BackgroundThe best way to monitor Chimeric Antigen Receptor (CAR)-T cells persistence and expansion in vivo after infusion, and the significance of observed fluctuations over time remains controversial. As living drugs, these therapies do not follow classical pharmacokinetic patterns. Therefore, reliable quantification of circulating CAR-T cells is essential to explore the relations between in vivo expansion and persistence on one hand, tumor response and occurrence of side-effects on the other hand; this work will prepare integration of this information in harmonized post-CAR-T Cells intervention algorithms. Conventional flow cytometry protocols rely on multi-step wash procedures that increase processing time and may reduce sensitivity. We here developed and validated a one-step no-wash flow cytometry assay for routine CAR-T monitoring in patients treated with approved autologous CAR-T Cells.MethodsCAR-T cell monitoring was implemented between 2021 and 2024 in patients treated with autologous CD19-directed CAR-T therapies at our institution with a classical two steps and wash flow cytometry method. Analytical validation included determination of detection and quantification limits, linearity, precision, and inter-laboratory reproducibility. In 2024, the classical method was considerably optimized to a one-step no-wash format to reduce manual handling and improve sensitivity. Clinical relevance was assessed in a cohort of 29 patients treated with axicabtagene ciloleucel, correlating CAR-T expansion metrics with clinical endpoints.ResultsThe optimized one-step no-wash assay markedly improved analytical sensitivity, achieving a limit of detection of 0.3 cells/µL and a lower limit of quantification of 1.0 cells/µL, versus 2.0 and 5.0 cells/µL, respectively, when using the previous two steps and wash protocol, while demonstrating a strong concordance (r² = 0.984). Inter-assay coefficients of variation remained below 9%, confirming maintained precision despite workflow simplification. In 29 patients treated with axicabtagene ciloleucel (axicel), peak CAR-T expansion significantly correlated with objective responses (p< 0.01) and occurrences of immune effector cell–associated neurotoxicity syndrome (ICANS) (p = 0.02), supporting the clinical relevance of flow cytometry–based CAR-T monitoring in routine practice.ConclusionThis one-step no-wash flow cytometry assay combines enhanced sensitivity with improved operational efficiency and provides clinically informative CAR-T cell monitoring in standard-of-care settings.

Abstract Objectives Inborn errors of immunity are rare genetic disorders that cause dysfunction of the immune system. Among these, familial haemophagocytic lymphohistiocytosis (FHL) involves defects in the perforin/granzyme pathway, which is essential for regulating immune responses. These conditions predispose individuals to haemophagocytic lymphohistiocytosis, a life‐threatening hyperinflammatory syndrome. FHL has traditionally been described in paediatric patients with a fatal outcome in the absence of early haematopoietic stem cell transplantation. However, some patients harbouring missense mutations may present with atypical or late‐onset symptoms, for which management remains unstandardised. Among these reported variants, the pathogenicity of the A91V PRF1 mutation remains the most controversial in the literature. Method We report clinical, biological and cytometric characteristics of two patients with a homozygous PRF1 A91V mutation who developed clinical features consistent with FHL2. Discussion We discuss the latest 2024 classifications to better characterise this group of disorders and their underlying genetic basis, with particular emphasis on atypical presentations and the A91V mutation, which may pose diagnostic and therapeutic challenges. Conclusion A91V PRF1 variant appears to represent a risk factor for the development of atypical FHL manifestations under divers triggers.

Innate immune cells appear to have an important implication in the resolution and/or the aggravation of the COVID-19 pathogenesis after infection with SARS-CoV-2. To better appreciate the role of these cells during COVID-19, changes in blood eosinophil, the neutrophil and monocyte count, and levels of surface protein markers have been reported. However, analyses at several timepoints of multiple surface markers on granulocytes and monocytes over a period of one month after a SARS-CoV-2 infection are missing. Therefore, in this study, we performed blood eosinophil, neutrophil, and monocyte phenotyping using a list of surface proteins and flow cytometry during a period of 30 days after the hospitalization of patients with severe SARS-CoV-2 infections. Blood cell counts were reported at seven different timepoints over the 30-day period as well as measures of multiple mediators in serum using a targeted multiplex assay approach. Our results indicate a 95% drop in the blood eosinophil count by D1, with eosinophils displaying a phenotype defined as CD69/CD63/CD125high and CCR3/CD44low during the early phases of hospitalization. Conversely, by D7 the neutrophil count increased significantly and displayed an immature, activated, and immunosuppressive phenotype (i.e., 3% of CD10/CD16low and CD10lowCD177high, 6.7% of CD11bhighCD62Llow, and 1.6% of CD16highCD62Llow), corroborated by enhanced serum proteins that are markers of neutrophil activation. Finally, our results suggest a rapid recruitment of non-classical monocytes leaving CD163/CD64high and CD32low monocytes in circulation during the very early phase. In conclusion, our study reveals potential very early roles for eosinophils and monocytes in the pathogenesis of COVID-19 with a likely reprogramming of eosinophils in the bone marrow. The exact roles of the pro-inflammatory neutrophils and the functions of the eosinophils and the monocytes, as well as these innate immune cell types, interplays need to be further investigated.

