Immune Function Assay Research Articles

Leveraging Covalency to Stabilize Ternary Complex Formation For Cell-Cell "Induced Proximity".

Recent advances in the field of translational chemical biology use diverse "proximity-inducing" synthetic modalities to elicit new modes of "event driven" pharmacology. These include mechanisms of targeted protein degradation and immune clearance of pathogenic cells. Heterobifunctional "chimeric" compounds like Proteolysis TArgeting Chimeras (PROTACs) and Antibody Recruiting Molecules (ARMs) leverage these mechanisms, respectively. Both systems function through the formation of reversible "ternary" or higher-order biomolecular complexes. Critical to function are key parameters, such as bifunctional molecule affinity for endogenous proteins, target residence time, and turnover. To probe the mechanism and enhance function, covalent chemical approaches have been developed to kinetically stabilize ternary complexes. These include electrophilic PROTACs and Covalent Immune Recruiters (CIRs), the latter designed to uniquely enforce cell-cell induced proximity. Inducing cell-cell proximity is associated with key challenges arising from a combination of steric and/or mechanical based destabilizing forces on the ternary complex. These factors can attenuate the formation of ternary complexes driven by high affinity bifunctional/proximity inducing molecules. This Account describes initial efforts in our lab to address these challenges using the CIR strategy in antibody recruitment or receptor engineered T cell model systems of cell-cell induced proximity. ARMs form ternary complexes with serum antibodies and surface protein antigens on tumor cells that subsequently engage immune cells via Fc receptors. Binding and clustering of Fc receptors trigger immune cell killing of the tumor cell. We applied the CIR strategy to convert ARMs to covalent chimeras, which "irreversibly" recruit serum antibodies to tumor cells. These covalent chimeras leverage electrophile preorganization and kinetic effective molarity to achieve fast and selective covalent engagement of the target ternary complex protein, e.g., serum antibody. Importantly, covalent engagement can proceed via diverse binding site amino acids beyond cysteine. Covalent chimeras demonstrated striking functional enhancements compared to noncovalent ARM analogs in functional immune assays. We revealed this enhancement was in fact due to the increased kinetic stability and not concentration, of ternary complexes. This finding was recapitulated using analogous CIR modalities that integrate peptidic or carbohydrate binding ligands with Sulfur(VI) Fluoride Exchange (SuFEx) electrophiles to induce cell-cell proximity. Mechanistic studies in a distinct model system that uses T cells engineered with receptors that recognize covalent chimeras or ARMs, revealed covalent receptor engagement uniquely enforces downstream activation signaling. Finally, this Account discusses potential challenges and future directions for adapting and optimizing covalent chimeric/bifunctional molecules for diverse applications in cell-cell induced proximity.

Use of a platform with lens-free shadow imaging technology to monitor natural killer cell activity

Natural killer (NK) cells are a crucial component of the innate immune system. This study introduces Cellytics NK, a novel platform for rapid and precise measurement of NK cell activity. This platform combines an NK-specific activation stimulator cocktail (ASC) and lens-free shadow imaging technology (LSIT), using optoelectronic components. LSIT captures digital hologram images of resting and ASC-activated NK cells, while an algorithm evaluates cell size and cytoplasmic complexity using shadow parameters. The combined shadow parameter derived from the peak-to-peak distance and width standard deviation rapidly distinguishes active NK cells from inactive NK cells at the single-cell level within 30 s. Here, the feasibility of the system was demonstrated by assessing NK cells from healthy donors and immunocompromised cancer patients, demonstrating a significant difference in the innate immunity index (I3). Cancer patients showed a lower I3 value (161%) than healthy donors (326%). I3 was strongly correlated with NK cell activity measured using various markers such as interferon-gamma, tumor necrosis factor-alpha, perforin, granzyme B, and CD107a. This technology holds promise for advancing immune functional assays, offering rapid and accurate on-site analysis of NK cells, a crucial innate immune cell, with its compact and cost-effective optoelectronic setup, especially in the post-COVID-19 era.

