Adaptive plasticity of natural interleukin-35-induced regulatory T cells (Tr35) that are required for T-cell immune regulation.

Background: IL-35 potently inhibits immune responses both in vivo and in vitro. However, the specific characteristics of IL-35-producing cells, including their developmental origin, cellular phenotype, and function, are unknown. Methods: By using a novel IL-35 reporter mouse (Ebi3-Dre-Thy1.1) and double transgenic fate-mapping reporter mice (35EbiT-Rosa26-rox-tdTomato reporter mice or Foxp3 fate-mapping system), we tracked and analyzed the differentiation and developmental trajectories of Tr35 cells in vivo. And then we investigated the therapeutic effects of OVA-specific Tr35 cells in an OVA-induced allergic airway disease model. Results: We identified a subset of cells, denoted Tr35 cells, that secrete IL-35 but do not express Foxp3. These cells have high expression of molecules associated with T-cell activation and can inhibit T-cell proliferation in vitro. Our analyses showed that Tr35 cells are a distinct subpopulation of cells that are independent of Tr1 cells. Tr35 cells exhibit a unique gene expression profile and tissue distribution. The presence of Thy1.1 (Ebi3) expression in Tr35 cells indicates their active secretion of IL-35. However, the proportion of ex-Tr35 cells (Thy1.1-) is significantly higher compared to Tr35 cells (Thy1.1+). This suggests that Tr35 cells possess the ability to regulate IL-35 expression rapidly in vivo. Tr35 cells downregulated the expression of the inflammatory cytokines IL-4, IFN-γ and IL-17A. However, once Tr35 cells lost IL-35 expression and became exTr35 cells, the expression of inflammatory cytokines was upregulated. Importantly, our findings indicate that Tr35 cells have therapeutic potential. In an OVA-induced allergic airway disease mouse model, Tr35 cell reinfusion significantly reduced airway hyperresponsiveness and histopathological airway and lung inflammation. Conclusions: We have identified a subset of Tregs, Tr35 cells, that are distinct from Tr1 cells. Tr35 cells can dynamically regulate the secretion of inflammatory cytokines by controlling IL-35 expression to regulate inflammatory immune responses.

The histone lysine methyltransferase MLL1 regulates the activation and functional specialization of regulatory T cells.

The activation and specialization of regulatory T cells (Tregs) are crucial for maintaining immune self-tolerance; however, the regulation of these processes by histone modifications is not fully understood. Here, we show that T cell-specific deletion of the lysine methyltransferase MLL1 results in a spontaneous lymphocyte proliferation phenotype in aged mice without disturbing the development of conventional T cells and Tregs. Treg-specific MLL1 ablation leads to a systemic autoimmune disease associated with Treg dysfunction. Moreover, RNA sequencing demonstrates that the induction of multiple genes involved in Treg activation, functional specialization, and tissue immigration is defective in MLL1-deficient Tregs. This dysregulation is associated with defects in H3K4 trimethylation at these genes' transcription start sites. Finally, using a T-bet fate-mapping mouse system, we determine that MLL1 is required to establish stable Th1-type Tregs. Thus, MLL1 is essential in optimal Treg function by providing a coordinated chromatin context for activation and specialization.

Identification and affinity enhancement of T-cell receptor targeting a KRASG12V cancer neoantigen.

Neoantigens derived from somatic mutations in Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), the most frequently mutated oncogene, represent promising targets for cancer immunotherapy. Recent research highlights the potential role of human leukocyte antigen (HLA) allele A*11:01 in presenting these altered KRAS variants to the immune system. In this study, we successfully generate and identify murine T-cell receptors (TCRs) that specifically recognize KRAS8-16G12V from three predicted high affinity peptides. By determining the structure of the tumor-specific 4TCR2 bound to KRASG12V-HLA-A*11:01, we conduct structure-based design to create and evaluate TCR variants with markedly enhanced affinity, up to 15.8-fold. This high-affinity TCR mutant, which involved only two amino acid substitutions, display minimal conformational alterations while maintaining a high degree of specificity for the KRASG12V peptide. Our research unveils the molecular mechanisms governing TCR recognition towards KRASG12V neoantigen and yields a range of affinity-enhanced TCR mutants with significant potential for immunotherapy strategies targeting tumors harboring the KRASG12V mutation.

