CD62L Expression Research Articles

Lymph-Node-Based CD3+ CD20+ Cells Emerge from Membrane Exchange between T Follicular Helper Cells and B Cells and Increase Their Frequency following Simian Immunodeficiency Virus Infection.

CD4+ T follicular helper (TFH) cells are key targets for human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) replication and contribute to the virus reservoir under antiretroviral therapy (ART). Here, we describe a novel CD3+ CD20+ double-positive (DP) lymphocyte subset, resident in secondary lymphoid organs of humans and rhesus macaques (RMs), that appear predominantly after membrane exchange between TFH and B cells. DP lymphocytes are enriched in cells displaying a TFH phenotype (CD4+ PD1hi CXCR5hi), function (interleukin 21 positive [IL-21+]), and gene expression profile. Importantly, expression of CD40L upon brief in vitro mitogen stimulation identifies, by specific gene-expression signatures, DP cells of TFH-cell origin versus those of B-cell origin. Analysis of 56 RMs showed that DP cells (i) significantly increase following SIV infection, (ii) are reduced after 12 months of ART in comparison to pre-ART levels, and (iii) expand to a significantly higher frequency following ART interruption. Quantification of total SIV-gag DNA on sorted DP cells from chronically infected RMs showed that these cells are susceptible to SIV infection. These data reinforce earlier observations that CD20+ T cells are infected and expanded by HIV infection, while suggesting that these cells phenotypically overlap activated CD4+ TFH cells that acquire CD20 expression via trogocytosis and can be targeted as part of therapeutic strategies aimed at HIV remission. IMPORTANCE The HIV reservoir is largely composed of latently infected memory CD4+ T cells that persist during antiretroviral therapy and constitute a major barrier toward HIV eradication. In particular, CD4+ T follicular helper cells have been demonstrated as key targets for viral replication and persistence under ART. In lymph nodes from HIV-infected humans and SIV-infected rhesus macaques, we show that CD3+ CD20+ lymphocytes emerge after membrane exchange between T cells and B cells and are enriched in phenotypic, functional, and gene expression profiles found in T follicular helper cells. Furthermore, in SIV-infected rhesus macaques, these cells expand following experimental infection and after interruption of ART and harbor SIV DNA at levels similar to those found in CD4+ T cells; thus, CD3+ CD20+ lymphocytes are susceptible to SIV infection and can contribute to SIV persistence.

B1-adrenergic blockers provide cardiac protection through extracellular traps inhibition during widespread inflammation

Sepsis is one of the leading causes of death worldwide, and no therapy is available other than intensive care treatments. Drug development attempts have mainly focused on controlling inflammation or treating dysfunction, without significant improvement in the last decades. Recently, interest has emerged in β1-adrenergic blockade, which has proved beneficial to both parameters. Unfortunately, the specific underlying mechanisms have not been addressed yet. Our study aimed to investigate on mice (adult Swiss male, 30-45g) lipopolyssacharide or cecal ligature and puncture models the effects of atenolol, a β1-adrenergic blocker, currently used in intensive care units for hypertension and arrhythmia. The results demonstrated a beneficial effect of treatment on survival and a preservation of cardiac function including left ventricular ejection fraction (33±2,8% in sepsis group versus 55±5,1% in sepsis-treated group, p<0,05) at a concentration reducing heart rate by approximately 10%.We wondered if beneficial results obtained with atenolol could be due to a modulation of extracellular traps. These components are essential in pathogen spreading control and are increased during sepsis. However, an excess of their production induces vascular occlusions and acute heart injuries.We demonstrated that neutrophils extracellular traps were increased in heart and blood during sepsis and decreased significantly following atenolol administration (blood: 1.1±0,3% in control group, 13,4±1.8% in sepsis group, 6.9±1.2% in sepsis-treated group). This beneficial effect of atenolol was not associated with other modulations of neutrophil function such as their ability to phagocyte (33,6±3,9% in sepsis group versus 25.6±3.6% in sepsis-treated group, p>0,05), to undergo apoptosis (Annexin V+/ L/D-: 13,2±2.1% in sepsis group versus 14.1±2.9% in sepsis-treated group, p>0,05) or to degranulate. Blood neutrophil phenotypes associated with their maturation state (expression of CD62L and CXCR2) or ability to migrate into tissue (expression of adhesion molecules PSGL1, LFA1, VLA4, PECAM1) was also similar between LPS and LPS-treated groups. Although, the modulation of extracellular traps production by atenolol was also observed in other immune cells population able to extrude chromatin fibers in extracellular space such as macrophages. These results suggest a specific beneficial effect of ß1-adrenergic blockers on extracellular traps formation associated with cardiac function preservation and ultimately survival without detrimental effect on heart rate during widespread inflammation. This work was supported by funding from INSERM and Paris-Saclay University-UVSQ. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluating immunological outcomes associated with histotripsy treatment in canine osteosarcoma: a comparative human oncology model