Background. Routine lymphocyte counting requires quality assurance validation using quality controls (QCs) that closely resemble fresh human blood leukocytes. This study aimed to evaluate a novel artificial product designed to mimic leukocyte light scatters and marker expression. Methods. FlowCytes and TruCytes, “artificial cell mimics” (Slingshot Biosciences, Emeryville, CA, USA), were tested on CE-IVD-certified systems, namely FACSCanto, FACSLyric (BD Biosciences), Navios, and DxFlex (Beckman Coulter), using routine staining, lysing, fixation, and no-wash procedures for T, B, and NK counting. Results. FlowCytes and TruCytes provided forward and side scatter profiles comparable to human leukocytes on the FACSCanto, FACSLyric, and DxFlex systems but not on Navios despite adapting the process to be slightly different from the manufacturer’s recommendations. TruCytes demonstrated robust immunolabeling of CD3, CD4, CD8, and CD19 on the FACSCanto, FACSLyric, and DxFlex systems, with fluorescence intensities and subset distributions being similar to those usually observed in fresh human blood. However, CD16 and CD56 labeling was inconsistent and depended on the antibody clones used. Regrettably, monocyte and granulocyte mimics lacked expression of CD4, CD16, and CD14. TruCytes also displayed significantly lower concentrations of TBNK lymphocyte subsets compared to healthy human blood. Conclusions. FlowCytes and TruCytes show promises as internal quality controls for T cell and B cell immunophenotyping, but not NK cells. They are compatible with most CE-IVD cytometers, even when using lysis/fixation/no-wash routine diagnosis procedures. Further multicentric studies are warranted to assess their performance relative to existing products, such as stabilized human blood.

Atopic dermatitis and other type 2 immune response diseases are often linked to elevated eosinophil levels in the blood. Although the role of eosinophils in atopic dermatitis pathophysiology is suspected, it remains unclear. The development of new treatments targeting the type 2 response, particularly cytokines involved in eosinophil activation and chemotaxis, makes it necessary to identify potential eosinophil profiles in atopic dermatitis that may respond to these treatments. A prospective study was conducted comparing blood eosinophil phenotypes in patients with moderate to severe atopic dermatitis (n = 19) without recent systemic treatment to healthy individuals (n = 19). The primary outcome was the membranous phenotypic signature of eosinophils, assessed by flow cytometry. Most patients with atopic dermatitis (84%) had early onset in childhood, a severe disease (mean SCORing Atopic Dermatitis of 57.5), and elevated blood eosinophil counts (310 per mu L in atopic dermatitis vs 120 in healthy individuals, P < 0.0001). Patients with atopic dermatitis exhibited lower CRTH2 on eosinophils but higher levels of human leukocyte antigen-DR isotype and Siglec-8 compared to healthy individuals. Other surface proteins showed no significant differences. Clustering analysis confirmed increased Siglec-8 in patients with atopic dermatitis. Additionally, patients with atopic dermatitis had higher serum levels of type 2 immune response markers such as eotaxin-2, IL-5, IL-3, and TARC. Circulating eosinophils in patients with atopic dermatitis show a distinct phenotypic profile, suggesting a role in atopic dermatitis pathophysiology and potential involvement in differential treatment responses. [GRAPHICS] .

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. AntieSARS coronavirus 2 IgG levels and cellular response (through interferon gamma 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. Enagnon Kazali Alidjinou, Clin Microbiol Infect 2023;29:258.e1-258.e4 (c) 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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).

