Cells In Pediatric Patients Research Articles

Evaluation of novel Epstein-Barr virus-derived antigen formulations for monitoring virus-specific T cells in pediatric patients with infectious mononucleosis

BackgroundInfection with the Epstein-Barr virus (EBV) elicits a complex T-cell response against a broad range of viral proteins. Hence, identifying potential differences in the cellular immune response of patients with different EBV-associated diseases or different courses of the same disorder requires interrogation of a maximum number of EBV antigens. Here, we tested three novel EBV-derived antigen formulations for their ability to reactivate virus-specific T cells ex vivo in patients with EBV-associated infectious mononucleosis (IM).MethodsWe comparatively analyzed EBV-specific CD4+ and CD8+ T-cell responses to three EBV-derived antigen formulations in 20 pediatric patients during the early phase of IM: T-activated EBV proteins (BZLF1, EBNA3A) and EBV-like particles (EB-VLP), both able to induce CD4+ and CD8+ T-cell responses ex vivo, as well as an EBV-derived peptide pool (PP) covering 94 well-characterized CD8+ T-cell epitopes. We assessed the specificity, magnitude, kinetics, and functional characteristics of EBV-specific immune responses at two sequential time points (v1 and v2) within the first six weeks after IM symptom onset (Tonset).ResultsAll three tested EBV-derived antigen formulations enabled the detection of EBV-reactive T cells during the early phase of IM without prior T-cell expansion in vitro. EBV-reactive CD4+ and CD8+ T cells were mainly mono-functional (CD4+: mean 64.92%, range 56.15-71.71%; CD8+: mean 58.55%, range 11.79-85.22%) within the first two weeks after symptom onset (v1) with IFN-γ and TNF-secreting cells representing the majority of mono-functional EBV-reactive T cells. By contrast, PP-reactive CD8+ T cells were primarily bi-functional (>60% at v1 and v2), produced IFN-γ and TNF and had more tri-functional than mono-functional components. We observed a moderate correlation between viral load and EBNA3A, EB-VLP, and PP-reactive CD8+ T cells (rs = 0.345, 0.418, and 0.356, respectively) within the first two weeks after Tonset, but no correlation with the number of detectable EBV-reactive CD4+ T cells.ConclusionsAll three EBV-derived antigen formulations represent innovative and generic recall antigens suitable for monitoring EBV-specific T-cell responses ex vivo. Their combined use facilitates a thorough analysis of EBV-specific T-cell immunity and allows the identification of functional T-cell signatures linked to disease development and severity.

Immunological characterization and comparison of children with COVID-19 from their adult counterparts at single-cell resolution.

The immunological characteristics that could protect children with coronavirus disease 2019 (COVID-19) from severe or fatal illnesses have not been fully understood yet. Here, we performed single-cell RNA sequencing (scRNA-seq) analysis on peripheral blood samples of 15 children (8 with COVID-19) and compared them to 18 adults (13 with COVID-19). The child-adult integrated single cell data indicated that children with the disease presented a restrained response to type I interferon in most of the major immune cell types, along with suppression of upstream interferon regulatory factor and toll-like receptor expression in monocytes, which was confirmed by in vitro interferon stimulation assays. Unlike adult patients, children with COVID-19 showed lower frequencies of activated proinflammatory CD14+ monocytes, possibly explaining the rareness of cytokine storm in them. Notably, natural killer (NK) cells in pediatric patients displayed potent cytotoxicity with a rich expression of cytotoxic molecules and upregulated cytotoxic pathways, whereas the cellular senescence, along with the Notch signaling pathway, was significantly downregulated in NK cells, all suggesting more robust cytotoxicity in NK cells of children than adult patients that was further confirmed by CD107a degranulation assays. Lastly, a modest adaptive immune response was evident with more naïve T cells but less activated and proliferated T cells while less naïve B cells but more activated B cells in children over adult patients. Conclusively, this preliminary study revealed distinct cell frequency and activation status of major immune cell types, particularly more robust NK cell cytotoxicity in PBMC that might help protect children from severe COVID-19.

