Highly Networked SARS-CoV-2 Peptides Elicit T Cell Responses with Enhanced Specificity.

Identifying SARS-CoV-2-specific T cell epitope-derived peptides is critical for the development of effective vaccines and measuring the duration of specific SARS-CoV-2 cellular immunity. In this regard, we previously identified T cell epitope-derived peptides within topologically and structurally essential regions of SARS-CoV-2 spike and nucleocapsid proteins by applying an immunoinformatics pipeline. In this study, we selected 30 spike- and nucleocapsid-derived peptides and assessed whether these peptides induce T cell responses and avoid major mutations found in SARS-CoV-2 variants of concern. Our peptide pool was highly specific, with only a single peptide driving cross-reactivity in people unexposed to SARS-COV-2, and immunogenic, inducing a polyfunctional response in CD4+ and CD8+ T cells from COVID-19 recovered individuals. All peptides were immunogenic and individuals recognized broad and diverse peptide repertoires. Moreover, our peptides avoided most mutations/deletions associated with all four SARS-CoV-2 variants of concern while retaining their physicochemical properties even when genetic changes are introduced. This study contributes to an evolving definition of individual CD4+ and CD8+ T cell epitopes that can be used for specific diagnostic tools for SARS-CoV-2 T cell responses and is relevant to the development of variant-resistant and durable T cell-stimulating vaccines.

Characterizing and correcting immune dysfunction in non-tuberculous mycobacterial disease.

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic, progressive, and growing worldwide health burden associated with mounting morbidity, mortality, and economic costs. Improvements in NTM-PD management are urgently needed, which requires a better understanding of fundamental immunopathology. Here, we examine temporal dynamics of the immune compartment during NTM-PD caused by Mycobacterium avium complex (MAC) and Mycobactereoides abscessus complex (MABS). We show that active MAC infection is characterized by elevated T cell immunoglobulin and mucin-domain containing-3 expression across multiple T cell subsets. In contrast, active MABS infection was characterized by increased expression of cytotoxic T-lymphocyte-associated protein 4. Patients who failed therapy closely mirrored the healthy individual immune phenotype, with circulating immune network appearing to 'ignore' infection in the lung. Interestingly, immune biosignatures were identified that could inform disease stage and infecting species with high accuracy. Additionally, programmed cell death protein 1 blockade rescued antigen-specific IFN-γ secretion in all disease stages except persistent infection, suggesting the potential to redeploy checkpoint blockade inhibitors for NTM-PD. Collectively, our results provide new insight into species-specific 'immune chatter' occurring during NTM-PD and provide new targets, processes and pathways for diagnostics, prognostics, and treatments needed for this emerging and difficult to treat disease.

Synthetic hookworm-derived peptides are potent modulators of primary human immune cell function that protect against experimental colitis in vivo.

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world's population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell-based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid-exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions.

Identification and Characterization of a Peptide from the Stony Coral Heliofungia actiniformis.

Marine organisms produce a diverse range of toxins and bioactive peptides to support predation, competition, and defense. The peptide repertoires of stony corals (order Scleractinia) remain relatively understudied despite the presence of tentacles used for predation and defense that are likely to contain a range of bioactive compounds. Here, we show that a tentacle extract from the mushroom coral, Heliofungia actiniformis, contains numerous peptides with a range of molecular weights analogous to venom profiles from species such as cone snails. Using NMR spectroscopy and mass spectrometry we characterized a 12-residue peptide (Hact-1) with a new sequence (GCHYTPFGLICF) and well-defined ß-hairpin structure stabilized by a single disulfide bond. The sequence is encoded within the genome of the coral and expressed in the polyp body tissue. The structure present is common among toxins and venom peptides, but Hact-1 does not show activity against select examples of Gram-positive and Gram-negative bacteria or a range of ion channels, common properties of such peptides. Instead, it appears to have a limited effect on human peripheral blood mononuclear cells, but the ecological function of the peptide remains unknown. The discovery of this peptide from H. actiniformis is likely to be the first of many from this and related species.

Autoreactive T effector memory differentiation mirrors ß cell function in type 1 diabetes.

In type 1 diabetes, cytotoxic CD8+ T cells with specificity for ß cell autoantigens are found in the pancreatic islets, where they are implicated in the destruction of insulin-secreting ß cells. In contrast, the disease relevance of ß cell-reactive CD8+ T cells that are detectable in the circulation, and their relationship to ß cell function, are not known. Here, we tracked multiple, circulating ß cell-reactive CD8+ T cell subsets and measured ß cell function longitudinally for 2 years, starting immediately after diagnosis of type 1 diabetes. We found that change in ß cell-specific effector memory CD8+ T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8+ T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer-specific protein of 37 kDa, and CD16, and reduced expression of CD28) compared with their CD57- counterparts, and network association modeling indicated that the dynamics of ß cell-reactive CD57+ effector memory CD8+ T cell subsets were strongly linked. Thus, coordinated changes in circulating ß cell-specific CD8+ T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig.

There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.

Peptide mimic for influenza vaccination using nonnatural combinatorial chemistry.

Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic "mimics" using subunits that do not exist in the natural world. We developed a platform based on D-amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus-specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery.

Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations.

Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination.

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

More tricks with tetramers: a practical guide to staining T cells with peptide-MHC multimers.

Analysis of antigen-specific T-cell populations by flow cytometry with peptide-MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of 'tricks' that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of 'boosting' staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T-cell receptor is over 20-fold weaker (K(D) > 1 mm) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti-cancer or autoimmune T-cell populations, which tend to bear lower affinity T-cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them.

Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs.

Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.

Reprogramming of various cell types to a beta-like state by Pdx1, Ngn3 and MafA.

The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver) than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype.