The CD4 Versus CD8 T Cell Fate Decision: A Multiomics-Informed Perspective.

The choice of developing thymocytes to become CD8+ cytotoxic or CD4+ helper T cells has been intensely studied, but many of the underlying mechanisms remain to be elucidated. Recent multiomics approaches have provided much higher resolution analysis of gene expression in developing thymocytes than was previously achievable, thereby offering a fresh perspective on this question. Focusing on our recent studies using CITE-seq (cellular indexing of transcriptomes and epitopes) analyses of mouse thymocytes, we present a detailed timeline of RNA and protein expression changes during CD8 versus CD4 T cell differentiation. We also revisit our current understanding of the links between T cell receptor signaling and expression of the lineage-defining transcription factors ThPOK and RUNX3. Finally, we propose a sequential selection model to explain the tight linkage between MHC-I versus MHC-II recognition and T cell lineage choice. This model incorporates key aspects of previously proposed kinetic signaling, instructive, and stochastic/selection models.

T cell antigen receptor recognition of antigen-presenting molecules.

The Major Histocompatibility Complex (MHC) locus encodes classical MHC class I and MHC class II molecules and nonclassical MHC-I molecules. The architecture of these molecules is ideally suited to capture and present an array of peptide antigens (Ags). In addition, the CD1 family members and MR1 are MHC class I-like molecules that bind lipid-based Ags and vitamin B precursors, respectively. These Ag-bound molecules are subsequently recognized by T cell antigen receptors (TCRs) expressed on the surface of T lymphocytes. Structural and associated functional studies have been highly informative in providing insight into these interactions, which are crucial to immunity, and how they can lead to aberrant T cell reactivity. Investigators have determined over thirty unique TCR-peptide-MHC-I complex structures and twenty unique TCR-peptide-MHC-II complex structures. These investigations have shown a broad consensus in docking geometry and provided insight into MHC restriction. Structural studies on TCR-mediated recognition of lipid and metabolite Ags have been mostly confined to TCRs from innate-like natural killer T cells and mucosal-associated invariant T cells, respectively. These studies revealed clear differences between TCR-lipid-CD1, TCR-metabolite-MR1, and TCR-peptide-MHC recognition. Accordingly, TCRs show remarkable structural and biological versatility in engaging different classes of Ag that are presented by polymorphic and monomorphic Ag-presenting molecules of the immune system.

Early T cell activation: integrating biochemical, structural, and biophysical cues.

T cells carry out the formidable task of identifying small numbers of foreign antigenic peptides rapidly and specifically against a very noisy environmental background of endogenous self-peptides. Early steps in T cell activation have thus fascinated biologists and are among the best-studied models of cell stimulation. This remarkable process, critical in adaptive immune responses, approaches and even seems to exceed the limitations set by the physical laws ruling molecular behavior. Despite the enormous amount of information concerning the nature of molecules involved in the T cell antigen receptor (TCR) signal transduction network, and the description of the nanoscale organization and real-time analysis of T cell responses, the general principles of information gathering and processing remain incompletely understood. Here we review currently accepted key data on TCR function, discuss the limitations of current research strategies, and suggest a novel model of TCR triggering and a few promising ways of going further into the integration of available data.

Using CombiCells, a platform for titration and combinatorial display of cell surface ligands, to study T-cell antigen sensitivity modulation by accessory receptors.

Understanding cellular decisions due to receptor-ligand interactions at cell-cell interfaces has been hampered by the difficulty of independently varying the surface density of multiple different ligands. Here, we express the synthetic binder protein SpyCatcher, designed to form spontaneous covalent bonds with interactors carrying a Spytag, on the cell surface. Using this, we show that addition of different concentrations and combinations of native Spytag-fused ligands allows for the combinatorial display of ligands on cells within minutes. We use this combinatorial display of cell surface ligands-called CombiCells-to assess T cell antigen sensitivity and the impact of T cell co-stimulation and co-inhibition receptors. We find that the T cell receptor (TCR) displayed greater sensitivity to peptides on major-histocompatibility complexes (pMHC) than synthetic chimeric antigen receptor (CARs) and bi-specific T cell engager (BiTEs) display to their target antigen, CD19. While TCR sensitivity was greatly enhanced by CD2/CD58 interactions, CAR sensitivity was primarily but more modestly enhanced by LFA-1/ICAM-1 interactions. Lastly, we show that PD-1/PD-L1 engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor-ligand interactions at cell-cell interfaces.

