Studying antigen-specific T cells through a streamlined, whole blood-based extracellular approach.

Techniques currently used for the study of antigen-specific T-cell responses are either poorly informative or require a heavy workload. Consequently, many perspectives associated with the broader study of such approaches remain mostly unexplored in translational research. However, these could benefit many fields including but not limited to infectious diseases, oncology, and vaccination. Herein, the main objective of this work was to develop a standardized flow cytometry-based approach that would combine ease of use together with a relevant study of antigen-specific T-cell responses so that they could be more often included in clinical research. To this extent, a streamlined approach relying on 1/ the use of whole blood instead of peripheral blood mononuclear cells and 2/ solely based on the expression of extracellular activation-induced markers (AIMs), called whole blood AIM (WAIM), was developed and further compared to more conventional techniques such as enzyme-linked immunospot (ELISpot) and flow cytometry-based intracellular cytokine staining (ICS). Based on a cohort of 20 individuals receiving the COVID-19 mRNA vaccine and focusing on SARS-CoV-2 and cytomegalovirus (CMV)-derived antigen T-cell-specific responses, a significant level of correlation between the three techniques was found. Based on the use of whole blood and on the expression of extracellular activation-induced markers (CD154, CD137, and CD107a), the WAIM technique appears to be very simple to implement and yet allows interesting patient stratification capabilities as the chosen combination of extracellular markers exhibited higher orthogonality than cytokines that are commonly considered in ICS (IFN-γ, TNF-α, and IL-2).

IL-33/ST2 pathway regulates neutrophil migration and predicts outcome in patients with severe alcoholic hepatitis.

BACKGROUND & AIMS: Severe alcoholic hepatitis (SAH) is associated with a high risk of infection. The IL-33/ST2 pathway is involved in sepsis control but data regarding its role in alcohol-related liver disease (ALD) are lacking. We aimed to characterize the role of IL-33/ST2 in the polymorphonuclear neutrophils (PMNs) of patients with ALD and SAH. METHODS: Serum and circulating neutrophils were collected from patients with SAH, alcoholic cirrhosis and healthy controls. We quantified IL-33/ST2 pathway activity and CXCR2 at baseline and after exposure to IL-33. We also determined the migration capacity of PMNs. RESULTS: The decoy receptor of IL-33 (soluble ST2 [sST2]) was increased in SAH vs. cirrhosis and controls, demonstrating the defect in this pathway during ALD. The sST2 level was associated with response to treatment, 2-month survival, infection-free survival and probability of infection in SAH. Endotoxemia was weakly correlated with sST2. GRK2, a negative regulator of CXCR2, was overexpressed in PMNs of patients with SAH and cirrhosis and was decreased by IL-33. CXCR2 levels on PMNs were lower in SAH vs. cirrhosis and controls. Treatment with IL-33 partially restored CXCR2 expression in SAH and cirrhosis. PMN migration upon IL-8 was lower in patients with SAH and cirrhosis vs. controls. Treatment with IL-33 partially restored migration in those with SAH and cirrhosis. Interestingly, the migration capacity of PMNs and the response to IL-33 were enhanced in responders to corticosteroids (Lille <0.45) compared to non-responders. CONCLUSION: The IL33/ST2 pathway is defective in SAH and predicts outcome. This defect is associated with decreased CXCR2 expression on the surface of PMNs and lower migration capacity, which can be corrected by IL-33, especially in patients responding to steroids. These results suggest that IL-33 has therapeutic potential for SAH and its infectious complications. LAY SUMMARY: The neutrophils of patients with severe alcoholic hepatitis are associated with a defect in the IL-33/ST2 pathway. This defect is associated with lower migration capacities in neutrophils and a higher probability of getting infected. Administration of IL-33 to the neutrophils at least partly restores this defect and may be effective at reducing the risk of infection in patients with severe alcoholic hepatitis.

