Monitoring immunE DysregulAtion foLLowing Immune checkpOint-inhibitioN (MEDALLION): protocol for an observational cancer immunotherapy cohort study.

BACKGROUND: Checkpoint inhibitors (CPIs) are widely used in cancer treatment, with transformative impacts on survival. They nonetheless carry a significant risk of toxicity in the form of immune-related adverse events (IrAEs), which may be sustained and life-altering. IrAEs may require high-dose and/or prolonged steroid use and represent a significant healthcare burden. They mimic immune-mediated inflammatory diseases (IMIDs) but understanding of their pathogenesis is limited. The MEDALLION project aims to determine targetable mechanisms of immune dysregulation in IrAE development, employing an immune monitoring approach to determine changes in circulating and tissue resident cells of CPI recipients who do/do not develop them and assessing the contribution of the microbiome in parallel. METHODS: MEDALLION is a non-randomised longitudinal cohort study aiming to recruit 66 cancer patient recipients of anti-PD1/PD-L1, anti-CTLA-4 or combination therapy. Eligible participants include those with malignant melanoma in the adjuvant or metastatic setting, mesothelioma and non-small cell lung carcinoma (NSCLC) treated in the metastatic setting. Comprehensive clinical evaluation is carried out alongside blood, skin swab and stool sampling at the time of CPI initiation (baseline) and during subsequent routine hospital visits on 6 occasions over a 10-month follow-up period. It is conservatively anticipated that one third of enrolled patients will experience a "significant IrAE" (SirAE), defined according to pre-determined criteria specific to the affected tissue/organ system. Those developing such toxicity may optionally undergo a biopsy of affected tissue where appropriate, otherwise being managed according to standard of care. Peripheral blood mononuclear cells will be analysed using multi-parameter flow cytometry to investigate immune subsets, their activation status and cytokine profiles. Stool samples and skin swabs will undergo DNA extraction for 16 S ribosomal RNA (rRNA) sequencing and internal transcribed spacer (ITS) gene sequencing to determine bacterial and fungal microbiome diversity, respectively, including species associated with toxicity. Stored tissue biopsies will be available for in situ and single-cell transcriptomic evaluation. Analysis will focus on the identification of biological predictors and precursors of SirAEs. DISCUSSION: The pathogenesis of IrAEs will be assessed through the MEDALLION cohort, with the potential to develop tools for their prediction and/or strategies for targeted prevention or treatment. TRIAL REGISTRATION: The study was registered on 18/09/2023 in the ISRCTN registry (43,419,676).

A prospective humoral immune monitoring study of kidney transplant recipients receiving three doses of SARS-CoV-2 mRNA vaccine.

Kidney transplant recipients (KTRs), like other solid organ transplant recipients display a suboptimal response to mRNA vaccines, with only about half achieving seroconversion after two doses. However, the effectiveness of a booster dose, particularly in generating neutralizing antibodies (NAbs), remains poorly understood, as most studies have mainly focused on non-neutralizing antibodies. Here, we have longitudinally assessed the humoral response to the SARS-CoV-2 mRNA vaccine in 40 KTRs over a year, examining changes in both anti-spike IgG and NAbs following a booster dose administered about 5 months post-second dose. We found a significant humoral response increase 5 months post-booster, a stark contrast to the attenuated response observed after the second dose. Of note, nearly a quarter of participants did not achieve protective plasma levels even after the booster dose. We also found that the higher estimated glomerular filtration rate (eGFR) correlated with a more robust humoral response postvaccination. Altogether, these findings underscore the effectiveness of the booster dose in enhancing durable humoral immunity in KTRs, as evidenced by the protective level of NAbs found in 65% of the patients 5 months post- booster, especially those with higher eGFR rates.

Immune monitoring in pediatric kidney transplant.