Abstract Osteosarcoma (OS), the most commonly occurring primary bone tumor in dogs and humans, has deficient treatment options and dismal survival outcomes. Spontaneously occurring OS in dogs shares numerous similarities with humans and has greater prevalence, allowing the dog to serve as an informative comparative oncology model. The objective of this study was to investigate the non-thermal focused ultrasound technique histotripsy as an innovative treatment option for OS. Histotripsy has the potential to both ablate the primary tumor and stimulate an antitumor immune response to mitigate metastatic disease, the leading cause of death in OS. To investigate the immunological outcomes associated with histotripsy and OS, we delivered histotripsy to client-owned canine OS patients (n=29) and utilized an in-vitro model for cross species comparisons between human, dog, and mouse. Histotripsy ablation led to a significantly greater expression of activation markers CD80 and CD62L on circulating monocytes, at 1- and 5-days post histotripsy (DPH), respectively. We observed a significantly greater production of TGFβ in circulating monocytes at 3 DPH compared to baseline. On an independent patient basis, we observed a greater population of CD5 +CD4 +and CD5 +CD8 +cells within the treated region of the tumor compared to untreated region and changes in genetic signature also indicated immune activation. Across species in-vitro we observed macrophage activation (CD80, CD62L, and CD86), and upregulation of genes associated with immune cell chemotaxis and pro-inflammatory response. Our results indicate that histotripsy ablation leads to immune activation and has strong potential to target both primary OS and metastatic disease. NIH (1R21 EB030182-01), Focused Ultrasound Foundation (PLL7GBVK and FUSF-RAP-823R1), and American Kennel Club (Canine Health Foundation No. 02773)

Targeting Pim2 improves effector function and longevity of T cells in cancer immunotherapy

Abstract The Provirus Integration sites for Moloney murine leukemia virus (Pim) kinase family has been studied extensively in tumorigenesis. We previously reported a key role of Pim2 in negatively regulating T-cell responses to alloantigen. We further investigated how Pim2 regulates T-cell mediated anti-tumor responses using murine models of adoptive cell therapy (ACT) and autologous hematopoietic cell transplantation (HCT). We found that Pim2 −/−Pmel CD8 T cells showed enhanced ability for controlling B16 melanoma growth following ACT. Pim2 −/−T cells were more potent to control C1498 leukemia growth after syngeneic HCT. Mechanistically, Pim2 −/−CD8 T cells exhibited increased effector cytokine production, metabolic activities, and unexpectedly CD62L expression than WT controls following activation. Activated Pim2 −/−CD8 T cells had less apoptosis and exhaustion during expansion in IL-2. In vivo, Pim2 −/−CD8 T cells differentiated more into effector, but less into exhausted or suppressive subsets in tumors. On the other hand, Pim2 −/−CD8 T cells exhibited increased longevity in spleen and tumor draining lymph nodes. Our data indicates that Pim2 negatively regulates CD8 T-cell anti-tumor activity by controlling their persistence in secondary lymphoid organs and effector function in tumors. Blocking Pim2 with a Pim2-specific inhibitor improved graft-versus-leukemia activity after autologous HCT and enhanced anti-melanoma effects after ACT. Pim2 inhibition also increased anti-tumor activity of human melanoma-specific T cells and CD19 CAR-T cells. Taken together, targeting Pim2 may serve as a novel strategy for improving cancer immunotherapy through enhancing differentiation and persistence of effector T cells. NIH R01 CA258440 and R21 CA263140 to Xue-Zhong Yu, MCW Center for Immunology Pilot Award to Yongxia Wu

Biological scaffold implants in a murine volumetric muscle loss trauma model cause time dependent CD62L and CD101 upregulation on “regulatory like” eosinophils.

Abstract Introduction: Previously, we reported a strong increase in the recruitment of eosinophils at injured muscle when treated with a biologic scaffold implant. As such, we sought to characterize the surface marker expression of these eosinophils to understand their role at the site of injury. Method: Volumetric muscle loss (VML) model was performed on C57BL/6 mice and was subsequently treated with a biologic scaffold (decellularized extracellular matrix). After 7 and 21 days, post-injury, implanted material along with injured tissue was dissected and converted to single cell suspension for flow cytometry. Results: Compared to uninjured mice, at both 7 and 21 days time point, mice that received biological scaffold featured significantly increased presence of eosinophils at the site of injury. These eosinophils had a ring-shaped nucleus which has been previously associated with regulatory eosinophils. Eosinophils at the site of injury showed time dependent upregulation of CD101 (Mean fold change from uninjured muscle after 7 days post implant was 2.73 and the same was 9.90 after 21 days, (p<0.0001)), and CD62L (Mean fold change from uninjured muscle after 7 days post implant was 3.65 and the same was 11.64 after 21 days(p<0.0001)) expression. Interestingly, the same pattern was not observed with blood eosinophils except that the blood eosinophils at 3-week timepoint were featuring a significant increase in CD62L expression compared with 1 week time point, suggesting potential return of homeostasis in circulatory compartment. Conclusion: Here, we describe time-dependent transformation in eosinophil phenotype in response to biologic scaffold implantation, including upregulation of adhesion molecule CD62L and a regulatory marker CD101.

