Shared Antigen-specific CD8⁺ T cell Responses Against the SARS-COV-2 Spike Protein in HLA A*02:01 COVID-19 Participants

We report here on antigens from the SARS-CoV-2 virus spike protein, that when presented by Class I MHC, can lead to cytotoxic CD8⁺ T cell anti-viral responses in COVID-19 patients. We present a method in which the SARS-CoV-2 spike protein is converted into a library of peptide antigen-Major Histocompatibility Complexes (pMHCs) as single chain trimers that contain the peptide antigen, the MHC HLA allele, and the β-2 microglobulin sub-unit. That library is used to detect the evolution of virus-specific T cell populations from two COVID-19 patients, at two time points over the course of infection. Both patients exhibit similar virus-specific T cell populations, but very different time-trajectories of those populations. These results can be used to track those virus-specific T cell populations over the course of an infection, thus providing deep insight into the variations in immune system trajectories observed in different COVID-19 patients

Effects of HLA single chain trimer design on peptide presentation and stability

MHC class I “single-chain trimer” molecules, coupling MHC heavy chain, β2-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation

Sensitive Detection and Analysis of Neoantigen-Specific T Cell Populations from Tumors and Blood

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines

Passivation of Quantum Dots and Nanoparticles with Short Lipoic Acid Derivatives to Afford Biocompatibility

The use of quantum dots (QDs) and medium-to-large nanoparticles (NPs) (20-100 nm diameter) in biosensing applications is hindered by issues concerning stability and biocompatibility. In this thesis I address this concern by developing a new procedure with which to assess how coating conditions for these particles are able to confer colloidal stability in various pH ranges and different NaCl concentrations, and resistance to nonspecific protein adsorption. The particles to be tested include CdSe QDs, Ag NPs (20, 40, and 60 nm in diameter), and Au NPs (20, 40, 60, 80, and 100 nm in diameter). Coating conditions consist of combinations of three lipoic acid derivatives, one of which contains a polyethylene glycol end group and the other two containing complementary zwitterionic groups. I found that the ability of the coating layer to confer the desired properties is strongly dependent not only on the composition of the particle, but also on its size. Aggregation was observed in larger NPs and was more prone to occur in the case of Au NPs. I also discovered that, for the ligands tested in this thesis, coatings consisting of a mixture of ligands is required for biocompatibility optimization, as the use of just one ligand for any case cannot bestow both colloidal stability and resistance to protein adsorption. The colloidal stability of the NPs is further convoluted by dependencies upon the type of salt utilized, the concentration of the salt, and the time allowed for aggregation. Thus, this thesis provides the procedural foundation for future tests to optimize these variables for QDs/NPs

Molecular Technologies for Antigen-Based Immunity

The presence and proliferation antigen-specific T cells is a defining characteristic of an adaptive immune response against various disease types (autoimmune, cancer, and infectious). The use of Class I and Class II peptide-major histocompatibility complex (pMHC) reagents to identify such cells, however, is technically difficult and expensive, and it has been challenging to refine synthesis protocols for higher yield and more efficient assembly to accommodate large-scale applications. This achievement would enable high-throughput capture of corresponding T cell receptors (TCR), which may be further used in clinical applications such as adoptive cell transfer therapies. Overcoming this hurdle requires the development and integration of various molecular technologies and analytical methods. Toward this end, the bulk of my thesis work, covered in Chapter 2, introduces these developments in the context of pMHCs, where the three subunits of each reagent are covalent linked together and expressed as a single protein. These single-chain trimer (SCT) technologies primarily consist of traditional DNA cloning and protein production techniques which have been streamlined for applications requiring output on the scale of 102-103 of reagents. This chapter serves as the foundation for much of the methodology discussed throughout the rest of my thesis, and thus should serve as a reference point. The generated constructs are also functionally validated here, and potential future research directions are outlined. In Chapter 3, I explore the use of this technology in the context of COVID-19 to enumerate antigen specificity of the CD8+ T cell immune response. Class I SCTs were constructed to present peptides across several SARS-CoV-2 protein domains, using various HLA alleles to match haplotyped participant blood samples. These reagents were then used to capture SARS-CoV-2-specific T cells through flow and nanoparticle cytometry to demonstrate HLA-dependent, domain-dependent immune responses. Identified TCRs were cloned into T cells for confirmation of antigen specificity and functional cytotoxicity. In Chapters 4 and 5, I explore potential pMHC applications in cancer antigen contexts, covering both tumor-associated and tumor-specific antigens. Through various collaborations across the west coast (UCLA, Parker Institute, Fred Hutchinson Cancer Research Center), I make use of the SCT platform to showcase new assays to discover and rank key tumor targets (Chapter 4). Finally, Chapter 5 is a reproduction of our lab’s published work concerning identification of antigen-specific CD8+ T cells from melanoma cancer patients. In summary, the adaptation of SCTs in a high-throughput format allows for the rapid enumeration of antigen-specific T-cell receptor sequences. As demonstrated in the contexts of COVID-19 and cancer, this SCT platform enables subsequent downstream applications, such as single-cell, antigen-specific immunophenotypic mapping/analysis and target discovery for personalized immunotherapies.</p

Entropic analysis of antigen-specific CDR3 domains identifies essential binding motifs shared by CDR3s with different antigen specificities.

Antigen-specific T cell receptor (TCR) sequences can have prognostic, predictive, and therapeutic value, but decoding the specificity of TCR recognition remains challenging. Unlike DNA strands that base pair, TCRs bind to their targets with different orientations and different lengths, which complicates comparisons. We present scanning parametrized by normalized TCR length (SPAN-TCR) to analyze antigen-specific TCR CDR3 sequences and identify patterns driving TCR-pMHC specificity. Using entropic analysis, SPAN-TCR identifies 2-mer motifs that decrease the diversity (entropy) of CDR3s. These motifs are the most common patterns that can predict CDR3 composition, and we identify essential motifs that decrease entropy in the same CDR3 α or β chain containing the 2-mer, and super-essential motifs that decrease entropy in both chains. Molecular dynamics analysis further suggests that these motifs may play important roles in binding. We then employ SPAN-TCR to resolve similarities in TCR repertoires against different antigens using public databases of TCR sequences

Effects of HLA single chain trimer design on peptide presentation and stability.

MHC class