Septic Shock Shapes B Cell Response toward an Exhausted-like/Immunoregulatory Profile in Patients.

Septic shock is accompanied by the development of immune dysfunctions whose intensity and duration are associated with increased risk of secondary infections and mortality. Although B lymphocytes play a pivotal role in the immune response to infections, no comprehensive exploration of circulating B cell status has been performed during the immunosuppressive phase of septic shock. Thus, our aim was to extensively characterize the phenotype and function of B cells in septic shock, including IL-10 production. Circulating B lymphocyte phenotype and function were evaluated by flow cytometry on fresh whole blood and after ex vivo stimulation in adult septic shock patients sampled at day 1, 3, and 6 after the onset of shock. The circulating B cell number was reduced in septic shock patients, whereas the B cell proportion among total lymphocytes was increased. The remaining circulating B lymphocytes presented with decreased MHC class II expression and increased CD21low CD95high exhausted-like phenotype but showed no change in maturation status. Circulating B cell functions were markedly altered after sepsis with reduced ex vivo activation and proliferation capacities. Finally, B cell response after septic shock was characterized by a clear plasmacytosis and an increased IL-10 production in remaining B cells from patients after ex vivo stimulation. During the sepsis-induced immunosuppression phase, B cell response is altered and is oriented toward an exhausted-like/immunoregulatory profile. Further studies are now needed to confirm the immunoregulatory properties of B lymphocytes and evaluate their role in sepsis-induced immunosuppression.

IL-7 Restores T Lymphocyte Immunometabolic Failure in Septic Shock Patients through mTOR Activation.

T lymphocyte alterations are central to sepsis pathophysiology, whereas related mechanisms remain poorly understood. We hypothesized that metabolic alterations could play a role in sepsis-induced T lymphocyte dysfunction. Samples from septic shock patients were obtained at day 3 and compared with those from healthy donors. T cell metabolic status was evaluated in the basal condition and after T cell stimulation. We observed that basal metabolic content measured in lymphocytes by nuclear magnetic resonance spectroscopy was altered in septic patients. Basal ATP concentration, oxidative phosphorylation (OXPHOS), and glycolysis pathways in T cells were decreased as well. After stimulation, T lymphocytes from patients failed to induce glycolysis, OXPHOS, ATP production, GLUT1 expression, glucose entry, and proliferation to similar levels as controls. This was associated with significantly altered mTOR, but not Akt or HIF-1α, activation and only minor AMPKα phosphorylation dysfunction. IL-7 treatment improved mTOR activation, GLUT1 expression, and glucose entry in septic patients' T lymphocytes, leading to their enhanced proliferation. mTOR activation was central to this process, because rapamycin systematically inhibited the beneficial effect of recombinant human IL-7. We demonstrate the central role of immunometabolism and, in particular, mTOR alterations in the pathophysiology of sepsis-induced T cell alterations. Our results support the rationale for targeting metabolism in sepsis with recombinant human IL-7 as a treatment option.

Decreased intra-lymphocyte cytokines measurement in septic shock patients: A proof of concept study in whole blood.

Functional testing protocols are thought to be the gold standard for the exploration of the immune system. However, in terms of routine analysis, they present numerous drawbacks and consequently their use is mainly limited to research applications. In the clinical context of septic shock, characterized by marked lymphocyte alterations, a new approach for lymphocyte intracellular cytokine measurement in whole blood upon was evaluated in a proof-of-concept study. Following lymphocyte activation, simultaneous intracellular labeling of Interferon-γ (IFN-γ), Tumor Necrosis Factor-α (TNF-α), and Interleukin-2 (IL-2) was performed in CD4+ and CD8+ T cells (identified by surface marking). The analysis was carried out by flow cytometry (6 colors). Results obtained in septic patients (n=22) were compared to those of healthy volunteers (n=8). Independently of lymphopenia, there were significant differences between groups. In particular there was significant decrease in the production of IL-2 and TNF-α in septic patients, while the production of IFN-γ was not significantly altered. Polyfunctional results showed that patients presented with increased percentages of triple negative lymphocytes. In contrast, volunteers had higher proportions of triple positive cells. The approach could be performed in a robust and consistent way, taking 4.5h to complete. Moreover, clear differences could be observed between clinical groups with this modified method. These characteristics illustrate the potential of this novel whole blood protocol for clinical applications. However, further research is required to determine the applicability compared to alternative test and to evaluate clinical performances in larger cohorts of patients.