Antiviral memory B cells exhibit enhanced innate immune response facilitated by epigenetic memory.

The long-lasting humoral immunity induced by viral infections or vaccinations depends on memory B cells with greatly increased affinity to viral antigens, which are evolved from germinal center (GC) responses. However, it is unclear whether antiviral memory B cells represent a distinct subset among the highly heterogeneous memory B cell population. Here, we examined memory B cells induced by a virus-mimicking antigen at both transcriptome and epigenetic levels and found unexpectedly that antiviral memory B cells exhibit an enhanced innate immune response, which appeared to be facilitated by the epigenetic memory that is established through the memory B cell development. In addition, T-bet is associated with the altered chromatin architecture and is required for the formation of the antiviral memory B cells. Thus, antiviral memory B cells are distinct from other GC-derived memory B cells in both physiological functions and epigenetic landmarks.

B cell-reactive triad of B cells, follicular helper and regulatory T cells at homeostasis.

Autoreactive B cells are silenced through receptor editing, clonal deletion and anergy induction. Additional autoreactive B cells are ignorant because of physical segregation from their cognate autoantigen. Unexpectedly, we find that follicular B cell-derived autoantigen, including cell surface molecules such as FcγRIIB, is a class of homeostatic autoantigen that can induce spontaneous germinal centers (GCs) and B cell-reactive autoantibodies in non-autoimmune animals with intact T and B cell repertoires. These B cell-reactive B cells form GCs in a manner dependent on spontaneous follicular helper T (TFH) cells, which preferentially recognize B cell-derived autoantigen, and in a manner constrained by spontaneous follicular regulatory T (TFR) cells, which also carry specificities for B cell-derived autoantigen. B cell-reactive GC cells are continuously generated and, following immunization or infection, become intermixed with foreign antigen-induced GCs. Production of plasma cells and antibodies derived from B cell-reactive GC cells are markedly enhanced by viral infection, potentially increasing the chance for autoimmunity. Consequently, immune homeostasis in healthy animals not only involves classical tolerance of silencing and ignoring autoreactive B cells but also entails a reactive equilibrium attained by a spontaneous B cell-reactive triad of B cells, TFH cells and TFR cells.

Increased antibody titers but induced T cell AICD and apoptosis response in COVID-19 convalescents by inactivated vaccine booster.

It is urgently needed to evaluate the necessity and benefits of booster vaccination against the coronavirus 2 of the severe acute respiratory syndrome (SARS-CoV-2) Omicron to facilitate clinical decision-making for 2019 coronavirus disease (COVID-19) convalescents. We conducted a multicenter, prospective clinical trial (registration number: ChiCTR2100045810) in the first patients with COVID-19 from 28 January 2020 to 20 February 2020 to assess the long-term durability of neutralizing antibodies against live Omicron BA.5 and further assess the efficiency and safety of CoronaVac in the convalescent group. A total of 96 COVID-19 convalescents were enrolled in this study. Neutralizing antibody titers in convalescents were significantly reduced in 9-10 months. A dose-refreshing vaccination in 28 convalescents with an antibody titer below 96 significantly induced neutralizing antibodies against live Omicron by 4.84-fold. Meanwhile, the abundance of naive T cells increased dramatically, and TEMRA and TEM cells gradually decreased after vaccination. Activation-induced cell death and apoptosis-related genes were significantly elevated after vaccination in all T-cell subtypes. One-dose booster vaccination was effective in inducing a robust antibody response against SARS-CoV-2 Omicron in COVID-19 convalescents with low antibody titers. However, vaccine-mediated T-cell consumption and regeneration patterns may be detrimental to the antiviral response.IMPORTANCEThe globally dominant coronavirus 2 of the severe acute respiratory syndrome (SARS-CoV-2) Omicron variant raises the possibility of repeat infections among 2019 coronavirus disease (COVID-19) convalescents with low neutralizing antibody titers. The importance of this multicenter study lies in its evaluation of the long-term durability of neutralizing antibodies in COVID-19 convalescents and the efficacy of a booster vaccination against the live Omicron. The findings suggest that a one-dose booster vaccination is effective in inducing a robust antibody response against SARS-CoV-2 Omicron in convalescents with low antibody titers. However, the study also highlights the potential detrimental effects on the antiviral response due to vaccine-mediated T-cell consumption and regeneration patterns. These results are crucial for facilitating clinical decision-making for COVID-19 convalescents and informing public health policies regarding booster vaccinations.

