Near-infrared duocarmycin photorelease from a Treg-targeted antibody-drug conjugate improves efficacy of PD-1 blockade in syngeneic murine tumor models.

Regulatory T cells (Tregs) play a crucial role in mediating immunosuppression in the tumor microenvironment. Furthermore, Tregs contribute to the lack of efficacy and hyperprogressive disease upon Programmed cell death protein 1 (PD-1) blockade immunotherapy. Thus, Tregs are considered a promising therapeutic target, especially when combined with PD-1 blockade. However, systemic depletion of Tregs causes severe autoimmune adverse events, which poses a serious challenge to Treg-directed therapy. Here, we developed a novel treatment to locally and predominantly damage Tregs by near-infrared duocarmycin photorelease (NIR-DPR). In this technology, we prepared anti-CD25 F(ab')2 conjugates, which site-specifically uncage duocarmycin in CD25-expressing cells upon exposure to NIR light. In vitro, CD25-targeted NIR-DPR significantly increased apoptosis of CD25-expressing HT2-A5E cells. When tumors were irradiated with NIR light in vivo, intratumoral CD25+ Treg populations decreased and Ki-67 and Interleukin-10 expression was suppressed, indicating impaired functioning of intratumoral CD25+ Tregs. CD25-targeted NIR-DPR suppressed tumor growth and improved survival in syngeneic murine tumor models. Of note, CD25-targeted NIR-DPR synergistically enhanced the efficacy of PD-1 blockade, especially in tumors with higher CD8+/Treg PD-1 ratios. Furthermore, the combination therapy induced significant anti-cancer immunity including maturation of dendritic cells, extensive intratumoral infiltration of cytotoxic CD8+ T cells, and increased differentiation into CD8+ memory T cells. Altogether, CD25-targeted NIR-DPR locally and predominantly targets Tregs in the tumor microenvironment and synergistically improves the efficacy of PD-1 blockade, suggesting that this combination therapy can be a rational anti-cancer combination immunotherapy.

Graft protective effects and donor-specific antibody suppression by CD4+CD25+Foxp3+ regulatory T cell induced by HMG-CoA reductase inhibitor rosuvastatin in a murine heart transplant model.

BACKGROUND: We previously demonstrated that the hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor (statins) play an important role in the regulation of alloimmune responses. However, little is known regarding the effects of statin on allograft protection or donor-specific antibodies (DSA). In this study, we investigated the graft-protective and immunomodulatory effects of rosuvastatin in a model of fully major histocompatibility complex-mismatched murine cardiac allograft transplantation. METHODS: CBA mice underwent transplantation of C57BL/6 (B6) hearts and received 50 and 500 µg/kg/day of rosuvastatin from the day of transplantation until seven days after the completion of transplantation. To confirm the requirement for regulatory T cells (Tregs), we administered an anti-interleukin-2 receptor alpha antibody (PC-61) to rosuvastatin-treated CBA recipients. Additionally, histological and fluorescent staining, cell proliferation analysis, flow cytometry, and DSA measurements were performed. RESULTS: CBA recipients with no treatment rejected B6 cardiac graft acutely (median survival time [MST], 7 days). CBA mice treated with 500 µg/kg/day of rosuvastatin prolonged allograft survival (MSTs, 77 days). Fluorescent staining studies showed that rosuvastatin-treated recipients had strong aggregation of CD4+Foxp3+ cells in the myocardium and around the coronary arteries of cardiac allografts two weeks after grafting. Flow cytometry studies performed two weeks after transplantation showed an increased number of splenic CD4+CD25+Foxp3+ T cells in rosuvastatin-treated recipients. The addition of rosuvastatin to mixed leukocyte cultures suppressed cell proliferation by increasing the number of CD4+CD25+Foxp3+ Tregs. Additionally, Tregs suppressed DSA production in rosuvastatin-treated recipients. CONCLUSION: Rosuvastatin treatment may be a complementary graft-protective strategy for suppressing DSA production in the acute phase, driven by the promotion of splenic and graft-infiltrating CD4+CD25+Foxp3+ Tregs.

The role of CD20+ T cells: Insights in human peripheral blood.