Depletion of Ly6G-Expressing Neutrophilic Cells Leads to Altered Peripheral T-Cell Homeostasis and Thymic Development in Neonatal Mice.

Newborns and especially preterm infants are much more susceptible to infections than adults. Due to immature adaptive immunity, especially innate immune cells play an important role in a newborn's infection defense. Neonatal neutrophils exhibit profound differences in their functionality compared to neutrophils of adults. In particular, neonates possess a relevant population of suppressive neutrophils, which not only inhibit but also specifically modulate the function of T-cells. In this study, we investigated whether neonatal neutrophils are already involved in T-cell development in the thymus. For this purpose, we used a newly developed model of antibody-mediated immune cell depletion in which we administered a depleting antibody to pregnant and then lactating dams. Using this method, we were able to sufficiently deplete Ly6G-positive neutrophils in offspring. We demonstrated that the depletion of neutrophils in newborn mice resulted in altered peripheral T-cell homeostasis with a decreased CD4+/CD8+ T-cell ratio and decreased expression of CD62L. Neutrophil depletion even affected T-cell development in the thymus, with increased double positive thymocytes and a decreased CD4+/CD8+ single positive thymocyte ratio. Altogether, we demonstrated a previously unknown mechanism mediating neutrophils' immunomodulatory effects in newborns.

Abstract 6639: Preclinical characterization and clinical biomarker studies with RP3, a novel oncolytic immunotherapy expressing a fusogenic protein, a human anti-CTLA-4 antibody, hCD40L and h4-1BBL

Abstract Introduction: RP3, a tumor-directed oncolytic immunotherapy (TDOI), is a selectively replication-competent HSV-1 that contains the codon-optimized sequence for the fusogenic GALV-GP-R- protein, an anti-CTLA-4 antibody-like molecule, CD40L, and 4-1BBL. The expression of GALV-GP-R- potentiates immunogenic tumor cell death through the induction of cell-to-cell fusion. CD40L enhances dendritic cell and macrophage activation and 4-1BBL promotes CD8+ T-cell activation and co-stimulation. The anti-CTLA-4 antibody-like molecule blocks the interaction of CTLA-4 with its ligands B7.1 and B7.2, further enhancing T cell activation. RP3 is currently being assessed in a Phase 1 clinical trial with or without nivolumab in patients with advanced solid tumors (NCT04735978). Methods: CD1 nude mice with A375 melanoma xenograft tumors (Group 1) were treated with a single intratumoral injection of 1 × 107 PFU/mL of RP3. Tumors and blood samples were collected 3 days later, and immunohistochemistry (IHC) was performed to detect CD40L, 4-1BBL and HSV-1. For anti-CTLA-4 detection, two doses of 107 PFU/mL of RP3 were injected intratumorally into C57BL/6 mice bearing 4434 murine BRAFV600E mutant melanoma tumors (Group 2). Tumors and blood samples were collected at 3, 6, 9, 12 and 15-days post-dosing and anti-CTLA-4 ELISA was performed. For clinical biomarker studies, tumor biopsies were collected pre- and post-RP3 treatment. CD8 and PD-L1 were characterized by IHC, and gene expression analysis (GEA) was performed. TCR analysis was also performed using PBMCs. Results: IHC analysis from Group 1 confirmed abundant and widespread expression of CD40-L and 4-1BBL, which were detected alongside with HSV-1 antigen. In the blood samples collected from Group 2, robust levels of anti-CTLA-4 antibody were detected, which stayed in detectable range up to 15 days after the 2nd RP3 injection. IHC analysis of patient tumor samples demonstrated a marked increase in CD8 T cells and PD-L1 expression. GEA of patient tumor biopsies demonstrated increased levels of genes associated with innate and adaptive immune activation and genes previously reported to be associated with responsiveness to anti-PD1 therapy. TCR sequencing of PBMCs and multiplex IHC analysis are ongoing and updated results will be presented. Conclusion: Preclinical experiments from mice models confirmed the robust expression of CD40-L, 4-1BBL and anti-CTLA-4 in RP3-injected tumors. Biomarker studies from patients demonstrate increases in CD8-T cell infiltration and PD-L1 expression post RP3 +/- nivolumab treatment, including increases in the expression of genes associated with response to anti-PD-1 therapy. These data indicate the potential for broad utility of RP3 in a range of tumor types, including in tumors that are not sensitive to PD-1 blockade. Citation Format: Praveen K. Bommareddy, Kevin Harrington, Victoria Roulstone, Joan Kyula, Aaron Clack, Linta Kuncheria, Sylwia Jones, Robert Coffin. Preclinical characterization and clinical biomarker studies with RP3, a novel oncolytic immunotherapy expressing a fusogenic protein, a human anti-CTLA-4 antibody, hCD40L and h4-1BBL. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6639.