A Structural Change in Butyrophilin upon Phosphoantigen Binding Underlies Phosphoantigen-Mediated Vγ9Vδ2 T Cell Activation.

Human Vγ9Vδ2 T cells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, triggering extracellular interactions with the Vγ9Vδ2 T cell receptor (TCR). Here, we examined the molecular basis of this "inside-out" triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ T cell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ T cell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ T cell and a target cell during "outside" signaling, as measured by single-cell force microscopy. Our findings provide insight into the "inside-out" triggering of Vγ9Vδ2 T cell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design.

γδ T cell antigen receptor polyspecificity enables T cell responses to a broad range of immune challenges.

γδ T cells are essential for immune defense and modulating physiological processes. While they have the potential to recognize large numbers of antigens through somatic gene rearrangement, the antigens which trigger most γδ T cell response remain unidentified, and the role of antigen recognition in γδ T cell function is contentious. Here, we show that some γδ T cell receptors (TCRs) exhibit polyspecificity, recognizing multiple ligands of diverse molecular nature. These ligands include haptens, metabolites, neurotransmitters, posttranslational modifications, as well as peptides and proteins of microbial and host origin. Polyspecific γδ T cells are enriched among activated cells in naive mice and the responding population in infection. They express diverse TCR sequences, have different functional potentials, and include the innate-like γδ T cells, such as the major IL-17 responders in various pathological/physiological conditions. We demonstrate that encountering their antigenic microbiome metabolite maintains their homeostasis and functional response, indicating that their ability to recognize multiple ligands is essential for their function. Human γδ T cells with similar polyspecificity also respond to various immune challenges. This study demonstrates that polyspecificity is a prevalent feature of γδ T cell antigen recognition, which enables rapid and robust T cell responses to a wide range of challenges, highlighting a unique function of γδ T cells.

Monomeric agonist peptide/MHCII complexes activate T-cells in an autonomous fashion.

Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non-stimulatory endogenous pMHCIIs is postulated to sensitize T-cells for the recognition of single antigens on the surface of dendritic cells and B-cells. When testing this premise with the use of advanced live cell microscopy, we observe pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR engagement of highly abundant endogenous pMHCIIs is low or non-existent and affects neither TCR engagement of rare agonist pMHCII in early and advanced synapses nor agonist-induced TCR-proximal signaling. Our findings highlight the capacity of single freely diffusing agonist pMHCIIs to elicit the full T-cell response in an autonomous and peptide-specific fashion with consequences for adaptive immunity and immunotherapeutic approaches.

A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45.

The Src Family kinase Lck sets a critical threshold for T cell activation because it phosphorylates the TCR complex and the Zap70 kinase. How a T cell controls the abundance of active Lck molecules remains poorly understood. We have identified an unappreciated role for a phosphosite, Y192, within the Lck SH2 domain that profoundly affects the amount of active Lck in cells. Notably, mutation of Y192 blocks critical TCR-proximal signaling events and impairs thymocyte development in retrogenic mice. We determined that these defects are caused by hyperphosphorylation of the inhibitory C-terminal tail of Lck. Our findings reveal that modification of Y192 inhibits the ability of CD45 to associate with Lck in cells and dephosphorylate the C-terminal tail of Lck, which prevents its adoption of an active open conformation. These results suggest a negative feedback loop that responds to signaling events that tune active Lck amounts and TCR sensitivity.

StackTTCA: a stacking ensemble learning-based framework for accurate and high-throughput identification of tumor T cell antigens.

BACKGROUND: The identification of tumor T cell antigens (TTCAs) is crucial for providing insights into their functional mechanisms and utilizing their potential in anticancer vaccines development. In this context, TTCAs are highly promising. Meanwhile, experimental technologies for discovering and characterizing new TTCAs are expensive and time-consuming. Although many machine learning (ML)-based models have been proposed for identifying new TTCAs, there is still a need to develop a robust model that can achieve higher rates of accuracy and precision. RESULTS: In this study, we propose a new stacking ensemble learning-based framework, termed StackTTCA, for accurate and large-scale identification of TTCAs. Firstly, we constructed 156 different baseline models by using 12 different feature encoding schemes and 13 popular ML algorithms. Secondly, these baseline models were trained and employed to create a new probabilistic feature vector. Finally, the optimal probabilistic feature vector was determined based the feature selection strategy and then used for the construction of our stacked model. Comparative benchmarking experiments indicated that StackTTCA clearly outperformed several ML classifiers and the existing methods in terms of the independent test, with an accuracy of 0.932 and Matthew's correlation coefficient of 0.866. CONCLUSIONS: In summary, the proposed stacking ensemble learning-based framework of StackTTCA could help to precisely and rapidly identify true TTCAs for follow-up experimental verification. In addition, we developed an online web server ( http://2pmlab.camt.cmu.ac.th/StackTTCA ) to maximize user convenience for high-throughput screening of novel TTCAs.

