Tumor-derived extracellular vesicles (EVs) are active contributors in metastasis and immunosuppression in tumor microenvironment. At least some of the EVs carry tumor surface molecules such as tumor-associated antigens (TAAs) and/or checkpoint inhibitors, and potentially could interact with T cells or CAR T cells. Upon contact with T cells, EVs could alter their phenotype and functions by triggering signaling through TCR or CAR reprogramming them to escape immune response. We hypothesize that EVs that possess TAA on the surface will probably interact with CAR T cells which can recognize and bind corresponding TAA. This interaction between EVs and CAR T cells may change the outcome of CAR T-based cancer immunotherapy since it should affect CAR T cells. Also, EVs could serve as adjuvants and antigenic components of antitumor vaccines. Herein, we isolated EVs from B cell precursor leukemia cell line (pre-B ALL) Nalm-6 and demonstrated that recognition and binding of CD19+EVs with CD19-CAR T cells strongly depends on the presence of CD19 antigen. CD19+EVs induce secretion of pro-inflammatory cytokines (IL-2 and IFN-y) and upregulated transcription of activation-related genes (IFNG, IFNGR1, FASLG, IL2) in CD19-CAR T cells. Tumor necrosis factor receptor superfamily (TNFRSF4 and TNFRSF9) and T-cell exhaustion markers (CTLA4, LAG3, TIM3 and PDCD1LG2) were also upregulated in CD19-CAR T cells after incubation with CD19+EVs. Long-term cultivation of CD19+ or PD-L1+EVs with CD19-CAR T cells led to increased terminal differentiation and functional exhaustion according to elevated expression of PD-1, TIGIT, CD57. In summary, our results suggest that chronic exposure of CD19-CAR T cells to CD19+EVs mediates activation and systemic exhaustion in antigen-specific manner, and this negative effect is accompanied by the impaired cytotoxic activity in vitro.
CD19 CAR T cells became a breakthrough therapy in pediatric relapsed and refractory B-lineage acute lymphoblastic leukemia. 1,2 Standard usage of autologous T cells as a starting population for CAR-T manufacturing is often limited by the functional state of a patient's T cells and negatively affected by previous chemotherapy and posttransplant immune suppression. Healthy donor-derived T cells for CAR-T production may solve the issue of poor T-cell quality but is restricted by the risks of graft-versus-host disease (GVHD) and rejection of the CAR-T product, especially in the setting of haploidentical family donors. [3][4][5] Use of virus-specific T cells for CAR-T manufacturing was proposed as an approach to limit the alloreactivity of donor-derived CAR T cells after allogeneic hematopoietic stem cell transplantation (HSCT). 6 Depletion of naïve (CD45RA + ) T cells reduces the frequency of alloreactive T cells and is used to process hematopoietic stem cell grafts and donor lymphocyte infusions to lower the risk of GVHD. 7,8 The potential mechanisms of reduced alloreactivity of the CD45RA-depleted T cells include limited diversity of the T-cell receptor repertoire, diminished proliferative capacity, and differential tissue homing. [9][10][11] Recently, the possibility of large-scale bioreactor-based manufacturing of the memory T-cell-derived CAR-T product was demonstrated. 12 The animal studies suggest that CD45RA-depleted fraction can be used to produce CAR T cells that are equipotent to conventionally generated CAR T cells in vivo and do not induce xenogeneic GVHD. 13 Encouraged by these reports, we validated the manufacturing of healthy donor-derived memory CAR T cells (CAR-Tm) based on automatic large-scale bioreactor processing.Five pediatric patients with relapsed and refractory B-lineage acute lymphoblastic leukemia were offered the therapy with CAR-Tm on a compassionate-use basis. In each case, an Institutional Review Board approval for named-patient use was provided. All 5 cases had B-ALL, relapsing after multiple lines of treatment, including HSCT (n = 5), autologous CD19 CAR-T (n = 4), and blinatumomab (n = 3) therapy (Figure 1F). Median age at treatment was 9 years old. At the time of allogeneic CAR-Tm application, disease burden was either overt leukemia (n = 3) or minimal residual disease-level disease (n = 2). CAR-Tm were derived from haploidentical familial HSCT donors, and in all cases, the same donor as for HSC graft was used. CAR-Tm products were applied after HSCT on days plus 42, plus 100, and plus 539 in 3 cases and simultaneously with the HSC grafts on day 0 in 2 cases (Figure 1A). In all cases, HSCT from haploidentical donors was performed based on the ex vivo αβ T-cell depletion platform as described before (Figure 1B). 8,14 In 2 cases, standard fludarabine (120 mg/m 2 ) and cyclophosphamide (750 mg/m 2 ) lymphodepletion was used (Figure 1A; supplemental Figure 1). In 2 cases, lymphodepletion was represented by the pre-HSCT conditioning as shown in Figure 1F. Specifically, the use of antith...