Comparative Efficacy of P-CAB vs Proton Pump Inhibitors for Grade C/D Esophagitis: A Systematic Review and Network Meta-analysis.

INTRODUCTION: Los Angeles grade C/D esophagitis is a severe manifestation of gastroesophageal reflux disease that require active treatment and close follow-up. Potassium competitive acid blockers (P-CAB) are promising alternatives to proton pump inhibitors (PPI). We aimed to compare the efficacy and safety of P-CAB and PPI in healing grade C/D esophagitis to aid clinical decision-making. METHODS: A systematic literature search was performed using PubMed, MEDLINE, and Cochrane Central Register of Controlled Trials. Randomized controlled trials were eligible for inclusion if efficacy of P-CAB and PPI in healing grade C/D esophagitis was reported. Pooled risk ratios and risk difference with 95% credible intervals were used to summarize estimated effect of each comparison. The benefit of treatments was ranked using the surface under the cumulative probability ranking score. RESULTS: Of 5,876 articles identified in the database, 24 studies were eligible. Studies included incorporated 3 P-CAB (vonoprazan, tegoprazan, and keverprazan) and 6 PPI (lansoprazole, esomeprazole, omeprazole, rabeprazole extended-release (ER), pantoprazole, and dexlansoprazole). Based on the failure to achieve mucosal healing, 20 mg of vonoprazan q.d. ranked the first among PPI in initial and maintained healing of grade C/D esophagitis (surface under the cumulative probability ranking score = 0.89 and 0.87, respectively). Vonoprazan had similar risk of incurring adverse events, severe adverse events, and withdrawal to drug when compared with PPI. For those who attempted lower maintenance treatment dose, 10 mg of vonoprazan q.d. was a reasonable choice, considering its moderate efficacy and safety. DISCUSSION: Vonoprazan has considerable efficacy in initial and maintained healing of grade C/D esophagitis compared with PPI, with moderate short-term and long-term safety.

Sustained store-operated calcium entry utilizing activated chromatin state leads to instability in iTregs.

Thymus-originated tTregs and in vitro induced iTregs are subsets of regulatory T cells. While they share the capacity of immune suppression, their stabilities are different, with iTregs losing their phenotype upon stimulation or under inflammatory milieu. Epigenetic differences, particularly methylation state of Foxp3 CNS2 region, provide an explanation for this shift. Whether additional regulations, including cellular signaling, could directly lead phenotypical instability requires further analysis. Here, we show that upon TCR (T cell receptor) triggering, SOCE (store-operated calcium entry) and NFAT (nuclear factor of activated T cells) nuclear translocation are blunted in tTregs, yet fully operational in iTregs, similar to Tconvs. On the other hand, tTregs show minimal changes in their chromatin accessibility upon activation, in contrast to iTregs that demonstrate an activated chromatin state with highly accessible T cell activation and inflammation related genes. Assisted by several cofactors, NFAT driven by strong SOCE signaling in iTregs preferentially binds to primed-opened T helper (TH) genes, resulting in their activation normally observed only in Tconv activation, ultimately leads to instability. Conversely, suppression of SOCE in iTregs can partially rescue their phenotype. Thus, our study adds two new layers, cellular signaling and chromatin accessibility, of understanding in Treg stability, and may provide a path for better clinical applications of Treg cell therapy.

Foxp3-mediated blockage of ryanodine receptor 2 underlies contact-based suppression by regulatory T cells.