SAT-017 QuantiFERON monitor as an immune function assay predictive of clinical events in liver transplant recipients: a validation study

SAT-017 QuantiFERON monitor as an immune function assay predictive of clinical events in liver transplant recipients: a validation study

Clinical implications of innate immune exhaustion in cystic fibrosis.

Lung disease progression in people with cystic fibrosis (pwCF) varies from one individual to another. Different immunological characteristics have been suggested to explain this variation, and we hypothesised that lung capacity may be associated with the innate immune response in pwCF. In an exploratory study, we aimed to investigate potential links between the innate immune response and lung function in pwCF using the standardised immune function assay TruCulture. In a single-centre study with combined cross-sectional and longitudinal data before and after intravenous antibiotics, blood was sampled from Pseudomonas aeruginosa-infected pwCF. Whole blood was analysed by TruCulture to reveal the unstimulated and stimulated cytokine release. Tobit regressions and Spearman's correlations were used to estimate the associations between lung function and cytokine release. We included 52 pwCF in the cross-sectional study and 24 in the longitudinal study. In the cross-sectional study, we found that compared to a healthy population, the release of toll-like receptor (TLR)3, TLR4- and TLR7/8-stimulated interferon-γ, and interleukin (IL)-12p40 was reduced. Although TLR3-stimulated IL-1β and IL-6 release increased with lung function, overall, cytokine release did not correlate well with lung function. In the longitudinal study, the cytokine release was modified by antibiotic treatment, but the cytokine release before antibiotic treatment did not associate with changes in lung function after treatment. The stimulated cytokine release could not predict lung function levels or changes in pwCF, but our data indicate that pwCF experience exhaustion in the innate immune response after years of chronic bacterial infection.

(626) - Immune Cell Function Assay as a Monitoring Tool While on Augmented or Unconventional Immunosuppression

(626) - Immune Cell Function Assay as a Monitoring Tool While on Augmented or Unconventional Immunosuppression

Abstract 2366: Balancing efficacy and toxicity of a fibroblast activation protein dependent tetravalent CD40 agonist antibody for cancer therapy

Abstract Background: CD40 agonist antibodies have been found to augment tumor antigen presentation and have shown potential as anti-tumor treatments in human clinical trials. The clinical success of CD40 agonist antibodies has been restricted due to their limited efficacy and on-target, off-tumor toxicity. We hypothesized that toxicity associated with CD40 agonist antibodies could be overcome by activating CD40 specifically through tumor-specific antigens. Additionally, limited efficacy can be improved by rational design of CD40 bispecific antibodies (BsAbs). Herein, we present FAPxCD40, a trivalent CD40 agonist BsAb targeting fibroblast activation protein (FAP) possessing an optimized therapeutic window. Method: We generated a series of FAPxCD40 bispecific antibodies with 2, 4, and 6 CD40 valences by using a high-affinity anti-FAP antibody and a crosslink-dependent CD40 agonist VHH antibody. These BsAbs were evaluated using multiple immune cell functional assays, including assays for dendritic cell (DC) maturation, B cell maturation, and CMV-reactive T cell recall. The in vivo therapeutic window of the FAPxCD40 bispecifics was compared in CD40 humanized mice with subcutaneously transplanted FAP-positive MC38 tumor xenografts. Results: We developed FAPxCD40 BsAbs with varying valences, including bivalent FAPxCD40-2, tetravalent FAPxCD40-4, and hexavalent FAPxCD40-6. FAPxCD40-2 showed strict FAP-dependent CD40 agonist activity in both DC and B cell maturation assays, while its CD40 agonist activity upon FAP-crosslinking was modest. FAPxCD40-4 exhibited efficient CD40 agonist activity upon FAP-crosslinking, but however, it also exhibited some undesired FAP-independent CD40 agonist activity. FAPxCD40-6 efficiently clustered CD40 but marginally benefited from FAP binding, making it unsuitable as a choice. In the humanized CD40 mice model, FAPxCD40-2 effectively eliminated systemic toxicity at all tested doses. Its antitumor activity, however, is modest with TGI (tumor growth inhibition) rate of 32.8 % at a dose of 6.96 mpk. Further escalation of the FAPxCD40-2 dosage to 13.92 mpk did not result in enhanced antitumor activity with TGI rate of 46.9 %, indicating that its CD40 agonist activity may have reached a plateau. In contrast, FAPxCD40-4 broke this plateau and induce a promising antitumor response at a dose of 1.3 mpk, resulting in a 92% TGI rate. Furthermore, no liver enzyme upregulation or platelet count decrease was observed under these conditions, indicating an ideal therapeutic window. In addition, during nonclinical toxicology studies conducted on cynomolgus monkeys, it was found that doses of FAPxCD40-4 up to 99 mg/kg administered weekly for a total of four doses were well-tolerated. Conclusion: Our study demonstrates that FAPxCD40-4, a tetravalent FAPxCD40 BsAb, exhibits efficient CD40 agonist activity with an optimized therapeutic window. Citation Format: Simeng Chen, Yuan Lin, Xiaoru Zhou, Xing Sun, Changyong Yang, Cheng Liao. Balancing efficacy and toxicity of a fibroblast activation protein dependent tetravalent CD40 agonist antibody for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2366.