Abstract 890: ADAM17 knockout NK or CAR NK cells augment antibody dependent cellular cytotoxicity (ADCC) and anti-tumor activity

Abstract Natural killer (NK) cells are a critical component of the innate immune system and do not elicit graft-versus-host disease, making them an attractive source of ‘off-the-shelf’ allogeneic cell therapy. NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Another important anti-tumor role of NK cells is via antibody-dependent cell mediated cytotoxicity (ADCC). The process is mediated by CD16a of NK cells, which binds the Fc portion of antibodies. After NK cell activation, the CD16a ectodomain is rapidly cleaved from the NK cell surface by a disintegrin, Metalloprotease-17 (ADAM17), that is produced by both tumor cells and activated NK cells. This dampens NK cell activity by preventing NK cell attachment to antibody-coated target cells and diminishes CD16a signaling. Inhibiting ADAM17-mediated CD16a cleavage leads to augmented ADCC activity and increased cytokine production by human NK cells. Other NK cell activating receptors are known to be regulated by a similar mechanism1. In this study, we have utilized CRISPR-Cas9 ribonucleoproteins (RNPs) to disrupt the ADAM17 gene in isolated peripheral blood NK cells from healthy donors. FACS and Amplicon NGS Sequencing data confirmed successful disruption of the gene. Edited NK cells were expanded using IL-2 and stimulation with NKSTIM, a modified K562 stimulatory cell line expressing a membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation. Furthermore, we transduced ADAM17 edited NK cells to express a CD70 CAR2 or CD19 CAR3 construct and mbIL-15. CAR expression was assessed by FACS. In vitro cytotoxicity was measured by IncuCyte. Using an artificial activation system (phorbol 12-myristate 13-acetate [PMA]), we showed that ADAM17 KO NK cells maintain dramatically higher surface expression of CD16a and CD62L than control NK cells. In IncuCyte cytotoxicity assays against Raji tumor cells in the presence of rituximab (anti-CD20), ADAM17 KO NK cells and ADAM17 KO CD19 CAR NK cells demonstrated higher cytotoxicity compared to control NK cells. Similarly, ADAM17 KO NK cells and ADAM17 KO CD70 CAR NK cells also showed enhanced cytotoxicity against 786-O tumor cells in the presence of anti-EGFR antibody, cetuximab. Furthermore, ADAM17/CISH/CBLB triple KO CD70 CAR NK cells exhibited enhanced antitumor efficacy in vivo in a 786-O RCC tumor and a HL60 AML xenograft model. Taken together, we show ADAM17 KO NK or ADAM17 KO CAR NK cells demonstrate improved ADCC and ADAM17 KO can enhance in vivo anti-tumor activity of CISH/CBLB KO CD70 CAR NK cells against relevant tumor models. These data support the further exploration of ADAM17 gene knockout NK cells for clinical application and to improve efficacy of therapeutic antibodies in combination with adoptive transfer of engineered NK cells. Citation Format: Chao Guo, Yanying Fan, Alexander Aronov, Nikki Kimura, Damie Juat, Jessica Sood, Ivan H. Chan, James B. Trager. ADAM17 knockout NK or CAR NK cells augment antibody dependent cellular cytotoxicity (ADCC) and anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 890.

SYNOVIAL FLUID NEUTROPHILS PLAY A ROLE IN THE PATHOPHYSIOLOGY OF KNEE OSTEOARTHRITIS: A PHENOTYPIC AND FUNCTIONAL STUDY

An increased number of neutrophils (NEUs) has long been associated with infections in the knee joints; their contribution to knee osteoarthritis (KOA) pathophysiology remains largely unexplored. This study aimed to compare the phenotypic and functional characteristics of synovial fluid (SF)-derived NEUs in KOA and knee infection (INF).Flow cytometric analysis, protein level measurements (ELISA), NEU oxidative burst assays, detection of NEU phagocytosis (pHrodoTM Green Zymosan BiparticlesTM Conjugate for Phagocytosis), morphological analysis of the SF-derived/synovial tissue NEUs, and cultivation of human umbilical vein endothelial cells (HUVECs) using SF supernatant were used to characterise NEUs functionally/morphologically.Results: Compared with INF NEUs, KOA NEUs were characterised by a lower expression of CD11b, CD54 and CD64, a higher expression of CD62L, TLR2 and TLR4, and lower production of inflammatory mediators and proteases, except CCL2.Functionally, KOA NEUs displayed an increased production of radical oxygen species and phagocytic activity compared with INF NEUs. Morphologically, KOA and INF cells displayed different cell sizes and morphology, histological characteristics of the surrounding synovial tissues and influence on endothelial cells. KOA NEUs were further subdivided into two groups: SF containing <10% and SF with 10%–60% of NEUs. Analyses of two KOA NEU subgroups revealed that NEUs with SF <10% were characterised by 1) higher CD54, CD64, TLR2 and TLR4 expression on their surface; 2) higher concentrations of TNF-α, sTREM-1, VILIP-1, IL-1RA and MMP-9 in SFs.Our findings reveal a key role for NEUs in the pathophysiology of KOA, indicating that these cells are morphologically and functionally different from INF NEUs. Further studies should explore the mechanisms that contribute to the increased number of NEUs and their crosstalk with other immune cells in KOA.This study was supported by the Ministry of Health of the Czech Republic (NU20-06-00269; NU21-06-00370).