T-cell engineered with a fully humanized B-cell maturation antigen-specific T-cell antigen coupler receptor effectively target multiple myeloma.

B-cell maturation antigen (BCMA) is a clinically validated target for multiple myeloma. T-cell engineered with chimeric antigen receptors (CARs) directed against BCMA have demonstrated robust therapeutic activity in clinical trials, but toxicities remain a significant concern for a subset of patients, supporting continued investigation of other engineered T-cell platforms that may offer equal efficacy with an improved toxicity profile. The authors recently described a BCMA-specific, T-cell-centric synthetic antigen receptor, the T-cell antigen coupler (TAC) receptor, that can be used to engineer T-cell with robust anti-myeloma activity. Here the authors describe the creation of a fully humanized BCMA-specific TAC receptor. Single-chain variable fragments (scFvs) were developed from BCMA-specific F(ab)s that were identified in a fully human phage display library. Twenty-four configurations of the F(ab)s were evaluated in a medium-throughput screening using primary T-cell, and a single F(ab), TRAC 3625, emerged as the most robust following in vitro and in vivo evaluation. An optimized BCMA-specific TAC receptor was developed through iterations of the BCMA-TAC design that evaluated a next-generation TAC scaffold sequence, different domains connecting the TAC to the 3625 scFv and different orientations of the TRAC 3625 heavy and light variable regions.

Non-Antigenic Modulation of Antigen Receptor (TCR) Cß-FG Loop Modulates Signalling: Implications of External Factors Influencing T-Cell Responses.

T-cell recognition of antigens is complex, leading to biochemical and cellular events that impart both specific and targeted immune responses. The end result is an array of cytokines that facilitate the direction and intensity of the immune reaction-such as T-cell proliferation, differentiation, macrophage activation, and B-cell isotype switching-all of which may be necessary and appropriate to eliminate the antigen and induce adaptive immunity. Using in silico docking to identify small molecules that putatively bind to the T-cell Cß-FG loop, we have shown in vitro using an antigen presentation assay that T-cell signalling is altered. The idea of modulating T-cell signalling independently of antigens by directly targeting the FG loop is novel and warrants further study.

Inherent reactivity of unselected TCR repertoires to peptide-MHC molecules.

The repertoire of αß T cell antigen receptors (TCRs) on mature T cells is selected in the thymus where it is rendered both self-tolerant and restricted to the recognition of major histocompatibility complex molecules presenting peptide antigens (pMHC). It remains unclear whether germline TCR sequences exhibit an inherent bias to interact with pMHC prior to selection. Here, we isolated TCR libraries from unselected thymocytes and upon reexpression of these random TCR repertoires in recipient T cell hybridomas, interrogated their reactivities to antigen-presenting cell lines. While these random TCR combinations could potentially have reacted with any surface molecule on the cell lines, the hybridomas were stimulated most frequently by pMHC ligands. The nature and CDR3 loop composition of the TCRß chain played a dominant role in determining pMHC-reactivity. Replacing the germline regions of mouse TCRß chains with those of other jawed vertebrates preserved reactivity to mouse pMHC. Finally, introducing the CD4 coreceptor into the hybridomas increased the proportion of cells that could respond to pMHC ligands. Thus, αß TCRs display an intrinsic and evolutionary conserved bias for pMHC molecules in the absence of any selective pressure, which is further strengthened in the presence of coreceptors.

Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation.

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.

Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70T293 and destabilization of the signalosome.

ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70T293A) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex.

Tolerogenic vaccines: Targeting the antigenic and cytokine niches of FOXP3+ regulatory T cells.