The changing landscape of infections in the lung transplant recipient.

Infections in lung transplant recipients remain a major challenge and can affect lung allograft function and cause significant morbidity and mortality. New strategies for the prevention and treatment of infection in lung transplantation have emerged and are reviewed. For important vaccine preventable infections (VPIs), guidance has been updated for at risk solid organ transplant (SOT) recipients. However, data on the efficacy of newer vaccines in lung transplant, including the respiratory syncytial virus (RSV) vaccine, are limited. Studies demonstrate improved vaccination rate with Infectious Diseases consultation during pretransplant evaluation. Two new antiviral agents for the treatment and prevention of cytomegalovirus (CMV) in SOT, letermovir and maribavir, are being incorporated into clinical care. CMV-specific cell-mediated immune function assays are more widely available. Antibiotics for the management of multidrug resistant pathogens and Burkholderia cepacia complex have been described in case series and case reports in lung transplant. Although new vaccines and novel therapies for preventing and treating infections are available, larger studies evaluating efficacy in lung transplant recipients are needed.

Identification of a shared gene signature and biological mechanism between diabetic foot ulcers and cutaneous lupus erythemnatosus by transcriptomic analysis.

Diabetic foot ulcers (DFU) and cutaneous lupus erythematosus (CLE) are both diseases that can seriously affect a patient's quality of life and generate economic pressure in society. Symptomatically, both DLU and CLE exhibit delayed healing and excessive inflammation; however, there is little evidence to support a molecular and cellular connection between these two diseases. In this study, we investigated potential common characteristics between DFU and CLE at the molecular level to provide new insights into skin diseases and regeneration, and identify potential targets for the development of new therapies. The gene expression profiles of DFU and CLE were obtained from the Gene Expression Omnibus (GEO) database and used for analysis. A total of 41 common differentially expressed genes (DEGs), 16 upregulated genes and 25 downregulated genes, were identified between DFU and CLE. GO and KEGG analysis showed that abnormalities in epidermal cells and the activation of inflammatory factors were both involved in the occurrence and development of DFU and CLE. Protein-protein interaction network (PPI) and sub-module analysis identified enrichment in seven common key genes which is KRT16, S100A7, KRT77, OASL, S100A9, EPGN and SAMD9. Based on these seven key genes, we further identified five miRNAs(has-mir-532-5p, has-mir-324-3p,has-mir-106a-5p,has-mir-20a-5p,has-mir-93-5p) and7 transcription factors including CEBPA, CEBPB, GLI1, EP30D, JUN,SP1, NFE2L2 as potential upstream molecules. Functional immune infiltration assays showed that these genes were related to immune cells. The CIBERSORT algorithm and Pearson method were used to determine the correlations between key genes and immune cells, and reverse key gene-immune cell correlations were found between DFU and CLE. Finally, the DGIbd database demonstrated that Paquinimod and Tasquinimod could be used to target S100A9 and Ribavirin could be used to target OASL. Our findings highlight common gene expression characteristics and signaling pathways between DFU and CLE, indicating a close association between these two diseases. This provides guidance for the development of targeted therapies and mutual interactions.