Comparative dose-responses of recombinant human IL-2 and IL-7 on STAT5 phosphorylation in CD4+FOXP3- cells versus regulatory T cells: a whole blood perspective.

Interleukin(IL)-2 and IL-7 are cytokines with important functions related to CD4(+) lymphocyte proliferation, differentiation and survival. Depending on doses, they theoretically activate regulatory (Treg) and/or effector T cells (Teff) and thus may be indicated with different therapeutic objectives. In this study we assessed ex vivo the differential dose-responses of CD4(+) T cell subsets (Treg versus CD4(+)FOXP3(-) cells) to recombinant human (rh) IL-2 and rhIL-7. Fresh whole blood from healthy donors was stimulated with increasing doses of cytokines. By using a novel flow cytometry procedure of intracellular signaling pathway staining (e.g., detection of STAT5 phosphorylation; a pivotal marker of cytokine-induced activation; in combination with intracellular FOXP3 staining), we were able to specifically measure Treg and CD4(+)FOXP3(-) cell responses in the same tube. Half maximal effective concentrations (EC50) were calculated. We observed a dose-response effect on Treg and CD4(+)FOXP3(-) cells for both cytokines. Interestingly, low doses of hIL-2 preferentially activated Treg (EC50 Treg = 0.15 pg/ml versus CD4(+)FOXP3(-) cells = 750 pg/ml - p < 0.0001) whereas low doses of rhIL-7 preferentially induced CD4(+)FOXP3(-) cell activation (EC50 Treg = 25 pg/ml and CD4(+)FOXP3(-) cells = 2.5 pg/ml - p < 0.0001). To our knowledge, this work is the first to show differential dose-response effects on CD4(+)FOXP3(-) cells versus Treg of rhIL-7 and rhIL-2 in one ex vivo whole blood single tube assay including two intracellular stainings (i.e., pSTAT5 and FOXP3). Beyond the confirmation of the dose-dependent differential effects of IL-2 versus IL-7 on CD4(+)FOXP3(-) cells/Treg, our results illustrate the value of this approach for monitoring drugs' activities by flow cytometry in daily clinical practice.

[Interleukin-7, a new immunoadjuvant for the treatment of septic shock]. / L'interleukine-7 comme thérapeutique immuno-adjuvante dans le traitement du choc septique.

Sepsis-induced immunosuppression is a new paradigm in sepsis pathophysiology. This up-to-date review integrates recent facts in the field. It focuses on immune dysfunctions described so far in septic patients (especially regarding T lymphocytes), on the mechanisms sustaining this immune failure, on the monitoring of the pro-/anti-inflammatory balance rapidly changing over time and on new promising therapeutic avenues emerging from those recent findings. Of them, the case of interleukin-7 is more specifically envisaged.

Decreased HLA-DR antigen-associated invariant chain (CD74) mRNA expression predicts mortality after septic shock.