Characterization and CRISPR/Cas9-mediated genetic manipulation of neutrophils derived from Hoxb8-ER-immortalized myeloid progenitors.

Neutrophils represent a first line of defense against a wide variety of microbial pathogens. Transduction with an estrogen receptor-Hoxb8 transcription factor fusion construct conditionally immortalizes myeloid progenitor cells (NeutPro) capable of differentiation into neutrophils. This system has been very useful for generating large numbers of murine neutrophils for in vitro and in vivo studies. However, some questions remain as to how closely neutrophils derived from these immortalized progenitors reflect primary neutrophils. Here we describe our experience with NeutPro-derived neutrophils as it relates to our studies of Yersinia pestis pathogenesis. NeutPro neutrophils have circular or multilobed nuclei, similar to primary bone marrow neutrophils. Differentiation of neutrophils from NeutPro cells leads to increased expression of CD11b, GR1, CD62L, and Ly6G. However, the NeutPro neutrophils expressed lower levels of Ly6G than bone marrow neutrophils. NeutPro neutrophils produced reactive oxygen species at slightly lower levels than bone marrow neutrophils, and the 2 cell types phagocytosed and killed Y. pestis in vitro to a similar degree. To further demonstrate their utility, we used a nonviral method for nuclear delivery of CRISPR/Cas9 guide RNA complexes to delete genes of interest in NeutPro cells. In summary, we have found these cells to be morphologically and functionally equivalent to primary neutrophils and useful for in vitro assays related to studies of bacterial pathogenesis.

Radiographic airway abnormalities in untreated early rheumatoid arthritis are associated with peripheral neutrophil activation

BackgroundThe role of the lung for the initiation and progression of rheumatoid arthritis (RA) is still unclear. Up to 10% of RA patients develop interstitial lung disease which remains a clinical challenge. Understanding early disease mechanisms is of great importance. The objective of this study was to determine whether there is an association between peripheral neutrophil phenotypes and presence of pulmonary abnormalities (PA) on chest high-resolution computed tomography (HRCT) in untreated early RA (ueRA).MethodsClinical data and blood were collected, and HRCT performed at diagnosis on 30 consecutive anti-citrullinated protein antibody (ACPA) and/or rheumatoid factor (RF) positive ueRA patients. HRCTs were evaluated for the presence of RA-associated parenchymal, airway and/or pleural abnormalities. Expression of phenotype markers on neutrophils were determined by flow cytometry. Levels of calprotectin, ACPA and RF were measured using immunoassays.ResultsThe frequency of having any PA was 60%. Airway abnormalities were present in 50%, parenchymal nodules in 43% and interstitial lung abnormalities (ILA) in 10%. Unsupervised multivariate data analysis showed clustering of any PA with neutrophil activation, parameters of inflammation and RF titres. In univariate analysis, the patients with PA displayed significantly increased CD11b and decreased CD62L expression on neutrophils (1.2-fold, p = 0.014; 0.8-fold, p = 0.012) indicating activation and significantly increased RF IgM titre and CRP (5.7-fold, p = 0.0025; 2.3-fold, p = 0.0035) as compared to no PA. Titres of RF, but not ACPA, correlated with expression of the neutrophil activation marker CD11b. A stratified analysis demonstrated that airway involvement was the PA subtype with the strongest association with neutrophil activation.ConclusionWe report a strong association between radiographic airway findings and activation of circulating neutrophils in early RA supporting a role of innate immunity and the lung at disease onset. Our results also indicate different contributions of RF and ACPA in the RA pathogenesis.