Immunotoxicity of stainless-steel nanoparticles obtained after 3D printing

This study aims to investigate the in vitro effects of nanoparticles (NPs) produced during the selective laser melting (SLM) of 316 L stainless steel metal powder on the immune response in a human blood model. Experimental data did not reveal effect on viability of 316 L NPs for the tested doses. Functional immune assays showed a significant immunosuppressive effect of NPs. There was moderate stimulation (117%) of monocyte phagocytic activity without significant changes in phagocytic activity and respiratory burst of granulocytes. A significant dose-dependent increase in the levels of the pro-inflammatory cytokine TNF-a was found in blood cultures treated with NPs. On the contrary, IL-8 chemokine levels were significantly suppressed. The levels of the pro-inflammatory cytokine IL-6 were reduced by only a single concentration of NPs. These new findings can minimise potential health risks and indicate the need for more research in this area.

Generation of Connexin-Expressing Stable Cell Pools.

Stable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressing a transgene of interest in cells under induction is a powerful way to (switch on and) study a gene function in both in vitro and in vivo assays. Taking advantage of the ability of lentivirus (LV) to promote transgene delivery, and genomic integration and expression in both dividing and nondividing cells, a doxycycline-inducible transfer vector expressing a bicistronic transgene was developed to study the function of connexins in HeLa DH cells. Here, delving on connexin 32 (Cx32), we report how to use the backbone of this vector as a tool to generate stable pools to study the function of a gene of interest (GOI), especially with assays involving Ca2+ imaging, employing the GCaMP6s indicator. We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vivo functional assays, including molecular, immune, and optical assays.

Development of a c-MET x CD137 bispecific antibody for targeted immune agonism in cancer immunotherapy

BackgroundTargeting the costimulatory receptor CD137 has shown promise as a therapeutic approach for cancer immunotherapy, resulting in anti-tumor efficacy demonstrated in clinical trials. However, the initial CD137 agonistic antibodies, urelumab and utomilumab, faced challenges in clinical trials due to the liver toxicity or lack of efficacy, respectively. Concurrently, c-MET has been identified as a highly expressed tumor-associated antigen (TAA) in various solid and soft tumors. MethodsIn this study, we aimed to develop a bispecific antibody (BsAb) that targets both c-MET and CD137, optimizing the BsAb format and CD137 binder for efficient delivery of the CD137 agonist to the tumor microenvironment (TME). We employed a monovalent c-MET motif and a trimeric CD137 Variable Heavy domain of Heavy chain (VHH) for the BsAb design. ResultsOur results demonstrate that the c-MET x CD137 BsAb provides co-stimulation to T cells through cross-linking by c-MET-expressing tumor cells. Functional immune assays confirmed the enhanced efficacy and potency of the c-MET x CD137 BsAb, as indicated by activation of CD137 signaling, target cell killing, and cytokine release in various tumor cell lines. Furthermore, the combination of c-MET x CD137 BsAb with Pembrolizumab showed a dose-dependent enhancement of target-induced T cell cytokine release. ConclusionOverall, the c-MET x CD137 BsAb exhibits a promising developability profile as a tumor-targeted immune agonist by minimizing off-target effects while effectively delivering immune agonism. It has the potential to overcome resistance to anti-PD-(L)1 therapies.