OCS Introduction. Lymphocytic hypereosinophilic syndrome (L-HES) is an indolent T-cell lymphoproliferative disorder characterized by chronic blood hypereosinophilia and eosinophil-related organ damage secondary to the production of eosinophilopoietic cytokines (including interleukin-5) by clonal T cells, among which CD3-CD4+ T cells are the most frequent subset. Both high exposure to oral corticosteroids (OCS), failure and/or toxicity of OCS-sparing therapies and the risk of progression to AITL (up to 5 - 10% of patients) underscore the unmet need for novel treatments targeting the clonal T-cells (and not only eosinophils). We previously reported that peripheral CD3-CD4+ T cells express C-C chemokine receptor 4 (CCR4). We hypothesized that mogamulizumab, an afucosylated monoclonal antibody targeting CCR4-positive cells through antibody-dependent cellular cytotoxicity, could be beneficial for refractory L-HES patients. Methods. We assessed both the expression of CCR4 by clonal CD3-CD4+ T cells in the skin and lymph nodes as well as theability of mogamulizumab to induce ADCC in vitro on sorted CD3-CD4+ T cells from two L-HES patients, using homologous sorted NK cells as effector cells. We then set up a compassionate use program of intravenous mogamulizumab (1.0 mg/kg weekly for the first 28-day cycle, then on days 1 and 15 from subsequent cycles) for refractory L-HES patients despite treatment with immunosuppressants and/or biologics targeting the IL-5 pathway. Complete blood response was defined as complete if the subset of CD3-CD4+ T cells was <0.5% of all lymphocytes, or as partial if the number of CD3-CD4+ T cells decreased by >50% without reaching complete response at the time of the last perfusion. Results. Significant CCR4 expression on tissue CD4+ T cells was evidenced, and NK cells indeed led to the lysis of CD3-CD4+ T-cells coated with mogamulizumab (10 μg/ml). Four patients with refractory L-HES (despite treatment with high-dose OCS, n=4; pegylated interferon alpha 2a; n=3; or mepolizumab, a single patient) and active disease entered the compassionate use program (Table 1). Despite substantial tapering in OCS doses, all cases showed clinical response. Blood responses (either partial or complete, two patients each) and sharp decrease in absolute eosinophil counts (AEC) were reported in all patients ( Figure 1). Both patients with lymph node enlargement rapidly improved. Although prolonged blood and clinical responses have been observed after discontinuation of mogamulizumab, relapses still occurred preceded by an increase in T-cell clone size and absolute eosinophil count (AEC). Rechallenging a relapse with a short course of mogamulizumab resulted in a complete remission for a further 12 months in case #1. In terms of safety, mogamulizumab was discontinued in two patients due to adverse events: one for recurrent infusion-related reactions and one for infectious events. Lymphopenia was reported in 3/4 cases but was preexisting in the 3 cases. Case #4 reported a serious adverse event related to a tumor lysis syndrome leading to the massive release of IL-5 and subsequent rebound of AEC. Hence lower doses of mogamulizumab, transient use of OCS and/or mepolizumab should be considered in patients with very high number of CD3-CD4+ T cells who are about to start mogamulizumab. Discussion. Mogamulizumab represents a new therapeutic option for L-HES patients with severe refractory HES, T-cell clone related symptoms and/or large or rapidly increasing clonal T-cell counts. In patients who achieve complete remission under mogamulizumab, whether fixed repeated (e.g., every 6 months) infusions could be beneficial to prevent relapses and transformation into high-grade lymphoma remains to be investigated. Figure legend. Hematological responses to mogamulizumab in four refractory lymphocytic HES patients. oral corticosteroids; pINF-α: pegylated interferon alpha; MEPO: mepolizumab; MOGA: mogamulizumab.

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. Copyright (C) 2022 The Authors. Published by Elsevier Ltd.

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 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 3 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. Repeated BNT162b2 booster doses or new specific vaccines might be considered for protecting such fragile patients. Trial Registration Details: The study was registered in ClinicalTrials.gov, with the identifier NCT04760704. Funding Information: 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). Declaration of Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Approval Statement: This study was performed in accordance with the Declaration of Helsinki principles for ethical research. The study was approved by the Ile-De-France V (ID-CRB 2021-A00119-32) ethics committee. All participants (and/or their legal representative if required) received detailed information and signed a consent form before participating in the study.

We quantified S1-specific IgG, neutralizing antibody titers, specific IFNγ secreting T cells and functionality of specific CD4+ and CD8+ T cells in 130 young adults (median age 44.0 years) and 106 older residents living in a long-term care facility (86.5 years) after 2 doses of BNT162b2. Three months after the first injection, humoral and cellular memory responses were dramatically impaired in the 54 COVID-19-naive older compared to the 121 COVID-19-naive younger adults. Notably, older participants’ neutralizing antibodies, detected in 76.5% (versus 100% in young adults, P < 0.0001), were ten times lower than the younger’s antibody titers (P < 0.0001). Antibody and T cell responses were greater among the 52 COVID-19-recovered than among the 54 COVID-19-naive older adults (P < 0.0001). Our study shows that 2 doses of BNT162b2 does not guarantee long-term protection against SARS-CoV-2 in the older. An additional dose should be considered to boost their specific memory response.