PD-1/PD-L1 Control of Antigen-Specifically Activated CD4 T-Cells of Neonates

Newborns are highly susceptible to infections; however, the underlying mechanisms that regulate the anti-microbial T-helper cells shortly after birth remain incompletely understood. To address neonatal antigen-specific human T-cell responses against bacteria, Staphylococcus aureus (S. aureus) was used as a model pathogen and comparatively analyzed in terms of the polyclonal staphylococcal enterotoxin B (SEB) superantigen responses. Here, we report that neonatal CD4 T-cells perform activation-induced events upon S. aureus/APC-encounter including the expression of CD40L and PD-1, as well as the production of Th1 cytokines, concomitant to T-cell proliferation. The application of a multiple regression analysis revealed that the proliferation of neonatal T-helper cells was determined by sex, IL-2 receptor expression and the impact of the PD-1/PD-L1 blockade. Indeed, the treatment of S. aureus-activated neonatal T-helper cells with PD-1 and PD-L1 blocking antibodies revealed the specific regulation of the immediate neonatal T-cell responses with respect to the proliferation and frequencies of IFNγ producers, which resembled in part the response of adults' memory T-cells. Intriguingly, the generation of multifunctional T-helper cells was regulated by the PD-1/PD-L1 axis exclusively in the neonatal CD4 T-cell lineage. Together, albeit missing memory T-cells in neonates, their unexperienced CD4 T-cells are well adapted to mount immediate and strong anti-bacterial responses that are tightly controlled by the PD-1/PD-L1 axis, thereby resembling the regulation of recalled memory T-cells of adults.

Changes in Adhesion and the Expression of Adhesion Molecules in PBMCs after Aneurysmal Subarachnoid Hemorrhage: Relation to Cerebral Vasospasm

Aneurysmal subarachnoid hemorrhage (aSAH) is a neurovascular disease produced by extravasation of blood to the subarachnoid space after rupture of the cerebral vessels. After bleeding, the immune response is activated. The role of peripheral blood mononuclear cells (PBMCs) in this response is a current subject of research. We have analysed the changes in PBMCs of patients with aSAH and their interaction with the endothelium, focusing on their adhesion and the expression of adhesion molecules. Using an in vitro adhesion assay, we observed that the adhesion of PBMCs of patients with aSAH is increased. Flow cytometry analysis shows that monocytes increased significantly in patients, especially in those who developed vasospasm (VSP). In aSAH patients, the expression of CD162, CD49d, CD62L and CD11a in T lymphocytes and of CD62L in monocytes increased. However, the expression of CD162, CD43, and CD11a decreased in monocytes. Furthermore, monocytes from patients who developed arteriographic VSP had lower expression of CD62L. In conclusion, our results confirm that after aSAH, monocyte count and adhesion of PBMCs increase, especially in patients with VSP, and that the expression of several adhesion molecules is altered. These observations can help predict VSP and to improve the treatment of this pathology.

Characteristics of immunological events in Epstein-Barr virus infection in children with infectious mononucleosis.

Epstein-Barr virus (EBV) infection occurs commonly in children and may cause acute infectious mononucleosis (AIM) and various malignant diseases. Host immune responses are key players in the resistance to EBV infection. We here assessed the immunological events and laboratory indicators of EBV infection, as well as determined the clinical usefulness of evaluating the severity and efficacy of antiviral therapy in AIM patients. We enrolled 88 children with EBV infection. The immune environment was defined by immunological events such as frequencies of lymphocyte subsets, phenotypes of T cells, and their ability to secrete cytokines, and so on. This environment was analyzed in EBV-infected children with different viral loads and in children in different phases of infectious mononucleosis (IM) from disease onset to convalescence. Children with AIM had higher frequencies of CD3+ T and CD8+ T cells, but lower frequencies of CD4+ T cells and CD19+ B cells. In these children, the expression of CD62L was lower and that of CTLA-4 and PD-1 was higher on T cells. EBV exposure induced granzyme B expression, but reduced IFN-γ secretion, by CD8+ T cells, whereas NK cells exhibited reduced granzyme B expression and increased IFN-γ secretion. The frequency of CD8+ T cells was positively correlated with the EBV DNA load, whereas the frequencies of CD4+ T cells and B cells were negatively correlated. During the convalescent phase of IM, CD8+ T cell frequency and CD62L expression on T cells were restored. Moreover, patient serum levels of IL-4, IL-6, IL-10, and IFN-γ were considerably lower throughout the convalescent phase than throughout the acute phase. Robust expansion of CD8+ T cells, accompanied by CD62L downregulation, PD-1 and CTLA-4 upregulation on T cells, enhanced granzyme B production, and impaired IFN-γ secretion, is a typical characteristic of immunological events in children with AIM. Noncytolytic and cytolytic effector functions of CD8+ T cells are regulated in an oscillatory manner. Furthermore, the AST level, number of CD8+ T cells, and CD62L expression on T cells may act as markers related to IM severity and the effectiveness of antiviral treatment.

Repeated injection of multivitamins and multiminerals during the transition period enhances immune response by suppressing inflammation and oxidative stress in cows and their calves.