A clinical trial of therapeutic vaccination in lymphoma with serial tumor sampling and single-cell analysis

AbstractIn situ vaccination (ISV) triggers an immune response to tumor-associated antigens at 1 tumor site, which can then tackle the disease throughout the body. Here, we report clinical and biological results of a phase 1/2 ISV trial in patients with low-grade lymphoma, combining an intratumoral toll-like receptor 9 (TLR9) agonist with local low-dose radiation and ibrutinib (an inhibitor of B- and T-cell kinases). Adverse events were predominately low grade. The overall response rate was 50%, including 1 complete response. All patients experienced tumor reduction at distant sites. Single-cell analyses of serial fine needle aspirates from injected and uninjected tumors revealed correlates of clinical response, such as lower CD47 and higher major histocompatibility complex class II expression on tumor cells, enhanced T-cell and natural killer cell effector function, and reduced immune suppression from transforming growth factor β and inhibitory T regulatory 1 cells. Although changes at the local injected site were more pronounced, changes at distant uninjected sites were more often associated with clinical responses. Functional immune response assays and tracking of T-cell receptor sequences provided evidence of treatment-induced tumor-specific T-cell responses. Induction of immune effectors and reversal of negative regulators were both important in producing clinically meaningful tumor responses. The trial was registered at www.clinicaltrials.gov as #NCT02927964.

Abstract 14306: Immune Function Assay by Race/Ethnicity Within the First Year After Heart Transplantation

Introduction: The immune function assay (IFA) is used in transplant recipients to measure the degree of lymphocyte-mediated cell immunity. Values <225 ng/mL are associated with an increased risk of infection and it is uncertain if race/ethnicity associates with differences in IFA values after heart transplantation. We tested the association between ethnicity/race with IFA results for heart transplant recipients for the first year after transplantation. Methods: A single-center cohort study of consecutive heart transplant recipients from 1/1/2014-8/31/2022 with an IFA obtained within 30-days prior to heart transplant and > 1 assay within the first year afterwards. We described the change of IFA in 90-day increments by self-reported race/ethnicity. Multivariable logistic regression models tested the association of patient-level clinical factors (age, sex, race, ethnicity, BMI, creatinine) with IFA values <225 ng/mL. Results: In the cohort, (121/315) (38%) had an IFA within 30-days prior to heart transplant. Most patients were white (46%), and male (74%). All groups had decreases in IFA value post-transplant. White non-Hispanic patients had changes of -64, -131, -143, and -107 in IFA, respectively. White Hispanic patients had changes of -117, -119, -58, and -72. Asian patients had changes of -250, -241, -102, and -260. Black patients had changes of -95, -87, -118, and -113. Black patients had the greatest prevalence of an immune assay <225 ng/mL during days 270-360 after transplantation. White non-Hispanic, White Hispanic, and Asian patients had the greatest prevalence of an IFA <225 ng/mL 90-180 days after transplantation. In our adjusted models, race was not associated with IFA values <225 ng/mL during any quarter within the first year after transplant (P >0.05). Conclusion: In this small study, non-white heart transplant patients on average had lower IFA values over the first-year post-transplant compared with white non-hispanic patients.

Abstract 14884: Variation of Immune Function Assay Within the First Year After Heart Transplantation

Introduction: The ImmuKnow® immune function assay (IFA) is used in heart transplant recipients to measure lymphocyte-mediated cell immunity via CD4 cell ATP production. Values <225 ng/mL are associated with increased infection risk; however, serial changes in the first year after heart transplant are not well reported. We evaluated IFA trends for heart transplant recipients over the first year after transplantation. Methods: This is a single-center cohort study of consecutive heart transplant recipients from 1/1/2014-8/31/2022 that had an IFA obtained within 30-days of transplant and >1 subsequent IFA within the first year after transplant. We described the relative and absolute change of mean IFA in 90-day increments through the first year after transplant, including sex specific differences. Multivariable logistic regression models tested the independent association of patient-level clinical characteristics factors with IFA values <225 ng/mL. Results: In the study cohort, (121/315) (38%) had an IFA within 30-days prior to transplant. Most patients were white non-Hispanic (46%) and male (74%). IFA values decreased from the pre-transplant baseline with mean absolute changes of 92 ng/mL (-24%), 120 ng/mL (-31%), 121 ng/mL (-32%), and 114 ng/mL (-28%) every 90 days after transplant, respectively (panel B). The highest prevalence of an IFA value <225 ng/mL occurred 90-180 days post-transplant in 55/103 (53%) patients. Female patients had a greater absolute and relative decrease in IFA values from baseline and each 90-day period (panel C). In exploratory analysis, at the time of transplant age >60 was associated with a lower risk of IFA values <225 during days 90-180 post-transplant (OR 0.24, p=0.02). A creatinine >1.4 mg/dL was associated with IFA values <225 during 90-180 (OR 3.15, p=0.02) and 180-270 (OR 3.44, p=0.03) days. Conclusion: Heart transplant recipients experienced ~25% reduction in IFA value from baseline in the first-year post-heart transplant.