Background Langerhans cell histiocytosis (LCH) involves abnormal proliferation of Langerhans cells (LC), which is typically driven by the BRAF V600E mutation. High-risk LCH has a poor prognosis. Procedure Fifteen children (5 girls, 10 boys) with BRAF V600E+ LCH received vemurafenib (initial dose median 40 mg/kg/day, range: 11–51.6 mg/kg/day) between March 2016 and February 2020. All patients had previous received LCH-directed chemotherapy. The median age at LCH onset was 2 months (range: 1–28 months) and the median age at the start of vemurafenib treatment was 22 months (range: 13–62 months). The median disease activity score (DAS) at the start of vemurafenib treatment was 12 points (range: 2–22 points). Results The median duration of vemurafenib therapy was 29 months (range: 2.4–45 months). All patients responded to treatment, with median DAS values of 4 points (range: 0–14 points) at week 4 and 1 point (range: 0–3 points) at week 26. Toxicities included skin/hair changes (93%) and non-significant QT prolongation (73%). Two patients died, including 1 patient who experienced hepatic failure after NSAID overdose and 1 patient who developed neutropenic sepsis. Electively stopping vemurafenib treatment resulted in relapse in 5 patients, and complete cessation was only possible for 1 patient. Digital droplet PCR for BRAF V600E using cell-free circulating DNA revealed that 7 patients had mutation statuses that fluctuated over time. Conclusion Our study confirms that vemurafenib treatment is safe and effective for young children with BRAF V600E+ multisystem LCH. However, treatment using vemurafenib does not completely eliminate the disease.
Relevance. Juvenile xanthogranuloma (JXG) is the most common form of non-Langerhans cell histiocytic disorder. Cutaneous forms of the disease spontaneously regress within a few years, while systemic forms of JXG require treatment and may pose a threat to the lives of patients. Due to the lack of unified approach to the treatment of multisystem forms of JXG, the question of effective therapy tactics remains unresolved. The most common approach is to use Langerhans cell histiocytosis (LCH) treatment regimens for JXG. With the understanding of the leading role of mutations in the MEK-ERK signaling pathway in the pathogenesis of JXG, targeted therapy, BRAF- and MEK-inhibitors, are increasingly being considered in the treatment of JXG.Clinical cases. We present two cases of multisystem JXG with central nervous system (CNS) lesions. The first patient with CNS and skin lesions was treated with chemotherapy, developed for the treatment of multisystem LCH, which allowed us to obtain an effect “active disease better” (AD better). The second JXG patient with brain, lungs, bones, and adrenal gland lesions, combined targeted therapy with BRAF- and MEKinhibitors, vemurafenib and cobimetinib, resulted in a “non active disease” (NAD) effect.Conclusion. Multisystem form of JXG with CNS involvement is a rare oncological disease, the therapy of which has not been developed. With the introduction of molecular genetic profiling technology, it became possible to obtain NAD effect using targeted therapy.
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