Periparturient dairy cows undergo major physiological and metabolic changes as well as immunosuppression, associated with decrease in plasma concentrations of various minerals and vitamins. The present study was conducted to investigate effects of repeated injections of vitamins and minerals on oxidative stress, innate and adaptive immune response in periparturient dairy cows and their offspring. Experiment was carried out on 24 peripartum Karan-Fries cows, randomly divided into four groups (n=6): control, Multi-mineral (MM), Multi-vitamin (MV) and Multi-minerals and Multi-vitamin (MMMV). Five ml of MM (Zinc 40 mg/ml, Manganese 10 mg/ml, Copper 15 mg/ml, Selenium 5 mg/ml) and five ml of MV (Vitamin E 5 mg/ml, Vitamin A 1000 IU/ml, B-Complex 5 mg/ml, and Vitamin D3 500 IU/ml) were injected intramuscularly (IM) to the MM and MV groups. MMMV group cows were injected with both. In all treatment groups, injections and blood sampling were carried out on 30th, 15th, 7th days before and after expected date of parturition and at calving. In calves, blood was collected at calving and on 1, 2, 3, 4, 7, 8, 15, 30 and 45 days post-calving. Colostrum/milk were collected at calving and at days 2, 4, and 8 post-calving. A lower percentage of total neutrophils and immature neutrophils, higher percentage of lymphocytes together with increased phagocytic activity of neutrophils and proliferative capacity of lymphocytes found in blood of MMMV cows/calves. Lower relative mRNA expression of TLRs and CXCRs and higher mRNA expression of GR-α, CD62L, CD11b, CD25 and CD44 found in blood neutrophils of MMMV groups. Total antioxidant capacity was higher, activity of antioxidant enzymes (SOD and CAT), TBARS levels were lower in the blood plasma of treated cows/calves. In both cows/calves, plasma pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-17A, IFN-γ and TNF-α) increased, whereas anti-inflammatory cytokines (IL-4 and IL-10) decreased in MMMV groups. Total immunoglobulins increased in colostrum/milk of MMMV injected cows and plasma of their calves. Results indicate that repeated injections of multivitamins and multiminerals to peripartum dairy cows could be a major strategy to improve immune response and decrease in inflammation and oxidative stress in transition dairy cows and their calves.

Trans-epithelial migration is essential for neutrophil activation during RSV infection.

The recruitment of neutrophils to the infected airway occurs early following respiratory syncytial virus (RSV) infection, and high numbers of activated neutrophils in the airway and blood are associated with the development of severe disease. The aim of this study was to investigate whether trans-epithelial migration is sufficient and necessary for neutrophil activation during RSV infection. Here, we used flow cytometry and novel live-cell fluorescent microscopy to track neutrophil movement during trans-epithelial migration and measure the expression of key activation markers in a human model of RSV infection. We found that when migration occurred, neutrophil expression of CD11b, CD62L, CD64, NE, and MPO increased. However, the same increase did not occur on basolateral neutrophils when neutrophils were prevented from migrating, suggesting that activated neutrophils reverse migrate from the airway to the bloodstream side, as has been suggested by clinical observations. We then combined our findings with the temporal and spatial profiling and suggest 3 initial phases of neutrophil recruitment and behavior in the airways during RSV infection; (1) initial chemotaxis; (2) neutrophil activation and reverse migration; and (3) amplified chemotaxis and clustering, all of which occur within 20 min. This work and the novel outputs could be used to develop therapeutics and provide new insight into how neutrophil activation and a dysregulated neutrophil response to RSV mediates disease severity.

Small molecules targeting the NADH-binding pocket of VDAC enhance T cell cytotoxicity and selectively inhibit cancer cell proliferation.

Adoptive cell therapy involves administration of cytotoxic immune cells. Upon activation of naïve T cells, CD8+ (cytotoxic) T cells increase both mitochondrial metabolism and glycolysis. Upregulated mitochondrial metabolism is essential for the activation of effector T cells. Voltage dependent anion channels (VDAC 1/2/3) regulate the flux of most metabolites entering and exiting mitochondria. VDAC opening increases mitochondrial metabolism. NADH binding to a VDAC pocket, conserved in all isoforms, closes the channel. The small molecules SC01 and SC18 bind to the VDAC-NADH binding pocket. We hypothesize that SC01 and SC18 stabilize VDAC in an open state enhancing T cell oxidative metabolism, survival and cytotoxicity. We used melanoma epitope gp100 reactive mouse splenic CD8+ T cells, HepG2 and SNU-449 human hepatocarcinoma cancer cells, and B16 mouse melanoma cells. We determined T cell activation (CD25 expression), proliferation (Ki67), cytokine secretion (IFNγ, TNFα), cytolytic activity (granzyme-B), and expression of memory markers CD44 and CD62L, by FACS. We assessed mitochondrial membrane potential (ΔΨm) and NADH by confocal microscopy. Cell proliferation and cell death were determined by cell count and propidium iodide uptake, respectively. VDAC isoform distribution in T cells was ∼54%, 24% and 22% for VDAC 1, 2 and 3, respectively. SC18 moderately decreased T cell ΔΨm and NADH. SC01 and SC18 did not affect proliferation or activation of splenic CD8+ T cells exposed to cognate antigen (gp100). By contrast, SC18 inhibited cell proliferation in SNU-449, HepG2, and B16 cells. SC18 also increased the secretion of IFNγ, TNF-α, and granzyme-B. Memory markers determined by CD44 and CD62L co-expression were unchanged. These data suggest a two-sided antitumor property of SC18, that enhances T cell cytotoxicity and selectively inhibits cancer cell proliferation.

