Targeting TGFß-activated kinase-1 activation in microglia reduces CAR T immune effector cell-associated neurotoxicity syndrome.

Cancer immunotherapy with chimeric antigen receptor (CAR) T cells can cause immune effector cell-associated neurotoxicity syndrome (ICANS). However, the molecular mechanisms leading to ICANS are not well understood. Here we examined the role of microglia using mouse models and cohorts of individuals with ICANS. CD19-directed CAR (CAR19) T cell transfer in B cell lymphoma-bearing mice caused microglia activation and neurocognitive deficits. The TGFß-activated kinase-1 (TAK1)-NF-κB-p38 MAPK pathway was activated in microglia after CAR19 T cell transfer. Pharmacological TAK1 inhibition or genetic Tak1 deletion in microglia using Cx3cr1CreER:Tak1fl/fl mice resulted in reduced microglia activation and improved neurocognitive activity. TAK1 inhibition allowed for potent CAR19-induced antilymphoma effects. Individuals with ICANS exhibited microglia activation in vivo when studied by translocator protein positron emission tomography, and imaging mass cytometry revealed a shift from resting to activated microglia. In summary, we prove a role for microglia in ICANS pathophysiology, identify the TAK1-NF-κB-p38 MAPK axis as a pathogenic signaling pathway and provide a rationale to test TAK1 inhibition in a clinical trial for ICANS prevention after CAR19 T cell-based cancer immunotherapy.

TaKeTiNa Music Therapy for Outpatient Treatment of Depression: Study Protocol for a Randomized Clinical Trial.

BACKGROUND/OBJECTIVES: Depression is a prevalent and debilitating illness that significantly affects psychological and physical well-being. Apart from conventional therapies such as psychotherapy and medication, individuals with depression often lack opportunities for activities that are generally perceived as enjoyable, such as music, meditation, and arts, which have demonstrated therapeutic effectiveness. TaKeTiNa music therapy has been employed as a therapeutic intervention for more than two decades. However, there is a notable absence of well-designed clinical trials investigating its antidepressant effects, a gap we aim to address in our current study. Furthermore, shifts in the progression of depression may manifest both psychologically, by influencing emotional states, and physiologically, by leading to alterations in lipid and sphingolipid metabolism, cortisol levels, and immune system function. Our study seeks to analyze the impact of TaKeTiNa music therapy on both levels. METHODS: This is a prospective monocentric randomized waitlist-controlled clinical trial. It investigates the influence of TaKeTiNa music therapy on patients with major depression in an outpatient setting. Therefore, interested persons are randomly assigned to two groups, an intervention group or a control group, after completing a screening procedure. The intervention group starts with an eight-week TaKeTiNa music therapy intervention. The waiting group receives the same therapy program after completing the follow-up period. Blood and saliva sampling as well as responses to questionnaires are obtained at specific time points. DISCUSSION: Our study investigates the effects of TaKeTiNa music therapy, a non-pharmacological antidepressant treatment option, on depressive symptoms. We also address functional and causal immunological changes; hormonal changes, such as changes in cortisol levels; and metabolic changes, such as changes in serum lipids and sphingolipids, during the course of depression. We expect that this study will provide evidence to expand the range of treatment options available for depression.

Preclinical Development of CAR T Cells with Antigen-Inducible IL18 Enforcement to Treat GD2-Positive Solid Cancers.

PURPOSE: Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN: We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS: Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS: These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022-501725-21-00).

Increased PD-1 Expression on Circulating T Cells Correlates with Inferior Outcome after Autologous Stem Cell Transplantation.

High-dose chemotherapy followed by autologous stem cell transplantation (auto-SCT) is a well-established treatment option for multiple myeloma and malignant lymphoma patients. It is able to induce long-term progression-free survival (PFS) in both patient groups and even provide a cure in patients with aggressive lymphoma. However, relapse is common and has been associated with the pace and quality of immunologic reconstitution after transplantation, as well as with immune cell exhaustion and immunometabolic defects. We aimed to analyze the dynamics of the prototypical exhaustion marker PD-1 on immune cells during reconstitution on high-dose chemotherapy followed by auto-SCT and its impact on PFS. We performed a comprehensive analysis of exhaustion and metabolic markers on immune cells from myeloma and lymphoma patients undergoing auto-SCT using flow cytometry and NanoString technologies. The expression levels of PD-1 were increased during early reconstitution after transplantation on T cells and natural killer (NK) cells, as well as on monocytes. However, while PD-1 expression in NK cells and monocytes normalized over time, PD-1 expression on T cells demonstrated a variable course. Of note, lymphoma patients with continuously increasing PD-1 expression on T cells after auto-SCT had an inferior median PFS of only 146 days, whereas the median PFS was not reached in the lymphoma patients without such a PD-1 expression pattern. T cells from patients with increased PD-1 expression after auto-SCT exhibited an immunometabolic (over)activation and exhausted phenotype compared to T cells from patients with a low PD-1 expression after transplantation, including higher levels of the glycolytic pacemaker enzyme hexokinase 2 and the inhibitory receptor CTLA-4. In addition, proliferating Ki-67+ T cells were more abundant in patients with high PD-1 expression on T cells compared to those with low expression after auto-SCT (11.9% versus 4.2%). PD-1 expression on T cells might serve as an adverse biomarker for lymphoma patients undergoing auto-SCT; however, further validation by larger prospective studies is required.