CD20+ T cells constitute a small subset of T cells. These are found among CD4+, CD8+, CD4+CD8+, CD4-CD8- T, and TCRγδ+ T cells, and have been poorly characterized. The aim of this study was to characterize peripheral blood (PB) CD20+ T cells and compare them to their PB CD20- T cell counterparts. PB from 17 healthy individuals was collected. The distribution of CD20+ T cells among maturation-associated T cells compartments (naïve, central memory, transitional memory, effector memory, and effector T cells), their polarization, activation status, and expression of immune-regulatory proteins were evaluated by flow cytometry. Their function was also assessed, by measuring IFN-γ, TNF-α, and IL-17 production. Compared with CD20- T cells, CD20+ T cells represent a higher proportion of transitional memory cells. Furthermore, CD20+ T cells display a proinflammatory phenotype, characterized by the expansion of Th1, Th1/17, and Tc1 cell subsets , associated to a high expression of activation (CD25) and exhaustion (PD-1) markers. In addition, the simultaneous production of the proinflammatory cytokines IFN-γ, TNF-α, and IL-17 was also detected in CD4+CD20+ T cells. Our results show that CD20+ T cells are phenotypically and functionally different from CD20- T cells, suggesting that these cells are a distinct subset of T cells.

Preparation and application of fluorescent monoclonal antibodies recognizing goat CD4+CD25+ regulatory T cells.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.

Depletion of regulatory T cells enhancing the anti-tumor effect of in situ vaccination in solid tumors.

The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the clinical treatment for tumor. However, the low response rate of ICIs remains the major obstacle for curing patients and effective approaches for patients with primary or secondary resistance to ICIs remain lacking. In this study, immune stimulating agent unmethylated CG-enriched (CpG) oligodeoxynucleotide (ODN) was locally injected into the tumor to trigger a robust immune response to eradicate cancer cells, while anti-CD25 antibody was applied to remove immunosuppressive regulatory T cells, which further enhanced the host immune activity to attack tumor systematically. The combination of CpG and anti-CD25 antibody obtained notable regression in mouse melanoma model. Furthermore, rechallenge of tumor cells in the xenograft model has resulted in smaller tumor volume, which demonstrated that the combinational treatment enhanced the activity of memory T cells. Remarkably, this combinational therapy presented significant efficacy on multiple types of tumors as well and was able to prevent relapse of tumor partially. Taken together, our combinational immunotherapy provides a new avenue to enhance the clinical outcomes of patients who are insensitive or resistant to ICIs treatments.

Conventional and unconventional T-cell responses contribute to the prediction of clinical outcome and causative bacterial pathogen in sepsis patients.

Sepsis is characterized by a dysfunctional host response to infection culminating in life-threatening organ failure that requires complex patient management and rapid intervention. Timely diagnosis of the underlying cause of sepsis is crucial, and identifying those at risk of complications and death is imperative for triaging treatment and resource allocation. Here, we explored the potential of explainable machine learning models to predict mortality and causative pathogen in sepsis patients. By using a modelling pipeline employing multiple feature selection algorithms, we demonstrate the feasibility of identifying integrative patterns from clinical parameters, plasma biomarkers, and extensive phenotyping of blood immune cells. While no single variable had sufficient predictive power, models that combined five and more features showed a macro area under the curve (AUC) of 0.85 to predict 90-day mortality after sepsis diagnosis, and a macro AUC of 0.86 to discriminate between Gram-positive and Gram-negative bacterial infections. Parameters associated with the cellular immune response contributed the most to models predictive of 90-day mortality, most notably, the proportion of T cells among PBMCs, together with expression of CXCR3 by CD4+ T cells and CD25 by mucosal-associated invariant T (MAIT) cells. Frequencies of Vδ2+ γδ T cells had the most profound impact on the prediction of Gram-negative infections, alongside other T-cell-related variables and total neutrophil count. Overall, our findings highlight the added value of measuring the proportion and activation patterns of conventional and unconventional T cells in the blood of sepsis patients in combination with other immunological, biochemical, and clinical parameters.