Lung-resident CD69+ST2+ TH2 cells mediate long-term type 2 memory to inhaled antigen in mice

Chronic airway diseases such as asthma are characterized by persistent type 2 immunity in the airways. We know little about the mechanisms that explain why type 2 inflammation continues in these diseases. We used mouse models to investigate the mechanisms involved in long-lasting immune memory. Naive mice were exposed intranasally to ovalbumin (OVA) antigen with Alternaria extract as an adjuvant. Type 2 memory was analyzed by parabiosis model, flow cytometry with invivo antibody labeling, and intranasal OVA recall challenge. Gene-deficient mice were used to analyze the mechanisms. In the parabiosis model, mice previously exposed intranasally to OVA with Alternaria showed more robust antigen-specific immune responses and airway inflammation than mice with circulating OVA-specific T cells. After a single airway exposure to OVA with Alternaria, CD69+ST2+ TH2-type T cells, which highly express type 2 cytokine messenger RNA and lack CD62L expression, appeared in lung tissue within 5 days and persisted for at least 84 days. When exposed again to OVA invivo, these cells produced type 2 cytokines quickly without involving circulating T cells. Development of tissue-resident CD69+ST2+ TH2 cells and long-term memory to an inhaled antigen were abrogated in mice deficient in ST2 or IL-33, but not TSLP receptor. CD69+ST2+ TH2 memory cells develop quickly in lung tissue after initial allergen exposure and persist for a prolonged period. The ST2/IL-33 pathway may play a role in the development of immune memory in lung to certain allergens.

Platelet count, temperature and pH value differentially affect hemostatic and immunomodulatory functions of platelets

Platelets are primarily recognized for their role in hemostasis, but also regulate immune responses by interacting with leukocytes. Their highly sensitive nature enables platelets to rapidly respond to micro-environmental changes, which is crucial under physiological condition but can jeopardize in vitro analyses. Thus, we tested how platelet count and changes in pH and temperatures, which are commonly experienced during inflammation and infection but also affected by ex vivo analyses, influence platelet-leukocyte interaction and immunomodulation.Reducing platelet count by up to 90 % slightly decreased platelet activation and platelet-leukocyte aggregate formation, but did not affect CD11b activation nor CD62L shedding of monocytes or neutrophils. Acidosis (pH 6.9) slightly elevated platelet degranulation and binding to innate leukocytes, though pH changes did not modulate leukocyte activation. While platelet responsiveness was higher at room temperature than at 37 °C, incubation temperature did not affect platelet-leukocyte aggregate formation. In contrast, platelet-mediated CD11b activation and CD62L expression increased with temperature.Our data thus demonstrate the importance of standardized protocols for sample preparation and assay procedure to obtain comparable data. Further, unspecific physiologic responses such as thrombocytopenia, acidosis or temperature changes may contribute to platelet dysfunction and altered platelet-mediated immunomodulation in inflammatory and infectious disease.

Activation-induced marker assays for identification of Trypanosoma cruzi-specific CD4 or CD8 T cells in chronic Chagas disease patients.

Antigen-specific T cells are central to the adaptive immune response against T. cruzi infection and underpin the efficacy of on-going vaccine strategies. In this context, the present study focuses on T-cell assays that define the parasite-specificity on the basis of upregulation of TCR stimulation-induced surface markers. For this purpose, we tested different dual marker combinations (OX40, CD25, CD40L, CD137, CD69, PD-L1, CD11a, CD49d, HLA-DR, CD38) to reliably identify activated CD4+ and CD8+ T-cell populations from PBMCs of chronic Chagas disease (CCD) patients after 12 or 24 h of stimulation with T. cruzi lysate. Results demonstrated that activation-induced markers (AIM) assays combining the expression of OX40, CD25, CD40L, CD137, CD69 and/or PD-L1 surface markers are efficient at detecting T. cruzi-specific CD4+ T cells in CCD patients, in comparison to non-infected donors, after both stimulation times. For CD8+ T cells, only PD-L1/OX40 after 24 h of antigen exposure resulted to be useful to track a parasite-specific response. We also demonstrated that the agnostic activation is mediated by different T. cruzi strains, such as Dm28c, CL Brener or Sylvio. Additionally, we successfully used this approach to identify the phenotype of activated T lymphocytes based on the expression of CD45RA and CCR7. Overall, our results show that different combinations of AIM markers represent an effective and simple tool for the detection of T. cruzi-specific CD4+ and CD8+ T cells.