308 Zinc Supplementation and Implant Abscess Effects on Growth Performance and Immune System of Growing Beef Steers

Abstract Zinc is beneficial in growth and immune system function in cattle. Angus-cross steers (n = 72; initial BW = 261 ± 14 kg) were used in this 58-day study to determine the effects of supplemental Zn on growth, trace mineral status, and circulating immune cell populations. On d 0, steers received a Component E-S with Tylan implant (Elanco Animal Health, Greenfield, IN) and were stratified by BW into twelve GrowSafe-equipped pens. Dietary treatments included: control (CON; no supplemental Zn), Zn100 (100 mg supplemental Zn/kg DM), and Zn150 (150 mg supplemental Zn/kg DM). Supplemental Zn was provided as Zn sulfate, and other trace minerals were supplemented at NASEM (2016) recommendations. Individual BW was recorded on days -1, 0, 12, 13, 27, 28, 56, and 57. Blood was collected from 9 steers per treatment on d 13 for immune cell population and function assays. Liver biopsies were conducted on 18 steers per treatment on d 14. On d 16, implant abscesses (ABSC) were evaluated, and all steers were treated with meloxicam (285 mg). Data were analyzed using the Mixed procedure of SAS (Cary, NC) with fixed effects of treatment and ABSC prevalence using steer as the experimental unit. Abscess prevalence among treatments was analyzed using Proc Glimmix. Sixty percent of steers had ABSC; ABSC prevalence was similar by treatment (P = 0.67). Steers without ABSC had greater (P = 0.04) ADG d 0 to 13 and tended to have greater (P = 0.09) ADG d 0 to 57. There was also a tendency (Trt × ABSC; P ≤ 0.09) for steers without ABSC to have greater ADG and G:F from d 14 to 27 in CON and Zn100 while within Zn150 steers without ABSC tended to have lesser ADG and G:F than ABSC steers. No other treatment by ABSC or treatment effects (P ≥ 0.14) for growth performance were observed. There were no treatment by ABSC effects for immune cell populations on d 13 (P ≥ 0.26). In Zn150 steers, activated natural killer and gamma delta T cells were increased (P ≤ 0.03) on d 13. Similarly, CD21+ MHC IIhi B cells tended to be increased in Zn150 steers (P = 0.08). Steers with ABSC had increased relative frequency of CD5+ and CD21+ B cells (P ≤ 0.05) and tended to have increased natural killer and CD21+ MHC IIhi B cells (P ≤ 0.09). In addition, steers without ABSC had greater liver Mn on d 14 than ABSC steers (P = 0.05). There were no other effects on liver trace mineral concentrations (P ≥ 0.17). These observations suggest Zn supplementation has greater impact on the cellular immune system, while ABSC have a greater effect on B cells and the adaptive immune system.

Hyper-inflammatory profile and immunoparalysis in patients with severe Legionnaires' disease.

