Systematic discovery of neoepitope-HLA pairs for neoantigens shared among patients and tumor types.

The broad application of precision cancer immunotherapies is limited by the number of validated neoepitopes that are common among patients or tumor types. To expand the known repertoire of shared neoantigen-human leukocyte antigen (HLA) complexes, we developed a high-throughput platform that coupled an in vitro peptide-HLA binding assay with engineered cellular models expressing individual HLA alleles in combination with a concatenated transgene harboring 47 common cancer neoantigens. From more than 24,000 possible neoepitope-HLA combinations, biochemical and computational assessment yielded 844 unique candidates, of which 86 were verified after immunoprecipitation mass spectrometry analyses of engineered, monoallelic cell lines. To evaluate the potential for immunogenicity, we identified T cell receptors that recognized select neoepitope-HLA pairs and elicited a response after introduction into human T cells. These cellular systems and our data on therapeutically relevant neoepitopes in their HLA contexts will aid researchers studying antigen processing as well as neoepitope targeting therapies.

Reduction in COVID-19 Vaccine Effectiveness against SARS-CoV-2 Variants in Seoul according to Age, Sex, and Symptoms: A Test-Negative Case-Control Study.

BACKGROUND: We evaluated vaccine effectiveness (VE) against infections with SARS-CoV-2 variants of concern in Seoul, the capital of the Republic of Korea, having the highest population density in the country, under real-world conditions. METHODS: We evaluated the reduction in the effectiveness of mRNA and viral-vector COVID-19 vaccines against infection by the SARS-CoV-2 delta variant in a subpopulation from April 2021 to July 2021 who visited screening clinics in Seoul using a test-negative case-control study design. Moreover, we conducted a case-control study matching the ten-year-old age group, sex, healthcare workers, and five districts of Seoul, which are considered confounding factors. RESULTS: The full VE in the pre-delta-dominant period was 95.0% (95% confidence interval [CI]: 91.2-97.2); however, it decreased to 61.1% (95% CI: 53.2-67.6) during the delta-dominant period. Notably, we found that COVID-19 VE was significantly decreased in individuals aged ≥80 years (52.9%, 95% CI: -9.9-79.8), men (50.6 %, 95% CI: 39.4-59.8), and asymptomatic individuals (49.8%, 95% CI: 36.5-60.3) during the widespread SARS-CoV-2 delta variant circulation. CONCLUSIONS: Vaccine-mediated protection drastically declined during the delta-dominant period and in vulnerable groups. This study suggests the requirement for additional countermeasures, such as the administration of a booster vaccine, in vulnerable groups based on age, sex, and symptomatic manifestation.

High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA.

Genome engineering of T lymphocytes, the main effectors of antitumor adaptive immune responses, has the potential to uncover unique insights into their functions and enable the development of next-generation adoptive T cell therapies. Viral gene delivery into T cells, which is currently used to generate CAR T cells, has limitations in regard to targeting precision, cargo flexibility, and reagent production. Nonviral methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, but complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a convenient and scalable nonviral method that allows precise gene edits and transgene integration in primary human T cells, using plasmid donor DNA template and Cas9-RNP. This method is highly efficient for single and multiplex gene manipulation, without compromising T cell function, and is thus valuable for use in basic and translational research.

Metabolism perturbation Causedby the overexpression of carbon monoxide dehydrogenase/Acetyl-CoA synthase gene complex accelerated gas to acetate conversion rate ofEubacterium limosumKIST612.

Microbial conversion of carbon monoxide (CO) to acetate is a promising upcycling strategy for carbon sequestration. Herein, we demonstrate that CO conversion and acetate production rates of Eubacterium limosum KIST612 strain can be improved by in silico prediction and in vivo assessment. The mimicked CO metabolic model of KIST612 predicted that overexpressing the CO dehydrogenase (CODH) increases CO conversion and acetate production rates. To validate the prediction, we constructed mutant strains overexpressing CODH gene cluster and measured their CO conversion and acetate production rates. A mutant strain (ELM031) co-overexpressing CODH, coenzyme CooC2 and ACS showed a 3.1 × increased specific CO oxidation rate as well as 1.4 × increased specific acetate production rate, compared to the wild type strain. The transcriptional and translational data with redox balance analysis showed that ELM031 has enhanced reducing potential from up-regulation of ferredoxin and related metabolism directly linked to energy conservation.

Methanol supply speeds up synthesis gas fermentation by methylotrophic-acetogenic bacterium, Eubacterium limosum KIST612.