Graft Protective and Intercellular Immunomodulatory Effects by Adoptive Transfer of an Agonistic Anti-BTLA mAb (3C10) Induced CD4+CD25+ Regulatory T Cells in Murine Cardiac Allograft Transplant Model.

BACKGROUND: We demonstrated that an agonistic anti-B and T lymphocyte attenuator antibody (3C10) prolonged cardiac survival by inducing regulatory T cells (Treg). However, the mechanisms of immune tolerance in the recipients remained unclear. In this study, we investigated the graft-protective and intercellular immunomodulatory effects of adoptive transfer (AT) of 3C10-induced Tregs in a murine cardiac allograft transplant model. METHODS: Thirty days after transplantation of a C57BL/6 heart into the primary 3C10-treated CBA recipients, splenic CD4+CD25+ cells from these recipients (3C10/AT group) or naïve CBA mice (no-treatment group) were adoptively transferred into secondary CBA recipients with a C57BL/6 heart. To confirm the requirement for 3C10-induced Tregs, we administered an anti-interleukin-2 receptor alpha antibody (PC-61) to secondary CBA recipients. Additionally, histologic and fluorescent staining, cell proliferation analysis, flow cytometry, and donor-specific antibody (DSA) measurements were performed. RESULTS: 3C10/AT-treated CBA recipients resulted in significantly prolonged allograft survival (median survival time [MST], >50 days). Allografts displayed prolonged function with preservation of vessel structure by maintaining high numbers of splenic CD4+CD25+Foxp3+ Treg and intramyocardial CD4+Foxp3+ cells. DSA levels were suppressed in 3C10/AT-treated CBA recipients. Moreover, PC-61 administration resulted in a shorter MSTs of cardiac allograft survivals, a detrimental increase in DSA production, and enhanced expression of programmed cell death (PD)-1. CONCLUSION: AT of 3C10-induced Tregs may be a promising graft-protective strategy to prolong allograft survival and suppress DSA production, driven by the promotion of splenic and graft-infiltrating Tregs and collaboration with PD-1+ T cells and Treg.

Regulatory T-cell frequency and function in acute myocardial infarction patients and its correlation with ventricular dysfunction.

A high percentage of patients with acute coronary syndrome develop heart failure due to the ischemic event. Regulatory T (Treg) cells are lymphocytes with suppressive capacity that control the immune response and include the conventional CD4+ CD25hi Foxp3+ cells and the CD4+ CD25var CD69+ LAP+ Foxp3- IL-10+ cells. No human follow-up studies focus on Treg cells' behavior after infarction and their possible relationship with ventricular function as a sign of postischemic cardiac remodeling. This study aimed to analyze, by flow cytometry, the circulating levels of CD69+ Treg cells and CD4+ CD25hi Foxp3+ cells, their IL-10+ production as well as their function in patients with acute myocardial infarction (AMI), and its possible relation with ventricular dysfunction. We found a significant difference in the percentage of CD4+ CD25hi Foxp3+ cells and IL-10+ MFI in patients with AMI at 72 hours compared with the healthy control group, and the levels of these cells were reduced 6 months post-AMI. Regarding the suppressive function of CD4+ CD25+ regulatory cells, they were dysfunctional at 3 and 6 months post-AMI. The frequency of CD69+ Treg cells was similar between patients with AMI at 72 hours postinfarction and the control groups. Moreover, the frequency of CD69+ Treg cells at 3 and 6 months postischemic event did not vary over time. Treg cells play a role in regulating inflammation after an AMI, and its function may be compromised in this pathology. This work is the first report to evaluate CD69+ Foxp3- Treg cells in AMI patients.

CD4+CD25+CD127loFOXP3+ cell in food allergy: Does it predict anaphylaxis?