Severe Legionnaires' disease (LD) can lead to multi-organ failure or death in 10%-30% of patients. Although hyper-inflammation and immunoparalysis are well described in sepsis and are associated with high disease severity, little is known about the immune response in LD. This study aimed to evaluate the immune status of patients with LD and its association with disease severity. A total of 92 hospitalized LD patients were included; 19 plasmatic cytokines and pulmonary Legionella DNA load were measured in 84 patients on the day of inclusion (day 0, D0). Immune functional assays (IFAs) were performed from whole blood samples collected at D2 and stimulated with concanavalin A [conA, n = 19 patients and n = 21 healthy volunteers (HV)] or lipopolysaccharide (LPS, n = 14 patients and n = 9 HV). A total of 19 cytokines (conA stimulation) and TNF-α (LPS stimulation) were quantified from the supernatants. The Sequential Organ Failure Assessment (SOFA) severity score was recorded at D0 and the mechanical ventilation (MV) status was recorded at D0 and D8. Among the 84 patients, a higher secretion of plasmatic MCP-1, MIP1-β, IL-6, IL-8, IFN-γ, TNF-α, and IL-17 was observed in the patients with D0 and D8 MV. Multiparametric analysis showed that these seven cytokines were positively associated with the SOFA score. Upon conA stimulation, LD patients had a lower secretion capacity for 16 of the 19 quantified cytokines and a higher release of IL-18 and MCP-1 compared to HV. IL-18 secretion was higher in D0 and D8 MV patients. TNF-α secretion, measured after ex vivo LPS stimulation, was significantly reduced in LD patients and was associated with D8 MV status. The present findings describe a hyper-inflammatory phase at the initial phase of Legionella pneumonia that is more pronounced in patients with severe LD. These patients also present an immunoparalysis for a large number of cytokines, except IL-18 whose secretion is increased. An assessment of the immune response may be relevant to identify patients eligible for future innovative host-directed therapies.

The next frontier: cytomegalovirus antiviral stewardship programs in solid organ transplant.

Cytomegalovirus (CMV) is a driver of negative patient and allograft outcomes after solid organ transplantation (SOT) and new tools are needed to circumvent these outcomes. We will review key elements of CMV antiviral stewardship in SOT, discuss the available evidence for CMV antiviral stewardship programs and feature areas for expansion in the current landscape of CMV management. CMV remains a common complication after SOT. While consensus guidelines provide recommendations for the prevention and treatment of CMV, a one-size-fits-all approach is not necessarily appropriate for all unique patients and posttransplant courses, types of SOT recipients and transplant centers. Additionally, consensus guidelines have not been updated since the approval of two new antiviral therapies for the treatment of CMV after SOT or emerging evidence for the incorporation of immune functional assays into clinical practice.From the models provided in recent literature, CMV antiviral stewardship programs have demonstrated efficacy by increasing successful treatment of viremia, optimizing and reducing unnecessary use of (val)ganciclovir for both prophylaxis and treatment, and preventing development of ganciclovir-resistant CMV infections. These models highlight the multidisciplinary approach required of CMV antiviral stewardship programs to provide standardization of management, including incorporation of new therapies and diagnostic tools. CMV antiviral stewardship programs represent a promising avenue to considerably improve the management of CMV after SOT. Future studies are needed to evaluate a potential positive impact on graft outcomes and patient survival.

BCG vaccination induces innate immune memory in γδ T cells in humans.

Bacillus Calmette-Guérin vaccine is well known for inducing trained immunity in myeloid and natural killer cells, which can explain its cross-protective effect against heterologous infections. Although displaying functional characteristics of both adaptive and innate immunity, γδ T-cell memory has been only addressed in a pathogen-specific context. In this study, we aimed to determine whether human γδ T cells can mount trained immunity and therefore contribute to the cross-protective effect of the Bacillus Calmette-Guérin vaccine. We investigated in vivo induction of innate memory in γδ T cells by Bacillus Calmette-Guérin vaccination in healthy human volunteers by combining single-cell RNA sequencing technology with immune functional assays. The total number of γδ T cells and membrane markers of activation was not influenced by Bacillus Calmette-Guérin vaccination. In contrast, Bacillus Calmette-Guérin changed γδ T cells' transcriptional programs and increased their responsiveness to heterologous bacterial and fungal stimuli, including lipopolysaccharide and Candida albicans, as simultaneously characterized by higher tumor necrosis factor and interferon γ production, weeks after vaccination. Human γδ T cells in adults display the potential to develop a trained immunity phenotype after Bacillus Calmette-Guérin vaccination.