FOXP3+ regulatory T cells (Tregs) constitute a critical barrier that enforces tolerance to both the self-peptidome and the extended-self peptidome to ensure tissue-specific resistance to autoimmune, allergic, and other inflammatory disorders. Here, we review intuitive models regarding how T cell antigen receptor (TCR) specificity and antigen recognition efficiency shape the Treg and conventional T cell (Tcon) repertoires to adaptively regulate T cell maintenance, tissue-residency, phenotypic stability, and immune function in peripheral tissues. Three zones of TCR recognition efficiency are considered, including Tcon recognition of specific low-efficiency self MHC-ligands, Treg recognition of intermediate-efficiency agonistic self MHC-ligands, and Tcon recognition of cross-reactive high-efficiency agonistic foreign MHC-ligands. These respective zones of TCR recognition efficiency are key to understanding how tissue-resident immune networks integrate the antigenic complexity of local environments to provide adaptive decisions setting the balance of suppressive and immunogenic responses. Importantly, deficiencies in the Treg repertoire appear to be an important cause of chronic inflammatory disease. Deficiencies may include global deficiencies in Treg numbers or function, subtle 'holes in the Treg repertoire' in tissue-resident Treg populations, or simply Treg insufficiencies that are unable to counter an overwhelming molecular mimicry stimulus. Tolerogenic vaccination and Treg-based immunotherapy are two therapeutic modalities meant to restore dominance of Treg networks to reverse chronic inflammatory disease. Studies of these therapeutic modalities in a preclinical setting have provided insight into the Treg niche, including the concept that intermediate-efficiency TCR signaling, high IFN-ß concentrations, and low IL-2 concentrations favor Treg responses and active dominant mechanisms of immune tolerance. Overall, the purpose here is to assimilate new and established concepts regarding how cognate TCR specificity of the Treg repertoire and the contingent cytokine networks provide a foundation for understanding Treg suppressive strategy.

iTTCA-Hybrid: Improved and robust identification of tumor T cell antigens by utilizing hybrid feature representation.

In spite of the repertoire of existing cancer therapies, the ongoing recurrence and new cases of cancer poses a challenging health concern that prompts for novel and effective treatment. Cancer immunotherapy represents a promising venue for treatment by harnessing the body's immune system to combat cancer. Therefore, the identification of tumor T cell antigen represents an exciting area to explore. Computational tools have been instrumental in the identification of tumor T cell antigens and it is highly desirable to attain highly accurate models in a timely fashion from large volumes of peptides generated in the post-genomic era. In this study, we present a reliable, accurate, unbiased and automated sequence-based predictor named iTTCA-Hybrid for identifying tumor T cell antigens. The iTTCA-Hybrid approach proposed herein employs two robust machine learning models (e.g. support vector machine and random forest) constructed using five feature encoding strategies (i.e. amino acid composition, dipeptide composition, pseudo amino acid composition, distribution of amino acid properties in sequences and physicochemical properties derived from the AAindex). Rigorous independent test indicated that the iTTCA-Hybrid approach achieved an accuracy and area under the curve of 73.60% and 0.783, respectively, which corresponds to 4% and 7% performance increase than those of existing methods thereby indicating the superiority of the proposed model. To the best of our knowledge, the iTTCA-Hybrid is the first free web server (Available at http://camt.pythonanywhere.com/iTTCA-Hybrid) for identifying tumor T cell antigens presented by the MHC class I. The proposed web server allows robust predictions to be made without the need to develop in-house prediction models.

T-cell selection in the thymus: a spatial and temporal perspective.

The ability of T cells to respond to a wide array of foreign antigens while avoiding reactivity to self is largely determined by cellular selection of developing T cells in the thymus. While a great deal is known about the cell types and molecules involved in T-cell selection in the thymus, our understanding of the spatial and temporal aspects of this process remain relatively poorly understood. Thymocytes are highly motile within the thymus and travel between specialized microenvironments at different phases of their development while interacting with distinct sets of self-peptides and peptide presenting cells. A knowledge of when, where, and how thymocytes encounter self-peptide MHC ligands at different stages of thymic development is key to understanding T-cell selection. In the past several years, our laboratory has investigated this topic using two-photon time-lapse microscopy to directly visualize thymocyte migration and signaling events, together with a living thymic slice preparation to provide a synchronized experimental model of T-cell selection in situ. Here, we discuss recent advances in our understanding of the temporal and spatial aspects of T-cell selection, highlighting our own work, and placing them in the context of work from other groups.

Next-generation amplicon TRB locus sequencing can overcome limitations of flow-cytometric Vß expression analysis and confirms clonality in all T-cell prolymphocytic leukemia cases.