The suppression mechanism of Tregs remains an intensely investigated topic. As our focus has shifted toward a model centered on indirect inhibition of DCs, a universally applicable effector mechanism controlled by the transcription factor forkhead box P3 (Foxp3) expression has not been found. Here, we report that Foxp3 blocked the transcription of ER Ca2+-release channel ryanodine receptor 2 (RyR2). Reduced RyR2 shut down basal Ca2+ oscillation in Tregs, which reduced m-calpain activities that are needed for T cells to disengage from DCs, suggesting a persistent blockage of DC antigen presentation. RyR2 deficiency rendered the CD4+ T cell pool immune suppressive and caused it to behave in the same manner as Foxp3+ Tregs in viral infection, asthma, hypersensitivity, colitis, and tumor development. In the absence of Foxp3, Ryr2-deficient CD4+ T cells rescued the systemic autoimmunity associated with scurfy mice. Therefore, Foxp3-mediated Ca2+ signaling inhibition may be a central effector mechanism of Treg immune suppression.

The splicing isoform Foxp3Δ2 differentially regulates tTreg and pTreg homeostasis.

Foxp3 is the master transcription factor for regulatory T cells (Tregs). Alternative splicing of human Foxp3 results in the expression of two isoforms: the full length and an exon 2-deleted protein. Here, AlphaFold2 predictions and in vitro experiments demonstrate that the N-terminal domain of Foxp3 inhibits DNA binding by moving toward the C terminus and that this movement is mediated by exon 2. Consequently, we find that Foxp3Δ2-bearing thymus-derived Tregs (tTregs) in the peripheral lymphoid organ are less sensitive to T cell receptor (TCR) stimulation due to the enhanced binding of Foxp3Δ2 to the Batf promoter and are hyporesponsive to interleukin-2 (IL-2). In contrast, among RORγt+ peripherally induced Tregs (pTregs) in the large intestine, Foxp3Δ2 pTregs express many more RORγt-related genes, conferring a competitive advantage. Together, our results reveal that alternative splicing of exon 2 generates an active form of Foxp3, which plays a differential role in regulating tTreg and pTreg homeostasis.

How autoreactive thymocytes differentiate into regulatory versus effector CD4+ T cells after avoiding clonal deletion.

Thymocytes bearing autoreactive T cell receptors (TCRs) are agonist-signaled by TCR/co-stimulatory molecules to either undergo clonal deletion or to differentiate into specialized regulatory T (Treg) or effector T (Teff) CD4+ cells. How these different fates are achieved during development remains poorly understood. We now document that deletion and differentiation are agonist-signaled at different times during thymic selection and that Treg and Teff cells both arise after clonal deletion as alternative lineage fates of agonist-signaled CD4+CD25+ precursors. Disruption of agonist signaling induces CD4+CD25+ precursors to initiate Foxp3 expression and become Treg cells, whereas persistent agonist signaling induces CD4+CD25+ precursors to become IL-2+ Teff cells. Notably, we discovered that transforming growth factor-ß induces Foxp3 expression and promotes Treg cell development by disrupting weaker agonist signals and that Foxp3 expression is not induced by IL-2 except under non-physiological in vivo conditions. Thus, TCR signaling disruption versus persistence is a general mechanism of lineage fate determination in the thymus that directs development of agonist-signaled autoreactive thymocytes.

Apolipoprotein E mediates cell resistance to influenza virus infection.

Viruses exploit host cell machinery to support their replication. Defining the cellular proteins and processes required for a virus during infection is crucial to understanding the mechanisms of virally induced disease and designing host-directed therapeutics. Here, we perform a genome-wide CRISPR-Cas9-based screening in lung epithelial cells infected with the PR/8/NS1-GFP virus and use GFPhi cell as a unique screening marker to identify host factors that inhibit influenza A virus (IAV) infection. We discovered that APOE affects influenza virus infection both in vitro and in vivo. Cell deficiency in APOE conferred substantially increased susceptibility to IAV; mice deficient in APOE manifested more severe lung pathology, increased virus load, and decreased survival rate. Mechanistically, lack of cell-produced APOE results in impaired cell cholesterol homeostasis, enhancing influenza virus attachment. Thus, we identified a previously unrecognized role of APOE in restraining IAV infection.