INTRODUCTION: Septic syndromes remain the leading cause of mortality in intensive care units (ICU). Septic patients rapidly develop immune dysfunctions, the intensity and duration of which have been linked with deleterious outcomes. Decreased mRNA expressions of major histocompatibility complex (MHC) class II-related genes have been reported after sepsis. We investigated whether their mRNA levels in whole blood could predict mortality in septic shock patients. METHODS: A total of 93 septic shock patients were included. On the third day after shock, the mRNA expressions of five MHC class II-related genes (CD74, HLA-DRA, HLA-DMB, HLA-DMA, CIITA) were measured by qRT-PCR and monocyte human leukocyte antigen-DR (mHLA-DR) by flow cytometry. RESULTS: A significant correlation was found among MHC class II related gene expressions. Among mRNA markers, the best prognostic value was obtained for CD74 (HLA-DR antigen-associated invariant chain). For this parameter, the area under the receiver operating characteristic curve (AUC) was calculated (AUC = 0.67, 95% confidence interval (CI) = 0.55 to 0.79; P = 0.01) as well as the optimal cut-off value. After stratification based on this threshold, survival curves showed that a decreased CD74 mRNA level was associated with increased mortality after septic shock (Log rank test, P = 0.0043, Hazard Ratio = 3.0, 95% CI: 1.4 to 6.5). Importantly, this association remained significant after multivariate logistic regression analysis including usual clinical confounders (that is, severity scores, P = 0.026, Odds Ratio = 3.4, 95% CI: 1.2 to 9.8). CONCLUSION: Decreased CD74 mRNA expression significantly predicts 28-day mortality after septic shock. After validation in a larger multicentric study, this biomarker could become a robust predictor of death in septic patients.

Targeted Metabolomics Analysis Suggests That Tacrolimus Alters Protection against Oxidative Stress.

Tacrolimus (FK506) is an immunosuppressant that is experiencing a continuous rise in usage worldwide. The related side effects are known to be globally dose-dependent. Despite numerous studies on FK506, the mechanisms underlying FK506 toxicity are still not well understood. It is therefore essential to explore the toxicity mediated by FK506. To accomplish this, we conducted a targeted metabolomic analysis using LC-MS on the plasma samples of patients undergoing FK506 treatment. The aim was to identify any associated altered metabolic pathway. Another anti-calcineurin immunosuppressive therapy, ciclosporin (CSA), was also studied. Increased plasma concentrations of pipecolic acid (PA) and sarcosine, along with a decrease in the glycine/sarcosine ratio and a tendency of increased plasma lysine was observed in patients under FK506 compared to control samples. Patients under CSA do not show an increase in plasma PA compared to the control samples, which does not support a metabolic link between the calcineurin and PA. The metabolomics changes observed in patients under FK506 highlight a possible link between FK506 and the action of an enzyme involved in both PA and sarcosine catabolism and oxidative pathway, the Peroxisomal sarcosine oxidase (PIPOX). Moreover, PA could be investigated as a potential biomarker of early nephrotoxicity in the follow-up of patients under FK506.

Serum neutralization of SARS coronavirus 2 Omicron sublineages BA.1 and BA.2 and cellular immune responses 3 months after booster vaccination.

OBJECTIVES: We investigated serum neutralizing activity against BA.1 and BA.2 Omicron sublineages and T cell response before and 3 months after administration of the booster vaccine in healthcare workers (HCWs). METHODS: HCWs aged 18-65 years who were vaccinated and received booster doses of the BNT162b2 vaccine were included. Anti-SARS coronavirus 2 IgG levels and cellular response (through interferon γ ELISpot assay) were evaluated in all participants, and neutralizing antibodies against Delta, BA.1, and BA.2 were evaluated in participants with at least one follow-up visit 1 or 3 months after the administration of the booster dose. RESULTS: Among 118 HCWs who received the booster dose, 102 and 84 participants attended the 1-month and 3-month visits, respectively. Before the booster vaccine dose, a low serum neutralizing activity against Delta, BA.1, and BA.2 was detectable in only 39/102 (38.2%), 8/102 (7.8%), and 12/102 (11.8%) participants, respectively. At 3 months, neutralizing antibodies against Delta, BA.1, and BA.2 were detected in 84/84 (100%), 79/84 (94%), and 77/84 (92%) participants, respectively. Geometric mean titres of neutralizing antibodies against BA.1 and BA.2 were 2.2-fold and 2.8-fold reduced compared with those for Delta. From 1 to 3 months after the administration of the booster dose, participants with a recent history of SARS coronavirus 2 infection (n = 21/84) had persistent levels of S1 reactive specific T cells and neutralizing antibodies against Delta and BA.2 and 2.2-fold increase in neutralizing antibodies against BA.1 (p 0.014). Conversely, neutralizing antibody titres against Delta (2.5-fold decrease, p < 0.0001), BA.1 (1.5-fold, p 0.02), and BA.2 (2-fold, p < 0.0001) declined from 1 to 3 months after the administration of the booster dose in individuals without any recent infection. DISCUSSION: The booster vaccine dose provided significant and similar response against BA.1 and BA.2 Omicron sublineages; however, the immune response declined in the absence of recent infection.