This study analyzed the effect of methanol on the metabolism of syngas components (i.e., H2 and CO) by the syngas fermenting acetogenic strain E. limosum KIST612. The culture characteristics and relevant proteomic expressions (as fold changes) were carefully analyzed under CO/CO2 and H2/CO2 conditions with and without methanol addition, as well as, under methanol/CO2 conditions. The culture characteristics (specific growth rate and H2 consumption rate) under H2/CO2 conditions were greatly enhanced in the presence of methanol, by 4.0 and 2.7 times, respectively. However, the promoting effect of methanol was not significant under CO/CO2 conditions. Proteomic fold changes in most enzyme expression levels in the Wood-Ljungdahl pathway and chemiosmotic energy conservation also exhibited high correspondence between methanol and H2/CO2 but not between methanol and CO/CO2. These findings suggest the advantages of methanol addition to H2/CO2 for biomass enhancement and faster consumption of gaseous substrates during syngas fermentation.

PD-L1 expression by dendritic cells is a key regulator of T-cell immunity in cancer.

Inhibiting the programmed death-1 (PD-1) pathway is one of the most effective approaches to cancer immunotherapy, but its mechanistic basis remains incompletely understood. Binding of PD-1 to its ligand PD-L1 suppresses T-cell function in part by inhibiting CD28 signaling. Tumor cells and infiltrating myeloid cells can express PD-L1, with myeloid cells being of particular interest as they also express B7-1, a ligand for CD28 and PD-L1. Here we demonstrate that dendritic cells (DCs) represent a critical source of PD-L1, despite being vastly outnumbered by PD-L1+ macrophages. Deletion of PD-L1 in DCs, but not macrophages, greatly restricted tumor growth and led to enhanced antitumor CD8+ T-cell responses. Our data identify a unique role for DCs in the PD-L1-PD-1 regulatory axis and have implications for understanding the therapeutic mechanism of checkpoint blockade, which has long been assumed to reflect the reversal of T-cell exhaustion induced by PD-L1+ tumor cells.

The Kinase Activity of Hematopoietic Progenitor Kinase 1 Is Essential for the Regulation of T Cell Function.

We examined hematopoietic protein kinase 1 (HPK1), whose reliance on scaffold versus kinase functions for negative immune cell regulation is poorly understood and critical to its assessment as a viable drug target. We identify kinase-dependent roles for HPK1 in CD8 T cells that restrict their anti-viral and anti-tumor responses by using HPK1 kinase-dead (HPK1.kd) knockin mice. Loss of HPK1 kinase function enhanced T cell receptor signaling and cytokine secretion in a T-cell-intrinsic manner. In response to chronic lymphocytic choriomeningitis virus (LCMV) infection or tumor challenge, viral clearance and tumor growth inhibition were enhanced in HPK1.kd mice, accompanied by an increase in effector CD8 T cell function. Co-blockade of PD-L1 further enhanced T effector cell function, resulting in superior anti-viral and anti-tumor immunity over single target blockade. These results identify the importance of HPK1 kinase activity in the negative regulation of CD8 effector functions, implicating its potential as a cancer immunotherapy target.

Foxp3-independent mechanism by which TGF-ß controls peripheral T cell tolerance.

Peripheral T cell tolerance is promoted by the regulatory cytokine TGF-ß and Foxp3-expressing Treg cells. However, whether TGF-ß and Treg cells are part of the same regulatory module, or exist largely as distinct pathways to repress self-reactive T cells remains incompletely understood. Using a transgenic model of autoimmune diabetes, here we show that ablation of TGF-ß receptor II (TßRII) in T cells, but not Foxp3 deficiency, resulted in early-onset diabetes with complete penetrance. The rampant autoimmune disease was associated with enhanced T cell priming and elevated T cell expression of the inflammatory cytokine GM-CSF, concomitant with pancreatic infiltration of inflammatory monocytes that triggered immunopathology. Ablation of the GM-CSF receptor alleviated the monocyte response and inhibited disease development. These findings reveal that TGF-ß promotes T cell tolerance primarily via Foxp3-independent mechanisms and prevents autoimmunity in this model by repressing the cross talk between adaptive and innate immune systems.

Regulation of the Immune Response by TGF-ß: From Conception to Autoimmunity and Infection.

Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine involved in both suppressive and inflammatory immune responses. After 30 years of intense study, we have only begun to elucidate how TGF-ß alters immunity under various conditions. Under steady-state conditions, TGF-ß regulates thymic T-cell selection and maintains homeostasis of the naïve T-cell pool. TGF-ß inhibits cytotoxic T lymphocyte (CTL), Th1-, and Th2-cell differentiation while promoting peripheral (p)Treg-, Th17-, Th9-, and Tfh-cell generation, and T-cell tissue residence in response to immune challenges. Similarly, TGF-ß controls the proliferation, survival, activation, and differentiation of B cells, as well as the development and functions of innate cells, including natural killer (NK) cells, macrophages, dendritic cells, and granulocytes. Collectively, TGF-ß plays a pivotal role in maintaining peripheral tolerance against self- and innocuous antigens, such as food, commensal bacteria, and fetal alloantigens, and in controlling immune responses to pathogens.

Cancer Immunosurveillance by Tissue-Resident Innate Lymphoid Cells and Innate-like T Cells.

Malignancy can be suppressed by the immune system in a process termed immunosurveillance. However, to what extent immunosurveillance occurs in spontaneous cancers and the composition of participating cell types remains obscure. Here, we show that cell transformation triggers a tissue-resident lymphocyte response in oncogene-induced murine cancer models. Non-circulating cytotoxic lymphocytes, derived from innate, T cell receptor (TCR)αß, and TCRγδ lineages, expand in early tumors. Characterized by high expression of NK1.1, CD49a, and CD103, these cells share a gene-expression signature distinct from those of conventional NK cells, T cells, and invariant NKT cells. Generation of these lymphocytes is dependent on the cytokine IL-15, but not the transcription factor Nfil3 that is required for the differentiation of tumor-infiltrating NK cells, and IL-15 deficiency, but not Nfil3 deficiency, results in accelerated tumor growth. These findings reveal a tumor-elicited immunosurveillance mechanism that engages unconventional type-1-like innate lymphoid cells and type 1 innate-like T cells.

TETs Link Hydrogen Sulfide to Immune Tolerance.

Demethylation of the Foxp3 locus maintains gene expression and Treg cell stability. Yang et al. (2015) show that the gasotransmitter hydrogen sulfide co-operates with growth factor TGF-ß and interleukin-2 to activate Tet-mediated DNA demethylation of Foxp3 to promote immune tolerance.

Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly.

UNLABELLED: Bacillus anthracis vegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show that slaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elaborating S-layers, is required for the efficient secretion and assembly of Sap and EA1. S-layer protein precursors cosediment with SlaQ, and SlaQ appears to facilitate Sap assembly. Purified SlaQ polymerizes and when mixed with purified Sap promotes the in vitro formation of tubular S-layer structures. A model is discussed whereby SlaQ, in conjunction with S-layer secretion factors SecA2 and SlaP, promotes localized secretion and S-layer assembly in B. anthracis. IMPORTANCE: S-layer proteins are endowed with the propensity for self-assembly into crystalline arrays. Factors promoting S-layer protein assembly have heretofore not been reported. We identified Bacillus anthracis SlaQ, a small cytoplasmic protein that facilitates S-layer protein assembly in vivo and in vitro.

Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells.

Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-ß. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1(low). We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease.

Secretion genes as determinants of Bacillus anthracis chain length.

Bacillus anthracis grows in chains of rod-shaped cells, a trait that contributes to its escape from phagocytic clearance in host tissues. Using a genetic approach to search for determinants of B. anthracis chain length, we identified mutants with insertional lesions in secA2. All isolated secA2 mutants exhibited an exaggerated chain length, whereas the dimensions of individual cells were not changed. Complementation studies revealed that slaP (S-layer assembly protein), a gene immediately downstream of secA2 on the B. anthracis chromosome, is also a determinant of chain length. Both secA2 and slaP are required for the efficient secretion of Sap and EA1 (Eag), the two S-layer proteins of B. anthracis, but not for the secretion of S-layer-associated proteins or of other secreted products. S-layer assembly via secA2 and slaP contributes to the proper positioning of BslO, the S-layer-associated protein, and murein hydrolase, which cleaves septal peptidoglycan to separate chains of bacilli. SlaP was found to be both soluble in the bacterial cytoplasm and associated with the membrane. The purification of soluble SlaP from B. anthracis-cleared lysates did not reveal a specific ligand, and the membrane association of SlaP was not dependent on SecA2, Sap, or EA1. We propose that SecA2 and SlaP promote the efficient secretion of S-layer proteins by modifying the general secretory pathway of B. anthracis to transport large amounts of Sap and EA1.