Background: Food allergy (FA), hence the incidence of food anaphylaxis, is a public health problem that has increased in recent years. There are still no biomarkers for patients with FA to predict severe allergic reactions such as anaphylaxis. Objective: There is limited information on whether regulatory T (Treg) cell levels are a biomarker that predicts clinical severity in cases presenting with FA, and which patients are at a greater risk for anaphylaxis. Methods: A total of 70 children were included in the study: 25 who had IgE-mediated cow’s milk protein allergy (CMPA) and presented with non-anaphylactic symptoms (FA/A−), 16 who had IgE-mediated CMPA and presented with anaphylaxis (FA/A+) (a total of 41 FA cases), and a control group consisting of 29 children without FA. The study was conducted by performing CD4+CD25+CD127loFOXP3+ cell flow cytometric analysis during resting at least 2 weeks after the elimination diet to FA subjects. Results: When the FA group was compared with healthy control subjects, CD4+CD25+CD127loFOXP3+ cell rates were found to be significantly lower in the FA group (p < 0.001). When the FA/A− and FA/A+ groups and the control group were compared in terms of CD4+CD25+CD127loFOXP3+ cell ratios, they were significantly lower in the FA/A− and FA/A+ groups compared to the control group (p < 0.001). Conclusions: Although there was no significant difference between the FA/A+ group and the FA/A− group in terms of CD4+CD25+CD127loFOXP3+ cells, our study is important, as it is the first pediatric study we know to investigate whether CD4+CD25+CD127loFOXP3+ cells in FA predict anaphylaxis (AU)

[The role of CD4+ CD25+ Treg in the mechanism of autoimmune auditory neuropathy in SD rats].

Objective: To investigate the role of CD4+CD25+regulatory cell (CD4+CD25+Treg) in auditory neuropathy (AN) using a rat model of autoimmune auditory neuropathy. Methods: The SD rats were immunized with P0 protein emulsified in complete Freunds adjuvant for 8 weeks. The number of CD4+CD25+Treg in peripheral blood and cochlea and the expression of Foxp3 gene in cochlea were detected respectively 2, 4, 6 and 8 weeks after the immunization with P0 protein in rats. Then CD4+CD25+Treg were transferred intravenously to the AN rats at 2, 4, 6 and 8 weeks of the immunization, respectively. The change of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were detected, and the morphological changes in the inner ear were investigated. Results: The number of CD4+CD25+Treg in the peripheral blood of AN rats decreased gradually after 2, 4, 6 and 8 weeks of P0 protein immunization. The number of CD4+CD25+Treg in cochlea gradually increased with the prolongation of immunization time, but the expression of Foxp3 gene in cochlea gradually decreased over time. After intravenous transplantation of CD4+CD25+Treg in AN rats, the threshold of ABR response decreased, and DPOAE had no significant change. The number of spiral ganglion neurons in cochlea increased, and hair cells had no significant change under electron microscope. Conclusions: The decrease in the number and function of CD4+CD25+Treg reduces its inhibitory effect on autoimmune response and promotes the occurrence of autoimmune auditory neuropathy in AN rats. Adoptive transfer of CD4+CD25+Treg can reduce the autoimmune response and promote the recovery of autoimmune auditory neuropathy.

The role of CD4+ CD25+ Treg in the mechanism of autoimmune auditory neuropathy in SD rats / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the role of CD4+CD25+regulatory cell (CD4+CD25+Treg) in auditory neuropathy (AN) using a rat model of autoimmune auditory neuropathy. Methods: The SD rats were immunized with P0 protein emulsified in complete Freunds adjuvant for 8 weeks. The number of CD4+CD25+Treg in peripheral blood and cochlea and the expression of Foxp3 gene in cochlea were detected respectively 2, 4, 6 and 8 weeks after the immunization with P0 protein in rats. Then CD4+CD25+Treg were transferred intravenously to the AN rats at 2, 4, 6 and 8 weeks of the immunization, respectively. The change of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were detected, and the morphological changes in the inner ear were investigated. Results: The number of CD4+CD25+Treg in the peripheral blood of AN rats decreased gradually after 2, 4, 6 and 8 weeks of P0 protein immunization. The number of CD4+CD25+Treg in cochlea gradually increased with the prolongation of immunization time, but the expression of Foxp3 gene in cochlea gradually decreased over time. After intravenous transplantation of CD4+CD25+Treg in AN rats, the threshold of ABR response decreased, and DPOAE had no significant change. The number of spiral ganglion neurons in cochlea increased, and hair cells had no significant change under electron microscope. Conclusions: The decrease in the number and function of CD4+CD25+Treg reduces its inhibitory effect on autoimmune response and promotes the occurrence of autoimmune auditory neuropathy in AN rats. Adoptive transfer of CD4+CD25+Treg can reduce the autoimmune response and promote the recovery of autoimmune auditory neuropathy.