Undetectable triglycerides related to the combined intake of ascorbic acid and tacrolimus.

Management of triglyceride (TG) levels is essential in intensive care units (ICU), especially to manage the risk of pancreatitis induced by propofol. However, some therapeutics in ICU such as intravenous ascorbic acid protocol, especially used in the context of Covid-19 could lead to false decrease of triglycerides by analytical disruption of Trinder reaction. We report here the case of a sample with unmeasurable triglyceride levels partly due to high plasma ascorbic acid levels. However, repeated measure on the same sample four days later revealed that interference mechanism on TG was still present whereas the level of ascorbic acid was very reduced by oxidation degradation. Hence, additional interference mechanism was suspected. After clinical investigation, we found that the patient had also received high doses of tacrolimus due to a transplant. As previous studies reported that tacrolimus treatment lead to a decrease of the measured plasma activity of lipoprotein lipase (LPL), we hypothesized that tacrolimus or related metabolites could also interfere by direct inhibition of LPL involved in TG analytical method used.

Impact of Ventilator-associated Pneumonia on Cerebrospinal Fluid Inflammation During Immunosuppression After Subarachnoid Hemorrhage: A Pilot Study.

INTRODUCTION: Brain injuries can cause systemic immunosuppression, which in turn can lead to infections that adversely affect the injured brain and worsen clinical outcomes. This study aimed to investigate whether systemic infection, such as ventilator-associated pneumonia (VAP), induce intracranial inflammation in patients with subarachnoid hemorrhage (SAH). METHODS: This prospective, observational study included 16 adults with SAH treated in the neuro-intensive care unit. Three paired cerebrospinal fluid samples (obtained from an external ventricular drain) and peripheral blood samples were obtained on days 1 to 3, 4 to 5, and 6 to 7 after SAH onset. Cell counts, cell phenotypes (monocyte HLA-DR, T regulatory cells, lymphocytes, and neutrophils), and inflammatory mediator levels were monitored. RESULTS: Six patients developed VAP in the context of systemic immunosuppression demonstrated by a reduction in monocyte HLA-DR expression, lymphopenia, increased percentages of circulating T regulatory cells, and increased proportions of immature and immunosuppressive neutrophil subsets. During VAP, there was de novo recruitment of leukocytes into the cerebrospinal fluid, preferentially neutrophils, which exacerbated intracranial inflammation. CONCLUSIONS: VAP increased intracranial inflammatory responses in patients with SAH despite the occurrence of systemic immunosuppression. A better understanding of cell trafficking and their pleiotropic functions in brain injury is needed to define the optimal strategies for preventing infections in patients with SAH.

Evaluation of a rapid semiquantitative lateral flow assay for the prediction of serum neutralizing activity against SARS-CoV-2 variants.