Photochemical Synthesis of Succinic Ester-Containing Phenanthridines from Diazo Compounds as 1,4-Dicarbonyl Precursors.

We disclosed herein a straightforward photochemical method for the construction of phenanthridines containing a synthetically useful succinate unit. The reaction occurred under visible-light irradiation with cheap eosin Y Na as photoredox catalyst and a diazo compound as the succinate precursor. Under the optimal reaction conditions, a wide range of phenanthridines were obtained in moderate to good yields. Note that the succinate units in the final heterocycles could be easily transformed into many valuable structures, such as γ-butyrolactone, dihydrofuran-2(3H)-one, and tetrahydrofuran. Mechanistic experiments were performed to support the proposed mechanism.

The Crohn Disease-associated ATG16L1T300A polymorphism regulates inflammatory responses by modulating TLR- and NLR-mediated signaling.

The mechanisms by which the ATG16L1T300A polymorphism affects cell function and causes an increased risk for the development of Crohn disease remain incompletely understood. Here we report that healthy individuals and mice bearing this polymorphism, even as heterozygotes, manifest enhanced TLR, and NLR cytokine and chemokine responses due to increased activation of NFKB. We elucidated the mechanism of the NFKB abnormality and found that in the ATG16L1T300A cell, there is enhanced polyubiquitination of TRAF6 or RIPK2 resulting from the accumulation of SQSTM1/p62. Indeed, knockout of Sqstm1 in autophagy-deficient cells almost completely normalized TRAF6 or RIPK2 polyubiquitination and NFKB activation in these cells. Thus, by identifying that autophagy is a pathway-intrinsic homeostatic mechanism that restricts excessive TLR- or NLR-mediated inflammatory signaling, our findings shed new light on how the ATG16L1T300A polymorphism sets the stage for the occurrence of Crohn disease.Abbreviations: 3-MA: 3-methyladenine; ATG16L1: autophagy related 16 like 1; ATG7: autophagy related 7; BMDM: bone marrow-derived macrophage; CD: Crohn disease; CXCL: C-X-C motif chemokine ligand; IBD: inflammatory bowel disease; iBMDM: immortalized mouse BMDM; IL1B/IL-1ß: interleukin 1 beta; IL6: interleukin 6; KI: knockin; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPS: lipopolysaccharide; MDP: muramyl dipeptide; MEF: mouse embryonic fibroblast; NFKB/NF-κB: nuclear factor kappa B; NFKBIA/IKBA: NFKB inhibitor alpha; NLR: NOD-like receptor; NOD: nucleotide-binding oligomerization domain containing; RIPK2: receptor interacting serine/threonine kinase 2; SNP: single nucleotide polymorphism; SQSTM1/p62: sequestosome 1; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; Ub: ubiquitin; WT: wild type.

Induction of Foxp3 and activation of Tregs by HSP gp96 for treatment of autoimmune diseases.

Upregulation and stabilization of Foxp3 expression in Tregs are essential for regulating Treg function and immune homeostasis. In this study, gp96 immunization showed obvious therapeutic effects in a Lyn -/- mouse model of systemic lupus erythematosus. Moreover, gp96 alleviated the initiation and progression of MOG-induced experimental autoimmune encephalomyelitis. Immunization of gp96 increased Treg frequency, expansion, and suppressive function. Gene expression profiling identified the NF-κB family member p65 and c-Rel as the key transcription factors for enhanced Foxp3 expression in Treg by gp96. Mutant gp96 within its Toll-like receptor (TLR) binding domain, TLR2 knockout mice, and mice with cell-specific deletion of MyD88, were used to demonstrate that gp96 activated Tregs and induced Foxp3 expression via a TLR2-MyD88-mediated NF-κB signaling pathway. Taken together, these results show that gp96 immunization restricted antibody-induced and Th-induced autoimmune diseases by integrating Treg expansion and activation, indicating its potential clinical usefulness against autoimmune diseases.