BACKGROUND: Long-term outcomes in pediatric kidney transplantation remain suboptimal, largely related to chronic rejection. Creatinine is a late marker of renal injury, and more sensitive, early markers of allograft injury are an active area of current research. METHODS: This is an educational review summarizing existing strategies for monitoring for rejection in kidney transplant recipients. RESULTS: We summarize supporting currently available clinical tests, including surveillance biopsy, donor specific antibodies, and donor-derived cell free DNA, as well as the potential limitations of these studies. In addition, we review the current avenues of active research, including transcriptomics, proteomics, metabolomics, and torque tenovirus levels. CONCLUSION: Advancing the use of noninvasive immune monitoring will depend on well-designed multicenter trials that include patients with stable graft function, include biopsy results on all patients, and can demonstrate both association with a patient-relevant clinical endpoint such as graft survival or change in glomerular filtration rate and a potential timepoint for intervention.

[Optimizing efficacy and security of CAR-T cells, and immune monitoring]. / Optimisation de l'efficacité et de la sécurité d'utilisation des lymphocytes CAR-T.

The immune system plays a critical role in the control and eradication of tumors. A better understanding of the anti-tumor immune mechanisms over the last decade has led to the development of immunotherapies, including cellular therapies such as those using CAR-T cells. These therapies have been remarkably effective in hematological malignancies. However, their application to solid tumors requires some optimization. Many efforts are being made in this regard, both to increase the efficacy of CAR-T cells, and to make them more secure. For the former goal, there is a need for the identification of new targets, better activation strategies, or arming T cells in a way that makes them able to overcome intra-tumoral barriers. For the latter goal, dose adjustment, locoregional administration or use of suicide genes are currently investigated as ways to mitigate the risks of this therapy. Together, these adjustments will permit larger applicability of CAR-T cells, in anti-tumor immunity, but also in the context of auto-immune diseases or fibrolytic therapies.

Title: Optimisation de l'efficacité et de la sécurité d'utilisation des lymphocytes CAR-T. Abstract: Le système immunitaire joue un rôle déterminant dans le contrôle et l'éradication des tumeurs. Une meilleure compréhension des mécanismes en jeu a permis le développement des immunothérapies, et notamment des thérapies par lymphocytes CAR-T. Ces thérapies ont montré une grande efficacité dans les maladies hématologiques, mais leur application aux tumeurs solides nécessite des optimisations pour améliorer leur efficacité et leur sécurité. Ces ajustements permettront une plus grande applicabilité des lymphocytes CAR-T, non seulement pour les traitements anti-tumoraux mais aussi pour le traitement de maladies auto-immunes ou fibreuses.

Potential biomarkers for immune monitoring after renal transplantation.

Renal transplantation represents the foremost efficacious approach for ameliorating end-stage renal disease. Despite the current state of advanced renal transplantation techniques and the established postoperative immunosuppression strategy, a subset of patients continues to experience immune rejection during both the early and late postoperative phases, ultimately leading to graft loss. Consequently, the identification of immunobiomarkers capable of predicting the onset of immune rejection becomes imperative in order to facilitate early intervention strategies and enhance long-term prognoses. Upon reviewing the pertinent literature, we identified several indicators that could potentially serve as immune biomarkers to varying extents. These include the T1/T2 ratio, Treg/Th17 ratio, IL-10/TNF-α ratio, IL-33, IL-34, IL-6, IL-4, other cytokines, and NOX2/4.

Tumor-specific T cells support chemokine-driven spatial organization of intratumoral immune microaggregates needed for long survival.