BACKGROUND: Neutralizing antibodies (NAbs) against SARS-CoV-2 have been shown to correlate with protection against infection. Simple tools such as lateral flow assays (LFA) that can accurately measure NAbs may be useful for monitoring anti-SARS-CoV-2 immunity in the future. OBJECTIVES: We assessed the performance of the ichroma™ COVID-19 nAb test, a rapid semiquantitative LFA, for the prediction of serum neutralizing activity against SARS-CoV-2 variants. STUDY DESIGN: Serum samples were collected from COVID-19 recovered patients and vaccinated individuals. The result of the ichroma assay was provided as inhibition rate, and was compared to anti-SARS-CoV-2 IgG levels, and NAbs against Alpha, Delta and Omicron variants. RESULTS: A total of 90 sera from recovered unvaccinated patients and 209 sera from the vaccine cohort were included in this study. In post-infection samples, the ichroma inhbition rate was found to be correlated with IgG levels (ρ = 0.83), and with anti-Alpha NAbs levels (ρ = 0.78). In the vaccine cohort, a good correlation was also observed between the ichroma inhibition rate and IgG levels (ρ = 0.84), as well as NAbs against Alpha (ρ = 0.62), Delta (ρ = 0.88) and Omicron (ρ = 0.74). An ichroma inhbition rate of 77.2%, 90.8% and 99.6% accurately predicted neutralization against Alpha, Delta and Omicron variants respectively. CONCLUSIONS: The ichroma™ COVID-19 nAb assay, with appropriate variant cut-offs, can be useful for the monitoring of anti-SARS-CoV-2 immunization and may provide a rapid prediction of protection, especially in individuals with significant levels of NAbs.

Immunogenicity of BNT162b2 vaccine booster against SARS-CoV-2 Delta and Omicron variants in nursing home residents: A prospective observational study in older adults aged from 68 to 98 years.

Background: The present study aimed to evaluate the persistent immunogenicity offered by a third dose of BNT162b2 against Delta and Omicron variants, in nursing home (NH) residents. Methods: In this monocenter prospective observational study, anti-spike IgG levels, S1 domain reactive T cell counts, serum neutralizing antibody titers against Delta and Omicron variants were compared before and up to three months after the BNT162b2 booster dose, in NH residents without COVID-19 (COVID-19 naive) or with COVID-19 prior to initial vaccination (COVID-19 recovered). Findings: 106 NH residents (median [interquartile range] age: 86·5 [81;91] years) were included. The booster dose induced a high increase of anti-spike antibody levels in all subjects (p < 0.0001) and a mild transient increase of specific T cells. Before the booster dose, Delta neutralization was detected in 19% (n = 8/43) and 88% (n = 37/42) of COVID-19 naive and COVID-19 recovered subjects, respectively. Three months after the booster dose, all NH residents developed and maintained a higher Delta neutralization (p < 0·0001). Before the booster dose, Omicron neutralization was detected in 5% (n = 2/43) and 55% (n = 23/42) of COVID-19 naive and COVID-19 recovered subjects, respectively, and three months after, in 84% and 95%, respectively. Neutralizing titers to Omicron were lower than to Delta in both groups with a 35-fold reduction compared to Delta. Interpretation: The booster dose restores high neutralization titers against Delta in all NH residents, and at a lower level against Omicron in a large majority of participants. Future studies are warranted to assess if repeated BNT162b2 booster doses or new specific vaccines might be considered for protecting such fragile patients against Omicron and/or future SARS-CoV-2 variants. Funding: French government through the Programme Investissement d'Avenir (I-SITE ULNE/ANR-16-IDEX-0004 ULNE) and the Label of COVID-19 National Research Priority (National Steering Committee on Therapeutic Trials and Other COVID-19 Research, CAPNET).

Myeloid cells in sepsis-acquired immunodeficiency.

