separate occasions. RESULTS: The mean difference between visits for axial length measurements was 0.008 mm +/- 0.04 (SD). The between visits repeatability for all refractive error measurements were <0.75 diopter (D). The mean difference between the subjective refraction and the Humphrey autorefractor for spherical equivalent was statistically significant under noncycloplegic conditions (-0.90 D, P<.0001) and cycloplegic conditions (-2.05 D, P<.0001). The mean difference between subjective refraction and Grand Seiko autorefraction measurements was not significant under noncycloplegic conditions (+0.05 D, 95% limits of agreement [LoA]=-0.99, 1.09; P=.52) conditions but was statistically significant, but not clinically relevant, under cycloplegic conditions (+0.17 D, 95% LoA=-0.73, 1.07; P=.03). CONCLUSIONS: Refractive error measurements after LASIK using the Grand Seiko autorefractor are reliable and agree well with subjective refraction measurements.

A survey of clinical prescribing philosophies for hyperopia.

BACKGROUND: Prescribing philosophies for hyperopic refractive error in symptom-free children vary widely because relatively little information is available regarding the natural history of hyperopic refractive error in children and because accommodation and binocular function closely related to hyperopic refractive error vary widely among children. We surveyed pediatric optometrists and ophthalmologists to evaluate typical prescribing philosophies for hyperopia. METHODS: Practitioners were selected from the American Academy of Optometry Binocular Vision, Perception, and Pediatric Optometry Section; the College of Vision Development; the pediatric and binocular vision faculty members of the colleges of optometry; and the American Association for Pediatric Ophthalmology and Strabismus. Surveys were mailed to 314 participants: 212 optometrists and 102 ophthalmologists. RESULTS: A total of 161 (75%) of the optometrists and 59 (57%) of the ophthalmologists responded. About one-third of optometrists surveyed prescribe optical correction for symptom-free 6-month-old infants with +3.00 D to +4.00 D hyperopia, but fewer than 5% of ophthalmologists prescribe at this level. Most eye care practitioners prescribe optical correction for symptom-free 2-year-old children with +5.00 D of hyperopia, and this criterion for hyperopia decreases with age. Most ophthalmologists (71.4%) prescribe the full amount of astigmatism and less than the full amount of cycloplegic spherical component, and most optometrists (71.6%) prescribe less than the full amount of both components. When prescribing less than the full amount of astigmatism, eye care practitioners do not tend to prescribe a specific proportion of the cycloplegic refractive error. CONCLUSION: Pediatric eye care providers show a lack of consensus on prescribing philosophies for hyperopic children.