T-cell receptor (TCR) ß repertoire analysis can distinguish monoclonal from polyclonal T-cell proliferations and crucially aid in the diagnosis of T-cell malignancies. TCR repertoire can be assessed either by flow cytometry (FCM), or by molecular genetic techniques. We compared the results of parallel analyses of Vß expression by FCM and TRB rearrangements by DNA-based next-generation sequencing (NGS) in 80 diagnostic peripheral blood samples of patients with T-cell prolymphocytic leukemia (T-PLL) for (1) the diagnosis of clonality and (2) the assessment of dominant Vß usage. FCM-based analysis of the surface expression was performed using the IOTest Beta Mark kit. The NGS-based analysis employed the multiplex Biomed-2 VB-JB primers. In all the samples, one or two clonal TRB rearrangements were detected by NGS. Although a dominant Vß domain usage was detected by FCM in only 41/80 (51%) samples, clonality was suspected in all of them. In a total of 12 cases, the FCM missed the clone detected by NGS, despite theoretical coverage by the antibodies, the functionality of the rearrangement, and the expression of TCRαß on the cell surface. Partly overlapping with those cases, FCM discovered predominant Vß usage in the T-PLL population that differed from the one detected by NGS in 10 cases. Overall, the concordant NGS and FCM results were obtained on 61/80 (76%) of samples. We conclude that NGS-based TRB analysis can overcome certain limitations of FCM-based analysis by the identification of both productive and nonproductive rearrangements and by covering the whole Vß spectrum. Currently available FCM analysis of Vß expression lacks this breadth but has advantages, such as parallel immunophenotyping and a more accurate quantification of the Vß usage. © 2018 International Society for Advancement of Cytometry.

[Expression of ßF1 and T cell receptor γ in T lymphoblastic lymphoma/leukemia].

Objective: To evaluate the expression of ßF1 and T cell receptor (TCR)γ in T lymphoblastic lymphoma/leukemia(T-LBL/ALL), and investigate the clinicopathological features. Methods: Fifty-one cases of T-LBL/ALL were collected at Guangdong General Hospital from 2010 to 2016, the expression of ßF1 and TCRγ was assessed by immunohistochemistry. Results: There were 13 cases of children and adolescents, and 38 cases of adults. The expression rates of ßF1 and TCRγ were 27.5%(14/51) and 15.7%(8/51) respectively. The proportion of adults in αß T-LBL/ALL, TCR-silent T-LBL/ALL and γδ T-LBL/ALL was 7/14, 79.3%(23/29)and 8/8 respectively, and the difference was significant (P=0.023). There was no statistical difference in sex, LDH, bone marrow involvement and Ann arbor stage among these three groups(P>0.05). γδ T-LBL/ALLs included 6 cases of CD4(-)/CD8(-) phenotype, whereas αß T-LBL/ALL included 7 cases of CD4(+) /CD8(+) phenotype. There was significant difference in CD4/CD8 expression among these three groups(P<0.01). Conclusions: γδ T-LBL/ALL occurred only in adults, with predominantly CD4(-)/CD8(-) phenotype. αß T-LBL/ALL occurred more common in children and adolescents, with predominantly CD4(+) /CD8(+) phenotype.

Immunogenicity and protective efficacy of recombinant alkaline shock protein 23 from Staphylococcus aureus in a murine model.

The diversity and severity of infections caused and the rapid emergence of antibiotic resistance necessitates the development of a vaccine against Staphylococcus aureus. None of the antigens tried as vaccine candidates so far has been translated into a clinically viable vaccine. Recent research data suggest that antigens with the potential to activate cell mediated immunity along with humoral immunity would be the key to the development of a vaccine. Alkaline shock protein 23, a membrane-anchored protein involved in the stress response, has been identified as a CD4+ T cell antigen from S. aureus. In the present study, we report the evaluation of immunogenicity and protective efficacy of a recombinant alkaline shock protein 23 from S. aureus in mouse models. The gene coding for the protein was cloned and expressed in Escherichia coli, purified using immobilized metal iron affinity chromatography, sequence-confirmed using mass spectrometry and intraperitoneally administered to BALB/c mice. Serum titers of IgG, IgG1, and IgG2a in response to the protein were measured on post-immunization days 21, 35 and 42 using indirect ELISA and compared to control mice injected with PBS. Our results showed that the protein induced significantly higher (p < 0.01) antibody responses in immunized mice compared to the control mice. The mean serum antibody titers of IgG, IgG1 and IgG2a three weeks after the last immunization were found to be 25600, 25600 and 12800 respectively. Moreover, we found that immunization with Asp23 protected mice from a lethal dose of S. aureus strain USA300.