BACKGROUND: The composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre-existing tumor-specific T cells. Here, the blueprint of the TIME associated with such an ongoing tumor-specific T-cell response was dissected in a unique prospective oropharyngeal squamous cell carcinoma (OPSCC) cohort, in which tumor-specific tumor-infiltrating T cells were detected (immune responsiveness (IR+)) or not (lack of immune responsiveness (IR-)). METHODS: A comprehensive multimodal, high-dimensional strategy was applied to dissect the TIME of treatment-naive IR+ and IR- OPSCC tissue, including bulk RNA sequencing (NanoString), imaging mass cytometry (Hyperion) for phenotyping and spatial interaction analyses of immune cells, and combined single-cell gene expression profiling and T-cell receptor (TCR) sequencing (single-cell RNA sequencing (scRNAseq)) to characterize the transcriptional states of clonally expanded tumor-infiltrating T cells. RESULTS: IR+ patients had an excellent survival during >10 years follow-up. The tumors of IR+ patients expressed higher levels of genes strongly related to interferon gamma signaling, T-cell activation, TCR signaling, and mononuclear cell differentiation, as well as genes involved in several immune signaling pathways, than IR- patients. The top differently overexpressed genes included CXCL12 and LTB, involved in ectopic lymphoid structure development. Moreover, scRNAseq not only revealed that CD4+ T cells were the main producers of LTB but also identified a subset of clonally expanded CD8+ T cells, dominantly present in IR+ tumors, which secreted the T cell and dendritic cell (DC) attracting chemokine CCL4. Indeed, immune cell infiltration in IR+ tumors is stronger, highly coordinated, and has a distinct spatial phenotypical signature characterized by intratumoral microaggregates of CD8+CD103+ and CD4+ T cells with DCs. In contrast, the IR- TIME comprised spatial interactions between lymphocytes and various immunosuppressive myeloid cell populations. The impact of these chemokines on local immunity and clinical outcome was confirmed in an independent The Cancer Genome Atlas OPSCC cohort. CONCLUSION: The production of lymphoid cell attracting and organizing chemokines by tumor-specific T cells in IR+ tumors constitutes a positive feedback loop to sustain the formation of the DC-T-cell microaggregates and identifies patients with excellent survival after standard therapy.

The importance of immune checkpoints in immune monitoring: A future paradigm shift in the treatment of cancer.

The growth and development of cancer are directly correlated to the suppression of the immune system. A major breakthrough in cancer immunotherapy depends on various mechanisms to detect immunosuppressive factors that inhibit anti-tumor immune responses. Immune checkpoints are expressed on many immune cells such as T-cells, regulatory B cells (Bregs), dendritic cells (DCs), natural killer cells (NKs), regulatory T (Tregs), M2-type macrophages, and myeloid-derived suppressor cells (MDSCs). Immune inhibitory molecules, including CTLA-4, TIM-3, TIGIT, PD-1, and LAG-3, normally inhibit immune responses via negatively regulating immune cell signaling pathways to prevent immune injury. However, the up-regulation of inhibitory immune checkpoints during tumor progression on immune cells suppresses anti-tumor immune responses and promotes immune escape in cancer. It has recently been indicated that cancer cells can up-regulate various pathways of the immune checkpoints. Therefore, targeting immune inhibitory molecules through antibodies or miRNAs is a promising therapeutic strategy and shows favorable results. Immune checkpoint inhibitors (ICIs) are introduced as a new immunotherapy strategy that enhance immune cell-induced antitumor responses in many patients. In this review, we highlighted the function of each immune checkpoint on different immune cells and therapeutic strategies aimed at using monoclonal antibodies and miRNAs against inhibitory receptors. We also discussed current challenges and future strategies for maximizing these FDA-approved immunosuppressants' effectiveness and clinical success in cancer treatment.

Early use of canakinumab to prevent mechanical ventilation in select COVID-19 patients: A retrospective, observational analysis.

INTRODUCTION: The fully-human monoclonal anti-interleukin (IL)-1ß antibody canakinumab may inhibit the production of inflammatory mediators in patients with coronavirus disease 2019 (COVID-19) and the hyperinflammatory response potentially leading to acute respiratory distress syndrome. OBJECTIVES: The goal of our retrospective, observational analysis was to evaluate the safety and efficacy of subcutaneous (s.c.) canakinumab in combination with our standard of care (SOC) treatment of selected patients with COVID-19 with respiratory failure and elevated reactive pro-inflammatory markers. METHODS: Eight participants received two doses of s.c. canakinumab 150 mg (or 2 mg/kg for participants weighing ≤40 kg) in addition to SOC. 12 patients received only SOC treatment. RESULTS: Canakinumab treatment reduced the need for mechanical ventilation and reduced proinflammatory markers, resulting in an amelioration of the final outcome, with respect to the control group who received SOC alone. The treatment was safe and well tolerated; no adverse events were reported. CONCLUSION: The use of canakinumab (300 mg, s.c.) in the early stage of COVID-19 with mild-to-moderate respiratory failure was superior to SOC at preventing clinical deterioration and may warrant further investigation as a treatment option for patients with COVID-19 who experience a hyperinflammatory response in the early stage of the disease.

