Brigitte Le Mauff

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.

Background: Anti-N-Methyl-d-aspartate receptor (NMDAR) autoimmune encephalitis (NMDAR AE) is an autoimmune disease characterized by severe psychiatric and neurological symptoms. While the pathogenic role of antibodies (Abs) directed against the GluN1 subunit of NMDAR is well described in this disease, the immune mechanisms involved in the generation of the autoimmune B cell response, especially the role of T helper cells, are poorly understood. Previously, we developed a B-cell-mediated mouse model of NMDAR AE by immunization with a GluN1_359–378 peptide that drives a series of symptoms that recapitulate AE such as anxiety behaviour and spatial memory impairment. Results: In this mouse model, we identified anti-GluN1-specific CD4⁺ but also CD8⁺ T cells in both spleen and meninges. T helper cells have a polyfunctional profile, arguing for a T and B cell crosstalk to generate anti-GluN1 pathogenic Abs. Interestingly, proteomic analysis of AE meninges showed enrichment of differentially expressed proteins in biological processes associated with B cell activation and cytokine signalling pathways. Conclusions: This study identified, for the first time, a potential contribution of T helper cells in the pathology of NMDAR AE and paved the way for the development of future tolerogenic approaches to treat relapses.

Background: The link between immediate hypersensitivity reactions (HSR) following the first cetuximab infusion and the IgE sensitization against anti-galactose-α-1,3-galactose (α-Gal) is now well-established. An automated Fluoroenzyme-Immunoassay (FEIA) is available and may facilitate the screening of patients with anti-α-Gal IgE before treatment. Methods: This study aimed to evaluate its performances as compared to a previously validated anti-cetuximab IgE ELISA, using 185 samples from two previously studied cohorts. Results: Despite 21.1% of discrepancies between the two techniques, FEIA discriminated better positive patients and similarly negative ones with a ≥ 0.525 kU_A/L threshold. Sensitivity was 87.5% for both tests, specificity was better for FEIA (96.3% vs ELISA: 82.1%). FEIA had a higher positive likelihood ratio (23.9 vs ELISA: 4.89) and a similar negative likelihood ratio (0.13 vs ELISA: 0.15). In our population, the risk of severe HSR following a positive test was higher with FEIA (56.7% vs ELISA: 19.6%) and similar following a negative test (0.7% vs ELISA: 0.8%). Conclusion: Although the predictive value of the IgE screening before cetuximab infusion remains discussed, this automated commercial test can identify high-risk patients and is suitable for routine use in laboratories. It could help avoiding cetuximab-induced HSR by a systematic anti-α-Gal IgE screening before treatment.

Multiple sclerosis is a chronic demyelinating disease of the central nervous system (CNS) with an autoimmune component. Among the recent disease-modifying treatments available, Natalizumab, a monoclonal antibody directed against the alpha chain of the VLA-4 integrin (CD49d), is a potent inhibitor of cell migration toward the tissues including CNS. It potently reduces relapses and active brain lesions in the relapsing remitting form of the disease. However, it has also been associated with a severe infectious complication, the progressive multifocal leukoencephalitis (PML). Using the standard protocol with an injection every 4 weeks it has been shown by a close monitoring of the drug that trough levels soon reach a plateau with an almost saturation of the target cell receptor as well as a down modulation of this receptor. In this review, mechanisms of action involved in therapeutic efficacy as well as in PML risk will be discussed. Furthermore the interest of a biological monitoring that may be helpful to rapidly adapt treatment is presented. Indeed, development of anti-NAT antibodies, although sometimes unapparent, can be detected indirectly by normalization of CD49d expression on circulating mononuclear cells and might require to switch to another drug. On the other hand a stable modulation of CD49d expression might be useful to follow the circulating NAT levels and apply an extended interval dose scheme that could contribute to limiting the risk of PML.

Cetuximab, a chimeric mouse-human IgG1 monoclonal antibody against the epidermal growth factor receptor, has proven effective in the treatment of metastatic colorectal cancer and squamous cell carcinoma of the head and neck. However, a high incidence of immediate hypersensitivity reactions (HSR) to cetuximab after the first infusion has been observed. We have developed a test for identification of patients likely to show treatment-related HSR to cetuximab. An enzyme-linked immunosorbent assay (ELISA) for detecting anti-cetuximab IgEs was developed and tested on serum samples collected from cancer patients before start of cetuximab treatment, and from healthy blood donors. Similar levels of anti-cetuximab IgE were detected in pre-treatment patient sera (24/92, 26.1%) and sera from healthy blood donors (33/117, 28.2%). HSR were observed in 14 out of the 92 patients (15.2%), and 8 of these (57.1%) were grade 3-4. Anti-cetuximab IgEs were detected in 7/8 of the patients (87.5%) with severe HSRs as compared wi...