CD19 CAR T-Cell Therapy in Autoimmune Disease - A Case Series with Follow-up.

BACKGROUND: Treatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission. METHODS: We evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology-European League against Rheumatism (ACR-EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded. RESULTS: The median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR-EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell-associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization. CONCLUSIONS: In this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.).

CD19 Chimeric Antigen Receptor T Cell Treatment: Unraveling the Role of B Cells in Systemic Lupus Erythematosus.

B cell generation of autoantibodies is a crucial step in the pathogenesis of systemic lupus erythematosus (SLE). After their differentiation in the bone marrow, B cells populate the secondary lymphatic organs, where they undergo further maturation leading to the development of memory B cells as well as antibody-producing plasmablasts and plasma cells. Targeting B cells is an important strategy to treat autoimmune diseases such as SLE, in which B cell tolerance is disturbed and autoimmune B cells and autoantibodies emerge. This review discusses the functional aspects of antibody- and cell-based B cell-depleting therapy in SLE. It thereby particularly focuses on lessons learned from chimeric antigen receptor (CAR) T cell treatment on the role of B cells in SLE for understanding B cell pathology in SLE. CAR T cells model a deep B cell depletion and thereby allow understanding the role of aberrant B cell activation in the pathogenesis of SLE. Furthermore, the effects of B cell depletion on autoantibody production can be better described, ie, explaining the concept of different cellular sources of (auto-) antibodies in the form of short-lived plasmablasts and long-lived plasma cells, which differ in their susceptibility to B cell depletion and require different targeted therapeutic approaches. Finally, the safety of deep B cell depletion in autoimmune disease is discussed.

Cytokine and reactivity profiles in SLE patients following anti-CD19 CART therapy.

Chimeric antigen receptor (CAR) T cells targeting CD19+ B cells have demonstrated efficacy in refractory systemic lupus erythematosus (SLE). Although initial clinical data suggest that anti-CD19 CAR T cell therapy is well tolerated and highly effective, the immunologic consequences of CAR T cell therapy in SLE patients remain unclear. We profiled serum in six refractory SLE patients prior to and 3 months following CAR T cell infusion. Three months post T cell infusion, the inflammatory cytokines IL-6 and TNFα decreased in patient sera. This was accompanied by elevations in serum IL-7 and BAFF. Furthermore, SLE-associated antibodies dropped profoundly in five of six patients. Last, consistent with other reports of CD19 CAR T therapy in B cell malignancies, we were able to show marginal impact of anti-CD19 CART therapy on pre-existing humoral immune responses in SLE patients. Together, these results provide insights into the mechanisms of efficacy of anti-CD19 CAR T cell therapy in SLE.

Severe hematotoxicity after CD19 CAR-T therapy is associated with suppressive immune dysregulation and limited CAR-T expansion.

Prolonged cytopenias after chimeric antigen receptor (CAR) T cell therapy are a significant clinical problem and the underlying pathophysiology remains poorly understood. Here, we investigated how (CAR) T cell expansion dynamics and serum proteomics affect neutrophil recovery phenotypes after CD19-directed CAR T cell therapy. Survival favored patients with "intermittent" neutrophil recovery (e.g., recurrent neutrophil dips) compared to either "quick" or "aplastic" recovery. While intermittent patients displayed increased CAR T cell expansion, aplastic patients exhibited an unfavorable relationship between expansion and tumor burden. Proteomics of patient serum collected at baseline and in the first month after CAR-T therapy revealed higher markers of endothelial dysfunction, inflammatory cytokines, macrophage activation, and T cell suppression in the aplastic phenotype group. Prolonged neutrophil aplasia thus occurs in patients with systemic immune dysregulation at baseline with subsequently impaired CAR-T expansion and myeloid-related inflammatory changes. The association between neutrophil recovery and survival outcomes highlights critical interactions between host hematopoiesis and the immune state stimulated by CAR-T infusion.