A Split-Cre system designed to detect simultaneous expression of two genes based on SpyTag/SpyCatcher conjugation and Split-GFP dimerization.

The Split-Cre system is a powerful tool for genetic manipulation and can be used to spatiotemporally control gene expression in vivo. However, the low activity of the reconstituted NCre/CCre recombinase in the Split-Cre system limits its application as an indicator of the simultaneous expression of a pair of genes of interest. Here, we describe two approaches for improving the activity of the Split-Cre system after Cre reconstitution based on self-associating split GFP (Split-GFP) and SpyTag/SpyCatcher conjugation. First, we created the Split-GFP-Cre system by constructing fusion proteins of NCre and CCre with the N-terminal and C-terminal subunits of GFP, respectively. Reconstitution of Cre by GFP-mediated dimerization of the two fusion proteins resulted in recombinase activity approaching that of full-length Cre in living cells. Second, to further increase recombinase activity at low levels of Split-Cre expression, the Split-Spy-GCre system was established by incorporating the sequences for SpyTag and SpyCatcher into the components of the Split-GFP-Cre system. As anticipated, covalent conjugation of the SpyTag and SpyCatcher segments improved Split-GFP dimerization to further increase Cre recombinase activity in living cells. The increased efficiency and robustness of this dual-split system (Split-Cre and Split-GFP) minimize the problems of incomplete double gene-specific KO or low labeling efficiency due to poor NCre/CCre recombinase activity. Thus, this Split-Spy-GCre system allows more precise gene manipulation of cell subpopulations, which will provide advanced analysis of genes and cell functions in complex tissue such as the immune system.

Ex-TFRs: A Missing Piece of the SLE Puzzle?

Systemic lupus erythematosus (SLE) is a chronic multi-organ autoimmune disease involving the production of a wide range of autoantibodies and complement activation. The production of these high-affinity autoantibodies requires T cell/B cell collaboration as well as germinal center (GC) formation. T follicular regulatory cells (TFRs) are functional specialized T regulatory cells (Tregs) that safeguard against both self-reactive T and B cells. However, recent evidence suggests that TFRs are not always stable and can lose Foxp3 expression to become pathogenic "ex-TFRs" that gain potent effector functions. In this review, we summarize the literature on intrinsic and extrinsic mechanisms of regulation of TFR stability and discuss the potential role of TFR reprogramming in autoantibody production and SLE pathogenesis.

Radical antegrade modular pancreatosplenectomy versus standard distal pancreatosplenectomy for pancreatic cancer, a dual-institutional analysis.

BACKGROUND: Radical antegrade modular pancreatosplenectomy (RAMPS) has been adopted by some surgeons in the treatment of left-sided pancreatic cancer (PDAC). Low disease incidence and heterogenous disease biology make robust prospective comparison of RAMPS and standard distal pancreatosplenectomy (DPS) difficult. METHODS: Consecutive cases of chemo-naïve patients undergoing open RAMPS and DPS for PDAC between 2010-2017 at two international high-volume pancreatectomy centers were compared. Cox proportional hazard modeling was utilized for multivariate analysis. RESULTS: We identified 193 DPS and 253 RAMPS during the study period. DPS was associated with higher rates of median estimated blood loss (500 vs. 300 cc, P<0.001), median total harvested lymph nodes (18 vs. 12, P<0.001) and R0 resection (94.3% vs. 88.9%, P=0.013). There were no differences in rates of postoperative pancreatic fistula (16.5% vs. 17.8%, P=1) or postoperative hemorrhage (5.9% vs. 3.6%, P=0.385) (DPS vs. RAMPS). After controlling for significant clinical pathological parameters, RAMPS was associated with non-superior recurrence-free survival (RFS) (HR 0.29; 95% CI, 0.07-1.27, P=0.101) and overall-survival (HR 1.03; 95% CI, 0.71-1.49, P=0.895) compared with DPS. Similar results were observed in node-positive patients. CONCLUSIONS: RAMPS is safe and effective in the treatment of PDAC, but is not associated with an improvement in either RFS or overall-survival over DPS.