High Expression of Tim-3 in Alveolar Echinococcosis Mediates Depletion of CD8+ T Cell Function.

OBJECTIVE: CD8+ T cells can participate in immune action by secreting various cytokines, which have a killing effect on certain viruses, tumor cells, and other antigenic substances. However, in studies such as chronic viral infections and some parasitic infections, CD8+ T lymphocyte showed functional depletion, and its immune dysfunction was an important reason for the persistence of infection. Tim-3 has been shown to be a negative regulator of CD8+ T cell function, causing depletion of CD8+ T cells in cancer and chronic infection. However, the relationship between Tim-3 and CD8+ T cells in Echinococcus multilocularis infection is not clear. METHODS: In this study, we analyzed peripheral blood CD8+ T cells from 62 alveolar echinococcosis (AE) patients and 30 healthy controls. RESULTS: Compared with the healthy control group, the proportion of CD8+ T cells in the peripheral blood of AE patients increased significantly, while the levels of perforin, granzyme B and IFN-γ in peripheral blood CD8+ T cell related factors of metabolically active alveolar echinococcosis (MAAE) patients decreased significantly. Later detection revealed that the expression of Tim-3 on CD8+ T cells in the peripheral blood of MAAE patients was significantly higher than that of metabolically inactive alveolar echinococcosis (MIAE) patients and healthy controls. The expression levels of function-related factors perforin, granzyme B and IFN-γ in CD8+ Tim-3+ T cell were significantly lower in the CD8+Tim-3- T cells of AE patients. In vitro, the secretion of CD8+ T cell-associated factors was significantly restored by inhibiting Tim-3 expression. CONCLUSION: Therefore, the depletion of CD8+ T lymphocyte in patients with alveolar echinococcosis disease is considered to be related to the high expression of Tim-3 on the surface.

Optimized workflow for human PBMC multiomic immunosurveillance studies.

Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021).

CXCR6 by increasing retention of memory CD8+ T cells in the ovarian tumor microenvironment promotes immunosurveillance and control of ovarian cancer.

PURPOSE: Resident memory CD8 T cells, owing to their ability to reside and persist in peripheral tissues, impart adaptive sentinel activity and amplify local immune response, and have beneficial implications for tumor surveillance and control. The current study aimed to clarify the less known chemotactic mechanisms that govern the localization, retention, and residency of memory CD8 T cells in the ovarian tumor microenvironment. EXPERIMENTAL DESIGN: RNA and protein expressions of chemokine receptors in CD8+ resident memory T cells in human ovarian tumor-infiltrating CD8+ T cells and their association with survival were analyzed. The role of CXCR6 on antitumor T cells was investigated using prophylactic vaccine models in murine ovarian cancer. RESULTS: Chemokine receptor profiling of CD8+CD103+ resident memory tumor-infiltrating lymphocytes in patients with ovarian cancer revealed high expression of CXCR6. Analysis of The Cancer Genome Atlas (TCGA) (ovarian cancer database revealed CXCR6 to be associated with CD103 and increased patient survival. Functional studies in mouse models of ovarian cancer revealed that CXCR6 is a marker of resident, but not circulatory, tumor-specific memory CD8+ T cells. CXCR6-deficient tumor-specific CD8+ T cells showed reduced retention in tumor tissues, leading to diminished resident memory responses and poor control of ovarian cancer. CONCLUSIONS: CXCR6, by promoting retention in tumor tissues, serves a critical role in resident memory T cell-mediated immunosurveillance and control of ovarian cancer. Future studies warrant exploiting CXCR6 to promote resident memory responses in cancers.

High-Throughput 3D In Vitro Tumor Vasculature Model for Real-Time Monitoring of Immune Cell Infiltration and Cytotoxicity.