In mouse chronic pancreatitis CD25+FOXP3+ regulatory T cells control pancreatic fibrosis by suppression of the type 2 immune response.

Chronic pancreatitis (CP) is characterized by chronic inflammation and the progressive fibrotic replacement of exocrine and endocrine pancreatic tissue. We identify Treg cells as central regulators of the fibroinflammatory reaction by a selective depletion of FOXP3-positive cells in a transgenic mouse model (DEREG-mice) of experimental CP. In Treg-depleted DEREG-mice, the induction of CP results in a significantly increased stroma deposition, the development of exocrine insufficiency and significant weight loss starting from day 14 after disease onset. In CP, FOXP3+CD25+ Treg cells suppress the type-2 immune response by a repression of GATA3+ T helper cells (Th2), GATA3+ innate lymphoid cells type 2 (ILC2) and CD206+ M2-macrophages. A suspected pathomechanism behind the fibrotic tissue replacement may involve an observed dysbalance of Activin A expression in macrophages and of its counter regulator follistatin. Our study identified Treg cells as key regulators of the type-2 immune response and of organ remodeling during CP. The Treg/Th2 axis could be a therapeutic target to prevent fibrosis and preserve functional pancreatic tissue.

Antibody-guided design and identification of CD25-binding small antibody mimetics using mammalian cell surface display.

Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). We have recently developed a method to create small antibody mimetics called FLuctuation-regulated Affinity Proteins (FLAPs), which consist of a small protein scaffold with a structurally immobilized target-binding peptide. In this study, to further develop this method, we established a novel screening system for FLAPs called monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a mAb guides selection in two manners. First, antibody-guided design allows construction of a peptide library that is relatively small in size, but sufficient to identify high-affinity binders in a single selection round. Second, in antibody-guided screening, the fluorescently labeled mAb is used to select mammalian cells that display FLAP candidates with high affinity for the target using fluorescence-activated cell sorting. We demonstrate the reliability and efficacy of MAGPIE using daclizumab, a mAb against human interleukin-2 receptor alpha chain (CD25). Three FLAPs identified by MAGPIE bound CD25 with dissociation constants of approximately 30 nM as measured by biolayer interferometry without undergoing affinity maturation. MAGPIE can be broadly adapted to any mAb to develop small antibody mimetics.

Prediction of anti-CD25 and 5-FU treatments efficacy for pancreatic cancer using a mathematical model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with rising incidence and with 5-years overall survival of less than 8%. PDAC creates an immune-suppressive tumor microenvironment to escape immune-mediated eradication. Regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSC) are critical components of the immune-suppressive tumor microenvironment. Shifting from tumor escape or tolerance to elimination is the major challenge in the treatment of PDAC. RESULTS: In a mathematical model, we combine distinct treatment modalities for PDAC, including 5-FU chemotherapy and anti- CD25 immunotherapy to improve clinical outcome and therapeutic efficacy. To address and optimize 5-FU and anti- CD25 treatment (to suppress MDSCs and Tregs, respectively) schedule in-silico and simultaneously unravel the processes driving therapeutic responses, we designed an in vivo calibrated mathematical model of tumor-immune system (TIS) interactions. We designed a user-friendly graphical user interface (GUI) unit which is configurable for treatment timings to implement an in-silico clinical trial to test different timings of both 5-FU and anti- CD25 therapies. By optimizing combination regimens, we improved treatment efficacy. In-silico assessment of 5-FU and anti- CD25 combination therapy for PDAC significantly showed better treatment outcomes when compared to 5-FU and anti- CD25 therapies separately. Due to imprecise, missing, or incomplete experimental data, the kinetic parameters of the TIS model are uncertain that this can be captured by the fuzzy theorem. We have predicted the uncertainty band of cell/cytokines dynamics based on the parametric uncertainty, and we have shown the effect of the treatments on the displacement of the uncertainty band of the cells/cytokines. We performed global sensitivity analysis methods to identify the most influential kinetic parameters and simulate the effect of the perturbation on kinetic parameters on the dynamics of cells/cytokines. CONCLUSION: Our findings outline a rational approach to therapy optimization with meaningful consequences for how we effectively design treatment schedules (timing) to maximize their success, and how we treat PDAC with combined 5-FU and anti- CD25 therapies. Our data revealed that a synergistic combinatorial regimen targeting the Tregs and MDSCs in both crisp and fuzzy settings of model parameters can lead to tumor eradication.