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 IFN gamma-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 IFN gamma(+) and IFN gamma+IL-2(+)TNF alpha(+) 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 p = 0.0018, respectively). However, COVID-19-recovered older participants (n = 51) had greater antibody and T-cell responses, including IFN gamma(+) and IFN gamma+IL-2(+)TNF alpha(+)-specific CD4(+) T cells (p < 0.0001), as well as TNF alpha(+)-specific CD8(+) T cells (p < 0.001), than COVID-19-naive older adults. We also observed that "inflammageing" and particularly high plasma levels of TNF alpha 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.

AbstractObjectivesAssessment of the adaptive immune response against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is crucial for studying long‐term immunity and vaccine strategies. We quantified IFNγ‐secreting T cells reactive against the main viral SARS‐CoV‐2 antigens using a standardised enzyme‐linked immunospot assay (ELISpot).MethodsOverlapping peptide pools built from the sequences of M, N and S viral proteins and a mix (MNS) were used as antigens. Using IFNγ T‐CoV‐Spot assay, we assessed T‐cell and antibody responses in mild, moderate and severe SARS‐CoV‐2 patients and in control samples collected before the outbreak.ResultsSpecific T cells were assessed in 60 consecutive patients (mild, n = 26; moderate, n = 10; and severe patients, n = 24) during their follow‐up (median time from symptom onset [interquartile range]: 36 days [28;53]). T cells against M, N and S peptide pools were detected in n = 60 (100%), n = 56 (93.3%), n = 55 patients (91.7%), respectively. Using the MNS mix, IFNγ T‐CoV‐Spot assay showed a specificity of 96.7% (95% CI, 88.5–99.6%) and a specificity of 90.3% (75.2–98.0%). The frequency of reactive T cells observed with M, S and MNS mix pools correlated with severity and with levels of anti‐S1 and anti‐RBD serum antibodies.ConclusionIFNγ T‐CoV‐Spot assay is a reliable method to explore specific T cells in large cohorts of patients. This test may become a useful tool to assess the long‐lived memory T‐cell response after vaccination. Our study demonstrates that SARS‐CoV‐2 patients developing a severe disease achieve a higher adaptive immune response.

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.

It has been suggested that the persistence of coxsackieviruses-B (CV-B) in pancreatic beta cells plays a role in the pathogenesis of type 1 diabetes (T1D). Yet, immunological effectors, especially natural killer (NK) cells, are supposed to clear virus-infected cells. Therefore, an evaluation of the response of NK cells to pancreatic beta cells persistently infected with CV-B4 was conducted. A persistent CV-B4 infection was established in 1.1B4 pancreatic beta cells. Infectious particles were found in supernatants throughout the culture period. The proportion of cells containing viral protein VP1 was low (< 5%), although a large proportion of cells harbored viral RNA (around 50%), whilst cell viability was preserved. HLA class I cell surface expression was downregulated in persistently infected cultures, but HLA class I mRNA levels were unchanged in comparison with mock-infected cells. The cytolytic activities of IL-2-activated non-adherent peripheral blood mononuclear cells (PBMCs) and of NK cells were higher towards persistently infected cells than towards mock-infected cells, as assessed by an LDH release assay. Impaired cytolytic activity of IL-2-activated non-adherent PBMCs from patients with T1D towards infected beta cells was observed. In conclusion, pancreatic beta cells persistently infected with CV-B4 can be lysed by NK cells, implying that impaired cytolytic activity of these effector cells may play a role in the persistence of CV-B in the host and thus in the viral pathogenesis of T1D.

In previous studies, we and others observed in patients undergoing HLA-matched hematopoietic cell transplantation that high proportion of donor-derived CD4+/CCR7+ T cells were associated with an increased risk of acute GVHD without any interference in relapse incidence. We investigated the impact of donor-derived CD4+/CCR7+ T cells on patient outcome in haploidentical settings where posttransplant cyclophosphamide is used. We analyzed T-cell subsets in grafts of 29 adult patients who underwent first haploidentical transplant following reduced intensity conditioning. The median CD4+/CCR7+ subset proportion was 69.2% among donor CD4+ T cells. With a median follow-up of 28.1 months (range: 11.0–44.3), 16 patients (55%) developed acute GVHD; this includes 5 patients with grade 3 acute GVHD. Fifty-four percent of patients who received > 69.2% of CD4+/CCR7+ T cells and 12% of patients who received < 69.2% CD4+/CCR7+ T cells developed acute GVHD (p = 0.028). In multivariate analysis, a high proportion of CD4+/CCR7+ T cells was the only factor that impacted acute GVHD (HR = 4.925, 95% CI [1.020–23.775], p = 0,047) with no impact on overall survival. Our results confirm the impact of a high proportion of CD4+/CCR7+ T cells on acute GVHD incidence in patients undergoing haploidentical transplant despite the use of posttransplant cyclophosphamide.