Recent advances in anticancer therapy have shown dramatic improvements in clinical outcomes, and adoptive cell therapy has emerged as a type of immunotherapy that can modulate immune responses by transferring engineered immune cells. However, a small percentage of responders and their toxicity remain as challenges. Three-dimensional (3D) in vitro models of the tumor microenvironment (TME) have the potential to provide a platform for assessing and predicting responses to therapy. This paper describes an in vitro 3D tumor model that incorporates clusters of colorectal cancer (CRC) cells around perfusable vascular networks to validate immune-cell-mediated cytotoxicity against cancer cells. The platform is based on an injection-molded 3D co-culture model and composed of 28 microwells where separate identical vascularized cancer models can be formed. It allows robust hydrogel patterning for 3D culture that enables high-throughput experimentation. The uniformity of the devices resulted in reproducible experiments that allowed 10× more experiments to be performed when compared to conventional polydimethylsiloxane (PDMS)-based microfluidic devices. To demonstrate its capability, primary natural killer (NK) cells were introduced into the vascularized tumor network, and their activities were monitored using live-cell imaging. Extravasation, migration, and cytotoxic activity against six types of CRC cell lines were tested and compared. The consensus molecular subtypes (CMS) of CRC with distinct immune responses resulted in the highest NK cell cytotoxicity against CMS1 cancer cells. These results show the potential of our vascularized tumor model for understanding various steps involved in the immune response for the assessment of adoptive cell therapy.

ACPA Antibodies Titer at the Time of Rheumatoid Arthritis Diagnosis Is Not Associated with Disease Severity.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory and destructive joint disease with the presence of autoantibodies, rheumatoid factor (RF), and anti-citrullinated protein antibodies (ACPA). The presence of RF or ACPA predicts RA severity. Data on the influence of ACPA titer on RA course are limited. OBJECTIVES: To determine the correlation between ACPA titers at the time of RA diagnosis to RA features and severity during 3 years of follow-up. METHODS: We performed a retrospective study of RA patients treated at our institution during the years 2006-2015 with known ACPA titers at RA diagnosis who completed at least 3 years of follow-up. Patients (N=133) were divided according to ACPA titer: seronegative (< 15 U/ml, n=55), weakly positive (15-49 U/ml, n=18), moderately positive (50-300 U/ml, n=29), and strongly positive (> 300 U/ml, n=31). Patient data, including disease activity score (DAS28), bone erosion on hand and/or foot X-rays, treatments with corticosteroids and disease-modifying-anti-rheumatic drugs (DMARDs), and hospitalizations, were recorded. Chi-square and Mann-Whitney method were used for statistical analysis. P < 0.05 was considered as statistically significant. RESULTS: Male gender, smoking, and RF positivity correlated with ACPA positivity and higher ACPA titers. There was no correlation between ACPA titer and the variables defined as representing RA severity: higher DAS28, bone erosions, hospitalizations, need for corticosteroids, and conventional and biological DMARDs. CONCLUSIONS: Titer of ACPA was not identified as a predictive factor for RA severity.

NLRP3 Deficiency in Hepatocellular Carcinoma Enhances Surveillance of NK-92 through a Modulation of MICA/B.

Human hepatocellular carcinoma (HCC) is the most common and even worse at prognosis. The patients with HCC which accompanied by other diseases, such as cirrhosis, can be limited in various treatments, such as chemotherapy, not HCC patients without other diseases. NLRP3 inflammasome plays an important role in the innate immune response, but emerging evidence has indicated that the NLRP3 inflammasome is implicated in all stages of cancer development. Various cells express NLRP3 protein through the autocrine or paracrine signaling in their environment, but NK cells do not. The expanding evidence shows that patients who suffer from liver cancers have a low frequency of natural killer (NK) cells, and the function of these cells is also impaired. Thus, we examined how the expression of NLRP3 in HCC cells affects cancer surveillance by NK cells in a state of a co-culture of both cells. When the expression of NLRP3 in HCC cells was ablated, MICA/B on the surface of HCC cells was upregulated through the lowered expression of matrix metalloproteinase. The expression of MICA on the surface of HCC cells interacted with the NKG2D receptor on NK-92 cells, which led to NK cytotoxicity. Furthermore, in a xenograft mice model, NLRP3 KO HCC cells delayed tumor development and metastasis as well as increased the sensitivity to NK cell cytotoxicity. Taken together, NLRP3 KO in HCC could enhance NK immunosurveillance through an interaction of NKG2D-MICA.