IL-10 Signaling Elicited by Nivolumab-Induced Activation of the MAP Kinase Pathway Does Not Fully Contribute to Nivolumab-Modulated Heterogeneous T Cell Responses.

Immune checkpoint inhibitor (ICI) therapy has revolutionized anti-cancer treatment for many late-stage cancer patients. However, ICI therapy has thus far demonstrated limited efficacy for most patients, and it remains unclear why this is so. Interleukin 10 (IL-10) is a cytokine that has been recognized as a central player in cancer biology with its ability to inhibit anti-tumor T cell responses. Recent studies suggest that IL-10 might also exert some intrinsic anti-tumor T cell responses, and clinical studies using recombinant IL-10 alone or in combination with ICI are underway. This paradoxical effect of IL-10 and its underlying mechanisms impacting ICI-modulated T cell responses remain poorly understood. In this study, using an in vitro mixed lymphocyte reaction assay, we found that treatment with ICIs such as the anti-programmed cell death receptor-1 (PD-1) mAb nivolumab elicits a strong expression of IL-10. While neutralization of IL-10 signaling with an anti-IL-10 specific mAb significantly decreases the production of IFN-γ by T cells in a cohort of donor cells, the opposite effect was observed in other donor cells. Similarly, neutralization of IL-10 signaling significantly decreases the expression of T cell activation markers Ki67 and CD25, as well as the production of Granzyme B in a cohort of donor cells, whereas the opposite effect was observed in others. Furthermore, we found that nivolumab and IL-10 differentially modulate the signal transducer and activator of transcription 3 (STAT3) and AKT serine-threonine kinase pathways. Finally, we found that nivolumab activates the mitogen-activated protein kinase (MAPK) pathway, which in turn is responsible for the observed induction of IL-10 production by nivolumab. These findings provide new insights into the mechanisms underlying anti-PD-1-modulated T cell responses by IL-10, which could lead to the discovery of novel combination treatments that target IL-10 and immune checkpoint molecules.

Effects of Salmonella enterica ser. Enteritidis and Heidelberg on host CD4+CD25+ regulatory T cell suppressive immune responses in chickens.

Poultry infected with Salmonella mount an immune response initially, however the immune responses eventually disappear leading the bird to be a carrier of Salmonella. The hypothesis of this study is that Salmonella infection induces T regulatory cell numbers and cytokine production and suppress host T cells locally in the gut to escape the host immune responses. An experiment was conducted to comparatively analyze the effect of S. enterica ser. Enteritidis (S. Enteritidis) and S. enterica ser. Heidelberg (S. Heidelberg) infection on CD4+CD25+ T regulatory cell properties in chickens. A total of 144 broiler chicks were randomly distributed into three experimental groups of non-infected control, S. Enteritidis infected and S. Heidelberg infected groups. Chickens were orally inoculated with PBS (control) or 5x106 CFU/mL of either S. Enteritidis or S. Heidelberg at 3 d of age. Each group was replicated in six pens with eight chickens per pen. Chickens infected with S. Enteritidis had 6.2, 5.4, and 3.8 log10 CFU/g, and chickens infected with S. Heidelberg had 7.1, 4.8, and 4.1 log10 CFU/g Salmonella in the cecal contents at 4, 11, and 32 dpi, respectively. Both S. Enteritidis and S. Heidelberg were recovered from the liver and spleen 4 dpi. At 4, 11, and 32 dpi, chickens infected with S. Enteritidis and S. Heidelberg had increased CD4+CD25+ cell numbers as well as IL-10 mRNA transcription of CD4+CD25+ cells compared to that in the control group. CD4+CD25+ cells from S. Enteritidis- and S. Heidelberg-infected chickens and restimulated with 1 µg antigen in vitro, had higher (P < 0.05) IL-10 mRNA transcription than the CD4+CD25+ cells from the non-infected controls Though at 4dpi, chickens infected with S. Enteritidis and S. Heidelberg had a significant (P < 0.05) increase in CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, the CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, were comparable to that in the control group at 11 and 32dpi identifying that the host inflammatory response against Salmonella disappears at 11 dpi. It can be concluded that S. Enteritidis and S. Heidelberg infection at 3 d of age induces a persistent infection through inducing CD4+CD25+ cells and altering the IL-10 mRNA transcription of CD4+CD25+ cell numbers and cytokine production in chickens between 3 to 32 dpi allowing chickens to become asymptomatic carriers of Salmonella after 18 dpi.