On May 2017, the World Health Organization recognized sepsis as a global health priority. Sepsis profoundly perturbs immune homeostasis by initiating a complex response that varies over time, with the concomitant occurrence of pro- and anti-inflammatory mechanisms. Sepsis deeply impacts myeloid cell response. Different mechanisms are at play, such as apoptosis, endotoxin tolerance, metabolic failure, epigenetic reprogramming, and central regulation. This induces systemic effects on circulating immune cells and impacts progenitors locally in lymphoid organs. In the bone marrow, a progressive shift toward the release of immature myeloid cells (including myeloid-derived suppressor cells), at the expense of mature neutrophils, takes place. Circulating dendritic cell number remains dramatically low and monocytes/macrophages display an anti-inflammatory phenotype and reduced antigen presentation capacity. Intensity and persistence of these alterations are associated with increased risk of deleterious outcomes in patients. Thus, myeloid cells dysfunctions play a prominent role in the occurrence of sepsis-acquired immunodeficiency. For the most immunosuppressed patients, this paves the way for clinical trials evaluating immunoadjuvant molecules (granulocyte-macrophage colony-stimulating factor and interferon gamma) aimed at restoring homeostatic myeloid cell response. Our review offers a summary of sepsis-induced myeloid cell dysfunctions and current therapeutic strategies proposed to target these defects in patients.

Monitoring CAR T-cells using flow cytometry.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is considered as a major scientific breakthrough in cancer immunotherapy. The success of adoptive CAR T-cell therapy for cancer has inspired researchers to expand indications into the area of solid tumors, autoimmune and infectious diseases. The most important factors influencing outcome and durability of the response after infusion of CAR T-cell are proliferation and persistence of this cell subset. It becomes therefore important to detect easily and monitor circulating CAR T-cells into blood samples. Approaches such as quantitative PCR (qPCR) or flow cytometry have been developed. The aim of this study was to set up and optimize a reachable flow cytometry technique using labeled CD19 protein for the measurement of CAR T-cells in infusion bag and patient's blood. METHODS: Patients receiving Yescarta in Cell Therapy Unit (Department of hematology, Lille university hospital, France) between April and October 2019 and healthy volunteers were included to set up the flow cytometry technique. RESULTS AND CONCLUSIONS: We assessed feasibility in clinic and suitability to routine workload of a flow cytometry technique to follow CAR T-cells in infusion bag and patient's blood. With only a few manual steps, the present protocol allows the technician to perform this technique among other routine tasks, meaning a time to results of <2 hr after sample reception. We were also able to assess CAR T-cell heterogenity in terms of CD4+ and CD8+ T lymphocytes within the subset. Moreover, this technique allows monitoring of both authority approved CD19 CAR T-cell.

Impaired Functional T-Cell Response to SARS-CoV-2 After Two Doses of BNT162b2 mRNA Vaccine in Older People.

Long-term care facility (LTCF) older residents display physiological alterations of cellular and humoral immunity that affect vaccine responses. Preliminary reports suggested a low early postvaccination antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The aim of this study was to focus on the specific T-cell response. We quantified S1-specific IgG, neutralizing antibody titers, total specific IFNγ-secreting T cells by ELISpot, and functionality of CD4+- and CD8+-specific T cells by flow cytometry, after two doses of the BNT162b2 vaccine in younger and older people, with and without previous COVID-19 infection (hereafter referred to as COVID-19-recovered and COVID-19-naive subjects, respectively). Frailty, nutritional, and immunosenescence parameters were collected at baseline in COVID-19-naive older people. We analyzed the immune response in 129 young adults (median age 44.0 years) and 105 older residents living in a LCTF (median age 86.5 years), 3 months after the first injection. Humoral and cellular memory responses were dramatically impaired in the COVID-19-naive older (n = 54) compared with the COVID-19-naive younger adults (n = 121). Notably, older participants' neutralizing antibodies were 10 times lower than the younger's antibody titers (p < 0.0001) and LCTF residents also had an impaired functional T-cell response: the frequencies of IFNγ+ and IFNγ+IL-2+TNFα+ cells among specific CD4+ T cells, and the frequency of specific CD8+ T cells were lower in COVID-19-naive older participants than in COVID-19-naive young adults (p < 0.0001 and p = 0.0018, respectively). However, COVID-19-recovered older participants (n = 51) had greater antibody and T-cell responses, including IFNγ+ and IFNγ+IL-2+TNFα+-specific CD4+ T cells (p < 0.0001), as well as TNFα+-specific CD8+ T cells (p < 0.001), than COVID-19-naive older adults. We also observed that "inflammageing" and particularly high plasma levels of TNFα was associated to poor antibody response in the older participants. In conclusion, our results show that the COVID-19-naive older people had low counts and impaired specific CD4+ and CD8+ T cells, in addition to impaired antibody response, and that specific studies are warranted to assess the efficiency of SARS-CoV-2 mRNA-based vaccines, as in other immunocompromised subjects. Our study also shows that, despite their physiological alterations of immunity, vaccination is highly efficient in boosting the prior natural memory response in COVID-19-recovered older people.

Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational study.

BACKGROUND: Haematopoietic stem cells expressing the CD34 surface marker have been posited as a niche for Mycobacterium tuberculosis complex bacilli during latent tuberculosis infection. Our aim was to determine whether M tuberculosis complex DNA is detectable in CD34-positive peripheral blood mononuclear cells (PBMCs) isolated from asymptomatic adults living in a setting with a high tuberculosis burden. METHODS: We did a cross-sectional study in Ethiopia between Nov 22, 2017, and Jan 10, 2019. Digital PCR (dPCR) was used to determine whether M tuberculosis complex DNA was detectable in PBMCs isolated from 100 mL blood taken from asymptomatic adults with HIV infection or a history of recent household or occupational exposure to an index case of human or bovine tuberculosis. Participants were recruited from HIV clinics, tuberculosis clinics, and cattle farms in and around Addis Ababa. A nested prospective study was done in a subset of HIV-infected individuals to evaluate whether administration of isoniazid preventive therapy was effective in clearing M tuberculosis complex DNA from PBMCs. Follow-up was done between July 20, 2018, and Feb 13, 2019. QuantiFERON-TB Gold assays were also done on all baseline and follow-up samples. FINDINGS: Valid dPCR data (ie, droplet counts >10 000 per well) were available for paired CD34-positive and CD34-negative PBMC fractions from 197 (70%) of 284 participants who contributed data to cross-sectional analyses. M tuberculosis complex DNA was detected in PBMCs of 156 of 197 participants with valid dPCR data (79%, 95% CI 74-85). It was more commonly present in CD34-positive than in CD34-negative fractions (154 [73%] of 197 vs 46 [23%] of 197; p<0·0001). Prevalence of dPCR-detected M tuberculosis complex DNA did not differ between QuantiFERON-negative and QuantiFERON-positive participants (77 [78%] of 99 vs 79 [81%] of 98; p=0·73), but it was higher in HIV-infected than in HIV-uninfected participants (67 [89%] of 75 vs 89 [73%] of 122, p=0·0065). By contrast, the proportion of QuantiFERON-positive participants was lower in HIV-infected than in HIV-uninfected participants (25 [33%] of 75 vs 73 [60%] of 122; p<0·0001). Administration of isoniazid preventive therapy reduced the prevalence of dPCR-detected M tuberculosis complex DNA from 41 (95%) of 43 HIV-infected individuals at baseline to 23 (53%) of 43 after treatment (p<0·0001), but it did not affect the prevalence of QuantiFERON positivity (17 [40%] of 43 at baseline vs 13 [30%] of 43 after treatment; p=0·13). INTERPRETATION: We report a novel molecular microbiological biomarker of latent tuberculosis infection with properties that are distinct from those of a commercial interferon-γ release assay. Our findings implicate the bone marrow as a niche for M tuberculosis in latently infected individuals. Detection of M tuberculosis complex DNA in PBMCs has potential applications in the diagnosis of latent tuberculosis infection, in monitoring response to preventive therapy, and as an outcome measure in clinical trials of interventions to prevent or treat latent tuberculosis infection. FUNDING: UK Medical Research Council.