Fetal Muse-based therapy prevents lethal radio-induced gastrointestinal syndrome by intestinal regeneration

BackgroundHuman multilineage-differentiating stress enduring (Muse) cells are nontumorigenic endogenous pluripotent-like stem cells that can be easily obtained from various adult or fetal tissues. Regenerative effects of Muse cells have been shown in some disease models. Muse cells specifically home in damaged tissues where they exert pleiotropic effects. Exposition of the small intestine to high doses of irradiation (IR) delivered after radiotherapy or nuclear accident results in a lethal gastrointestinal syndrome (GIS) characterized by acute loss of intestinal stem cells, impaired epithelial regeneration and subsequent loss of the mucosal barrier resulting in sepsis and death. To date, there is no effective medical treatment for GIS. Here, we investigate whether Muse cells can prevent lethal GIS and study how they act on intestinal stem cell microenvironment to promote intestinal regeneration.MethodsHuman Muse cells from Wharton’s jelly matrix of umbilical cord (WJ-Muse) were sorted by flow cytometry using the SSEA-3 marker, characterized and compared to bone-marrow derived Muse cells (BM-Muse). Under gas anesthesia, GIS mice were treated or not through an intravenous retro-orbital injection of 50,000 WJ-Muse, freshly isolated or cryopreserved, shortly after an 18 Gy-abdominal IR. No immunosuppressant was delivered to the mice. Mice were euthanized either 24 h post-IR to assess early small intestine tissue response, or 7 days post-IR to assess any regenerative response. Mouse survival, histological stainings, apoptosis and cell proliferation were studied and measurement of cytokines, recruitment of immune cells and barrier functional assay were performed.ResultsInjection of WJ-Muse shortly after abdominal IR highly improved mouse survival as a result of a rapid regeneration of intestinal epithelium with the rescue of the impaired epithelial barrier. In small intestine of Muse-treated mice, an early enhanced secretion of IL-6 and MCP-1 cytokines was observed associated with (1) recruitment of monocytes/M2-like macrophages and (2) proliferation of Paneth cells through activation of the IL-6/Stat3 pathway.ConclusionOur findings indicate that a single injection of a small quantity of WJ-Muse may be a new and easy therapeutic strategy for treating lethal GIS.

Differentiating infection, colonisation, and sterile inflammation in critical illness: the emerging role of host-response profiling.

Infection results when a pathogen produces host tissue damage and elicits an immune response. Critically ill patients experience immune activation secondary to both sterile and infectious insults, with overlapping clinical phenotypes and underlying immunological mechanisms. Patients also undergo a shift in microbiota with the emergence of pathogen-dominant microbiomes. Whilst the combination of inflammation and microbial shift has long challenged intensivists in the identification of true infection, the advent of highly sensitive molecular diagnostics has further confounded the diagnostic dilemma as the number of microbial detections increases. Given the key role of the host immune response in the development and definition of infection, profiling the host response offers the potential to help unravel the conundrum of distinguishing colonisation and sterile inflammation from true infection. This narrative review provides an overview of current approaches to distinguishing colonisation from infection using routinely available techniques and proposes matrices to support decision-making in this setting. In searching for new tools to better discriminate these states, the review turns to the understanding of the underlying pathobiology of the host response to infection. It then reviews the techniques available to assess this response in a clinically applicable context. It will cover techniques including profiling of transcriptome, protein expression, and immune functional assays, detailing the current state of knowledge in diagnostics along with the challenges and opportunities. The ultimate infection diagnostic tool will likely combine an assessment of both host immune response and sensitive pathogen detection to improve patient management and facilitate antimicrobial stewardship.