Two novel human anti-CD25 antibodies with antitumor activity inversely related to their affinity and in vitro activity.

High tumor regulatory T (Treg) cell infiltration is associated with poor prognosis of many cancers. CD25 is highly expressed on tumor Treg cells and is a potential target for Treg deletion. Previously characterized anti-CD25 antibodies appear to have limited efficacy in tumor inhibition. Here we identified two human anti-CD25 antibodies, BA9 and BT942, which did not prevent the activation of IL-2R signaling pathway by IL-2. BT942 had weaker binding and cytotoxic activity to human CD25-expressing cell lines than BA9. But both demonstrated significant tumor growth inhibition in early and late-stage animal cancer models. BT942 resulted in a higher expansion of CD8+ T cells and CD4+ T cells in tumor microenvironment in mouse MC38 model compared to BA9. BT942 also demonstrated significant higher tumor growth inhibition and higher expansion of CD8+ T cells and CD4+ T cells in combination with an anti-PD1 antibody. Pharmacokinetic study of BT942 in cynomolgus monkeys demonstrated a half-life of 206.97 ± 19.03 h. Structural analysis by cryo-EM revealed that BT942 recognizes an epitope on opposite side of the CD25-IL-2 binding site, consistent with no IL-2 signaling blockade in vitro. BT942 appears to be an excellent candidate for cancer immunotherapy.

Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL.

Blood transfusion practices affect CD4+ CD25+ FOXP3+ regulatory T cells/T helper-17 cells and the clinical outcome of geriatric patients with hip fracture.

Hip fracture (HF) is common among older individuals and associated with high mortality, poor vitality and functional impairment. HF patients suffer whole body immunological changes and that lead to severe consequences, including immobilization, physical impairment and a high risk of complications. The objective of this study was to decipher the pattern of dynamic immunological changes, especially in two major T cell subsets, CD4+ CD25+ FOXP3+ regulatory T (Treg) cells and T helper-17 (Th17) cells, and their balance, during the hospital stay and to observe whether blood transfusion could influence these cells and clinical patietns' prognosis. In this study, ninety-eight consecutive HF patients were initially enrolled, and finally fifty-one patients qualified for the study, and correlation analysis of their clinical parameters was carried out to predict the meaning of their distribution in clinical practice. Our results showed that the frequency of Tregs gradually decreased, while the frequency of Th17 cells slowly increased in HF patients who received blood transfusion. The Treg frequency was inversely correlated with the level of hemoglobin (Hb), and Th17 cell frequency was positively related to fluctuations in Hb levels in HF patients after trauma. HF patients with a better prognosis and survival time showed decreased a Treg frequency and a decreased Treg/Th17 ratio. Transfusion helped reverse the imbalance in the frequencies of Tregs and Th17 cells and the Treg/Th17 ratio and especially contributed to a better outcome in HF patients with moderate-to-severe anemia. In conclusion, a higher frequency of peripheral blood Tregs and a higher Treg/Th17 ratio may be associated with unfavorable outcomes in HF patients, and blood transfusion may benefit moderate-to-severe HF patients rebalance their immune response.