Immune Monitoring Assay for Extracorporeal Photopheresis Treatment Optimization After Heart Transplantation.

Background: Extracorporeal photopheresis (ECP) induces immunological changes that lead to a reduced risk of transplant rejection. The aim of the present study was to determine optimum conditions for ECP treatment by analyzing a variety of tolerance-inducing immune cells to optimize the treatment. Methods: Ten ECP treatments were applied to each of 17 heart-transplant patients from month 3 to month 9 post-HTx. Blood samples were taken at baseline, three times during treatment, and four months after the last ECP treatment. The abundance of subsets of tolerance-inducing regulatory T cells (Tregs) and dendritic cells (DCs) in the samples was determined by flow cytometry. A multivariate statistical model describing the immunological status of rejection-free heart transplanted patients was used to visualize the patient-specific immunological improvement induced by ECP. Results: All BDCA+ DC subsets (BDCA1+ DCs: p < 0.01, BDCA2+ DCs: p < 0.01, BDCA3+ DCs: p < 0.01, BDCA4+ DCs: p < 0.01) as well as total Tregs(p < 0.01) and CD39+ Tregs(p < 0.01) increased during ECP treatment, while CD62L+ Tregs decreased (p < 0.01). The cell surface expression level of BDCA1 (p < 0.01) and BDCA4 (p < 0.01) on DCs as well as of CD120b (p < 0.01) on Tregs increased during the study period, while CD62L expression on Tregs decreased significantly (p = 0.04). The cell surface expression level of BDCA2 (p = 0.47) and BDCA3 (p = 0.22) on DCs as well as of CD39 (p = 0.14) and CD147 (p = 0.08) on Tregs remained constant during the study period. A cluster analysis showed that ECP treatment led to a sustained immunological improvement. Conclusions: We developed an immune monitoring assay for ECP treatment after heart transplantation by analyzing changes in tolerance-inducing immune cells. This assay allowed differentiation of patients who did and did not show immunological improvement. Based on these results, we propose classification criteria that may allow optimization of the duration of ECP treatment.

Clinical remission and subsequent relapse in patients with juvenile idiopathic arthritis: predictive factors according to therapeutic approach.

BACKGROUND: Juvenile idiopathic arthritis constitutes a significant cause of disability and quality of life impairment in pediatric and adult patients. The aim of this study was to ascertain clinical remission (CR) and subsequent relapse in juvenile idiopathic arthritis (JIA) patients, according to therapeutic approach and JIA subtype. Evidence in literature regarding its predictors is scarce. METHODS: We conducted an observational, ambispective study. Patients diagnosed of JIA, treated with synthetic and/or biologic disease modifying antirheumatic drugs (DMARD) were included and followed-up to December 31st, 2015. Primary outcome was clinical remission defined by Wallace criteria, both on and off medication. In order to ascertain CR according to therapeutic approach, DMARD treatments were divided in four groups: 1) synthetic DMARD (sDMARD) alone, 2) sDMARD combined with another sDMARD, 3) sDMARD combined with biologic DMARD (bDMARD), and 4) bDMARD alone. RESULTS: A total of 206 patients who received DMARD treatment were included. At the time the follow-up was completed, 70% of the patients in the cohort had attained CR at least once (144 out of 206), and 29% were in clinical remission off medication (59 out of 206). According to treatment group, CR was more frequently observed in patients treated with synthetic DMARD alone (53%). Within this group, CR was associated with female sex, oligoarticular persistent subtypes, ANA positivity, Methotrexate treatment and absence of HLA B27, comorbidities and DMARD toxicity. 124 DMARD treatments (62%) were withdrawn, 64% of which relapsed. Lower relapse rates were observed in those patients with persistent oligoarticular JIA (93%) when DMARD dose was tapered before withdrawal (77%). CONCLUSIONS: More than two thirds of JIA patients attained CR along the 9 years of follow-up, and nearly one third achieved CR off medication. Females with early JIA onset, lower active joint count and ANA positivity were the ones achieving and sustaining remission more frequently, especially when receiving synthetic DMARD alone and in the absence of HLA B27, comorbidities or previous DMARD toxicity.