Identification and characterization of T-cell receptors with therapeutic potential showing conserved specificity against all SARS-CoV 2 strains.

INTRODUCTION: Treatment of severe COVID-19 disease can be challenging in immunocompromized patients due to newly emerging virus variants of concern (VOC) escaping the humoral response. Thus, T cells recognizing to date unmutated epitopes are not only relevant for patients' immune responses against VOC, but might also serve as a therapeutic option for patients with severe COVID-19 disease in the future, e.g. following allogenic stem cell transplantation. METHODS: To this purpose, the activation, cytokine profile and specificity of T-cell clones against unmutated and omicron Spike (S)-protein was analyzed, HLA restriction was determined and most promising T-cell receptor (TCR) was introduced into allogeneic T cells via CRISPR/Cas9-mediated orthotopic TCR replacement. Finally, T-cell responses of engineered T cells was determined and durability of the TCR replacement measured. PERSPECTIVE: SARS-CoV-2 specific engineered T cells recognizing a genomically stable region of the S-protein of all SARS-CoV 2 variants were successfully generated. Such transgenic T cells exhibit favorable effector functions and provide a treatment option of immunocompromised COVID-19 patients.

Case Report: IBD-like colitis following CAR T cell therapy for diffuse large B cell lymphoma.

Chimeric antigen receptor (CAR) T cell therapy has become a new mainstay in the treatment of several hematologic malignancies, but the spectrum of associated complications is still incompletely defined. Here, we report the case of a 70-year-old female patient treated with tisagenlecleucel for diffuse large B cell lymphoma (DLBCL), who developed chronic diarrhea with characteristics of inflammatory bowel disease (IBD)-like colitis. CAR T cells were substantially enriched in the colon lamina propria and other diagnoses were ruled out. Thus, we conclude that IBD-like colitis in this patient was associated to CAR T cell therapy and needs to be considered as a rare potential complication.

Accumulation of T-cell-suppressive PD-L1high extracellular vesicles is associated with GvHD and might impact GvL efficacy.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the only curative treatment option for a number of hemato-oncological disorders. In fact, allo-HSCT is considered as one of the most successful immunotherapies as its clinical efficacy is based on the donor T-cells' capacity to control residual disease. This process is known as the graft-versus-leukemia (GvL) reaction. However, alloreactive T-cells can also recognize the host as foreign and trigger a systemic potentially life-threatening inflammatory disorder termed graft-versus-host disease (GvHD). A better understanding of the underlying mechanisms that lead to GvHD or disease relapse could help us to improve efficacy and safety of allo-HSCT. In recent years, extracellular vesicles (EVs) have emerged as critical components of intercellular crosstalk. Cancer-associated EVs that express the immune checkpoint molecule programmed death-ligand 1 (PD-L1) can suppress T-cell responses and thus contribute to immune escape. At the same time, it has been observed that inflammation triggers PD-L1 expression as part of a negative feedback network.Here, we investigated whether circulating EVs following allo-HSCT express PD-L1 and tested their efficacy to suppress the ability of (autologous) T-cells to effectively target AML blasts. Finally, we assessed the link between PD-L1 levels on EVs to (T-)cell reconstitution, GvHD, and disease relapse.We were able to detect PD-L1+ EVs that reached a peak PD-L1 expression at 6 weeks post allo-HSCT. Development of acute GvHD was linked to the emergence of PD-L1high EVs following allo-HSCT. Moreover, PD-L1 levels correlated positively with GvHD grade and declined (only) on successful therapeutic intervention. T-cell-inhibitory capacity was higher in PD-L1high EVs as compared with their PD-L1low counterparts and could be antagonized using PD-L1/PD-1 blocking antibodies. Abundance of T-cell-suppressive PD-L1high EVs appears to also impact GvL efficacy as patients were at higher risk for relapse. Finally, patients of PD-L1high cohort displayed a reduced overall survival.Taken together, we show that PD-L1-expressing EVs are present following allo-HSCT. PD-L1 levels on EVs correlate with their ability to suppress T-cells and the occurrence of GvHD. The latter observation may indicate a negative feedback mechanism to control inflammatory (GvHD) activity. This intrinsic immunosuppression could subsequently promote disease relapse.