CD4⁺CD25⁺Foxp3⁺ regulatory T cell formation requires more specific recognition of a self-peptide than thymocyte deletion.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are generated during thymocyte development and play a crucial role in preventing the immune system from attacking the body's cells and tissues. However, how the formation of these cells is directed by T-cell receptor (TCR) recognition of self-peptide:major histocompatibility complex (MHC) ligands remains poorly understood. We show that an agonist self-peptide with which a TCR is strongly reactive can induce a combination of thymocyte deletion and CD4(+)CD25(+)Foxp3(+) Treg cell formation in vivo. A weakly cross-reactive partial agonist self-peptide could similarly induce thymocyte deletion, but failed to induce Treg cell formation. These studies indicate that CD4(+)CD25(+)Foxp3(+) Treg cell formation can require highly stringent recognition of an agonist self-peptide by developing thymocytes. They also refine the "avidity" model of thymocyte selection by demonstrating that the quality of the signal mediated by agonist self-peptides, rather than the overall intensity of TCR signaling, can be a critical factor in directing autoreactive thymocytes to undergo CD4(+)CD25(+)Foxp3(+) Treg cell formation and/or deletion during their development.

Architects at the bacterial surface - sortases and the assembly of pili with isopeptide bonds.

The cell wall envelope of Gram-positive bacteria can be thought of as a surface organelle for the assembly of macromolecular structures that enable the unique lifestyle of each microorganism. Sortases - enzymes that cleave the sorting signals of secreted proteins to form isopeptide (amide) bonds between the secreted proteins and peptidoglycan or polypeptides - function as the principal architects of the bacterial surface. Acting alone or with other sortase enzymes, sortase construction leads to the anchoring of surface proteins at specific sites in the envelope or to the assembly of pili, which are fibrous structures formed from many protein subunits. The catalysis of intermolecular isopeptide bonds between pilin subunits is intertwined with the assembly of intramolecular isopeptide bonds within pilin subunits. Together, these isopeptide bonds endow these sortase products with adhesive properties and resistance to host proteases.

Thymocyte deletion can bias Treg formation toward low-abundance self-peptide.

Autoreactive CD4+ T cells can undergo deletion and/or become CD25+Foxp3+ Treg as they develop intrathymically, but how these alternative developmental fates are specified based on interactions with self-peptide(s) is not understood. We show here that thymocytes expressing an autoreactive TCR can be subjected to varying degrees of deletion that correlate with the amount of self-peptide. Strikingly, among thymocytes that evade deletion, similar proportions acquire Foxp3 expression. These findings provide evidence that Foxp3+ Treg can develop among members of a cohort of autoreactive thymocytes that have evaded deletion by a self-peptide, and that deletion and Treg formation can act together to bias the Treg repertoire toward low-abundance self-peptide(s).

Sortase D forms the covalent bond that links BcpB to the tip of Bacillus cereus pili.

Bacillus cereus and other Gram-positive bacteria elaborate pili via a sortase D-catalyzed transpeptidation mechanism from major and minor pilin precursor substrates. After cleavage of the LPXTG sorting signal of the major pilin, BcpA, sortase D forms an amide bond between the C-terminal threonine and the amino group of lysine within the YPKN motif of another BcpA subunit. Pilus assembly terminates upon sortase A cleavage of the BcpA sorting signal, resulting in a covalent bond between BcpA and the cell wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the minor pilin, is cleaved by sortase D but not by sortase A. The C-terminal threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby positioning BcpB at the tip of pili. Thus, unique attributes of the sorting signals of minor pilins provide Gram-positive bacteria with a universal mechanism ordering assembly of pili.

Cell wall anchor structure of BcpA pili in Bacillus anthracis.

Assembly of pili in Gram-positive bacteria and their attachment to the cell wall envelope are mediated by sortases. In Bacillus cereus and its close relative Bacillus anthracis, the major pilin protein BcpA is cleaved between the threonine and the glycine of its C-terminal LPXTG motif sorting signal by the pilin-specific sortase D. The resulting acyl enzyme intermediate is relieved by the nucleophilic attack of the side-chain amino group of lysine within the YPKN motif of another BcpA subunit. Cell wall anchoring of assembled BcpA pili requires sortase A, which also cleaves the LPXTG sorting signal of BcpA between its threonine and glycine residues. We show here that sortases A and D require only the C-terminal sorting signal of BcpA for substrate cleavage. Unlike sortase D, which accepts the YPKN motif as a nucleophile, sortase A forms an amide bond between the BcpA C-terminal carboxyl group of threonine and the side-chain amino group of diaminopimelic acid within the cell wall peptidoglycan of bacilli. These results represent the first demonstration of a cell wall anchor structure for pili, which are deposited by sortase A into the envelope of many different microbes.