Immunological repertoire linked to PSTPIP1-associated myeloid-related inflammatory (PAMI) syndrome.

BACKGROUND: Mutations along PSTPIP1 gene are associated to two specific conditions, PAPA syndrome and PAMI syndrome, both autoinflammatory disorders associated to disturbances in cytoskeleton formation. Immunological aspects of PAMI syndrome has not yet been reported neither the clinical impact on therapeutical decisions. METHODS: Clinical data of patients records were retrospectively accessed. Genomic DNA were extracted and sequenced following standard procedures. Peripheral lymphocytes were quantified in T, B e FOXP3 phenotypes. RESULTS: We describe two related patients with PAMI syndrome harboring the usual E250K mutation. Anti-IL1 therapy could partially control the disease in the index patient. A broad spectrum of immunological effects as well as an aberrant expression of FOXP3 could be observed. CONCLUSIONS: Here we report two related brazilian patients with PAMI syndromes harboring the E250K mutation in PSTPIP1, their immunological aspects and the therapeutical response to canakinumab.

Converting an Anti-Mouse CD4 Monoclonal Antibody into an scFv Positron Emission Tomography Imaging Agent for Longitudinal Monitoring of CD4+ T Cells.

Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4+ T cells by immuno-PET. We tracked CD4+ and CD8+ T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4+ and CD8+ T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4+ and CD8+ T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents.

[Immunomonitoring of patients treated with CAR-T cells for hematological malignancy: Guidelines from the CARTi group and the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)]. / Suivi immunologique des patients traités par cellules CAR-T pour hémopathie maligne: recommandations du groupe CARTi et de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

CAR-T cells represent a new anti-tumor immunotherapy which has shown its clinical efficacy in B-cell malignancies. The results of clinical trials carried out in this context have shown that certain immunological characteristics of patients before (at the time of apheresis) and after the administration of the treatment, or of the CAR-T cells themselves, are correlated with the response to the treatment or to its toxicity. However, to date, there are no recommendations on the immunological monitoring of patients treated in real life. The objectives of this workshop were to determine, based on data from the literature and the experience of the centers, the immunological analyses to be carried out in patients treated with CAR-T cells. The recommendations relate to the characterization of the patient's immune cells at the time of apheresis, the characterization of the injected CAR-T cells, as well as the monitoring of the CAR-T cells and other parameters of immune reconstitution in the patient after administration of the treatment. Harmonization of practices will allow clinical-biological correlation studies to be carried out in patients treated in real life with the aim of identifying factors predictive of response and toxicity. Such data could have a major medico-economic impact by making it possible to identify the patients who will optimally benefit from these expensive treatments.

Lipofection with Synthetic mRNA as a Simple Method for T-Cell Immunomonitoring.

The quantification of T-cell immune responses is crucial for the monitoring of natural and treatment-induced immunity, as well as for the validation of new immunotherapeutic approaches. The present study presents a simple method based on lipofection of synthetic mRNA in mononuclear cells as a method to determine in vitro T-cell responses. We compared several commercially available transfection reagents for their potential to transfect mRNA into human peripheral blood mononuclear cells and murine splenocytes. We also investigated the impact of RNA modifications in improving this method. Our results demonstrate that antigen-specific T-cell immunomonitoring can be easily and quickly performed by simple lipofection of antigen-coding mRNA in complex immune cell populations. Thus, our work discloses a convenient solution for the in vitro monitoring of natural or therapy-induced T-cell immune responses.