Changes in T-cell subsets, preexisting cytopenias and hyperferritinaemia correlate with cytopenias after BCMA targeted CAR T-cell therapy in relapsed/refractory multiple myeloma: Results from a prospective comprehensive biomarker study.

Biomarkers for cytopenias following CAR T-cell treatment in relapsed/refractory (RR) multiple myeloma (MM) are not completely defined. We prospectively analysed 275 sequential peripheral blood (PB) samples from 58 RRMM patients treated with BCMA-targeted CAR T cells, and then divided them into three groups: (i) baseline (before leukapheresis), (ii) ≤day+30, and (iii) >day+30 after CAR T-cell therapy. We evaluated laboratory data and performed flow cytometry to determine the (CAR) T-cell subsets. Baseline hyperferritinaemia was a risk factor for long-lasting grade ≥3 anaemia (r = 0.47, p < 0.001) and thrombocytopenia (r = 0.44, p = 0.002) after CAR T-cell therapy. Low baseline haemoglobin (Hb) and PLT were associated with long-lasting grade ≥3 anaemia (r = -0.56, p < 0.001) and thrombocytopenia (r = -0.44, p = 0.002) respectively. We observed dynamics of CAR-negative T-cell subsets following CAR T-cell infusion. In the late phase after CAR T-cell therapy (>day+30), CD4Tn frequency correlated with anaemia (r = 0.41, p = 0.0014) and lymphocytopenia was related to frequencies of CD8+ T cells (r = 0.72, p < 0.001) and CD8Teff (r = 0.64, p < 0.001). CD4Tcm frequency was correlated with leucocytopenia (r = -0.49, p < 0.001). In summary, preexisting cytopenias and hyperferritinaemia indicated long duration of grade ≥3 post-CAR T-cell cytopenias. Prolonged cytopenia may be related to immune remodelling with a shift in the CAR-negative T-cell subsets following CAR T-cell therapy.

Effects of Alemtuzumab on (Auto)antigen-Specific Immune Responses.

Alemtuzumab (anti-CD52 mAb) leads to a long-lasting disease activity suppression in patients with relapsing forms of multiple sclerosis (MS). In this study, we examined the change of the immune cell repertoire and the cellular reactivity after treatment with alemtuzumab. We analyzed the number of IFN-γ-secreting cells in presence of several peptides which had been eluted from the central nervous system (CNS) of MS patients and are possible targets of autoreactive T cells in MS. The patients showed a stabilized disease activity measured in clinical parameters and lesion formation after the treatment. We detected a reduction of the number of IFN-γ-secreting cells in the presence of every tested self-antigen. The number of IFN-γ-secreting cells was also reduced in the presence of non-self-antigens. We also found a clear change in the immune cell repertoire. After an almost complete depletion of all lymphocytes, the cell specificities showed different reconstitution patterns, resulting in different cell fractions. The percentage of CD4+ T cells was clearly reduced after therapy, whereas the fractions of B and NK cells were elevated. When we evaluated the number of IFN-γ-secreting cells in relation to the number of present CD4+ T cells, we still found a significant reduction. We conclude that the reduction of IFN-γ-secreting cells by alemtuzumab is not only due to a reduction of the CD4+ T cell fraction within the peripheral blood mononuclear cell (PBMC) compartment but might also be caused by functional changes or a shift in the distribution of different subtypes in the CD4+ T cell pool.

IL-3 promotes the development of experimental autoimmune encephalitis.

Little is known about the role of IL-3 in multiple sclerosis (MS) in humans and in experimental autoimmune encephalomyelitis (EAE). Using myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE, we show that CD4+ T cells are the main source of IL-3 and that cerebral IL-3 expression correlates with the influx of T cells into the brain. Blockade of IL-3 with monoclonal antibodies, analysis of IL-3 deficient mice, and adoptive transfer of leukocytes demonstrate that IL-3 plays an important role for development of clinical symptoms of EAE, for migration of leukocytes into the brain, and for cerebral expression of adhesion molecules and chemokines. In contrast, injection of recombinant IL-3 exacerbates EAE symptoms and cerebral inflammation. In patients with relapsing-remitting MS (RRMS), IL-3 expression by T cells is markedly upregulated during episodes of relapse. Our data indicate that IL-3 plays an important role in EAE and may represent a new target for treatment of MS.

Peripheral Blood Mononuclear Cells: Isolation, Freezing, Thawing, and Culture.

The work with peripheral blood mononuclear cells (PBMCs), which comprise lymphocytes and monocytes, is indispensable in immunological diagnostics and research. The isolation of PBMCs takes advantage of differences in cell density of the different blood components. Density gradient centrifugation of diluted whole blood layered over a density gradient medium yields PBMCs; two subsequent washing steps remove remaining platelets. To store the cells for future assays, they can be frozen and thawed when required. Dimethyl sulfoxide (DMSO) serves as a cryoprotectant for freezing PBMCs, but must be removed by washing after thawing, as it can become toxic to the cells on longer exposure.

Antigen Presentation, Autoantigens, and Immune Regulation in Multiple Sclerosis and Other Autoimmune Diseases.

Antigen presentation is in the center of the immune system, both in host defense against pathogens, but also when the system is unbalanced and autoimmune diseases like multiple sclerosis (MS) develop. It is not just by chance that a major histocompatibility complex gene is the major genetic susceptibility locus in MS; a feature that MS shares with other autoimmune diseases. The exact etiology of the disease, however, has not been fully understood yet. T cells are regarded as the major players in the disease, but most probably a complex interplay of altered central and peripheral tolerance mechanisms, T-cell and B-cell functions, characteristics of putative autoantigens, and a possible interference of environmental factors like microorganisms are at work. In this review, new data on all these different aspects of antigen presentation and their role in MS will be discussed, probable autoantigens will be summarized, and comparisons to other autoimmune diseases will be drawn.