Identification and Functional Validation of Neoantigen-Specific T Cells in Pediatric Patients with Fusion-Derived Acute Leukemias

Leukemia emerges from the accumulation of genetic mutations in hematopoietic cells, resulting in formation of altered proteins. Some of these mutant proteins (neoantigens) are consequently processed and presented as neoepitopes through major histocompatibility complex (MHC) molecules expressed on the surface of leukemia cells, permitting tumor-specific immune targeting. While adoptive T cell therapies against high mutation burden tumors have shown promising results, less is known about leveraging tumor-specific T cells as an effective approach in pediatric patients, including high-risk leukemia. In this study, peripheral blood or bone marrow samples were obtained from 34 leukemia patients aged 0 -14 years old. The patient tumors had founder fusion genes, including 15 patients with acute lymphoblastic leukemia (ALL), 15 patients with acute myeloid leukemia (AML), and 4 patients with mixed phenotype acute leukemia (MPAL). Samples were collected at the time of diagnosis or relapse. While leukemia blasts were the most abundant cellular component in these samples, the frequency of lymphocytes ranged between 1%- 25%, with a median of 3.5%. KMT2A rearrangements were expressed with either AFF1, MLLT1, MLLT3, MLLT4, MLLT6, or MLLT10 fusion partner in 73.5% of the patients' samples in this study. This included (14/15) ALL, (10/15) AML, and (1/4) MPAL patients . T cells were enriched and expanded for a total of 14 days using a rapid expansion protocol culture system. This approach was successful on all tested samples irrespective of their mutation backgrounds or initial lymphocyte count, with a range of 133-4957fold increases in total cell number and average frequencies of 59.7% and 26.1% for CD4+ and CD8+ T cell, respectively. We further screened the T cell response by examining the expression of 4-1BB, OX40, and IFN-g upon coculture with their autologous leukemia cells, and additionally validated the T cell killing activity against the blasts by a cell killing assay using standard protocol. We found that the expanded T cells exhibited a robust immune activation when cocultured with the autologous leukemia cells from ALL patients with KMT2A rearrangements, indicating an endogenous cell response in all patients in the ALL cohort (100%). However, we observed a considerably reduced reactivity in the samples from the AML and MPAL patients. Interestingly, we observed that such diminished reactivity was associated with a marked downregulation in the expression of MHC class I and II molecules, and that pretreating the leukemia blasts with IFN-γ prior to coculturing with the expanded T cells was sufficient to induce the expression of MHC molecules and subsequent activation of the expanded T cells. Single-cell TCR sequencing of the sorted, tumor-reactive T cells identified the major clonotypes among these reactive T cell clones and the top 4 - 6 clonotypes were selected from 5 patients to evaluate reactivity against the leukemia blasts and examine their specificity against the corresponding driving fusions. We further generated TCR-expressing clones and subsequently cocultured each clone with leukemia blasts to validate their antigen recognition and reactivity by cytotoxicity assay. Interestingly, and in line with the findings from investigations on solid tumors, the leukemia- reactive T cells were consistently characterized by higher expression of both PD1 and CD39 (PD1 hi CD39 +), indicating the capability of utilizing both markers for the enrichment of T cell prior to expansion and subsequent implementation. Despite the high-risk prognosis and low mutation burden in our patient cohort, our findings demonstrate that patient T cells contain endogenous neoantigen-specific clones which can be therapeutically leveraged for targeting leukemia blasts. These results support the utilization of adoptive T cell therapy in the context of hematological malignancies especially given the less restrictive spatial segregation between tumor-specific T cells and leukemia cells compared to solid tumors, where T cell infiltration is a prerequisite for antitumor immunity. Overall, these findings suggest the possibility of leveraging leukemia-associated lymphocytes, fusion oncogene vaccines, and TCR-based therapy as novel immunotherapeutic approaches for mitigating pediatric leukemia, particularly in high-risk patients.

PB0267 High-Dose Dexamethasone Corrects the Elevated TCRαβ+ Double-Negative T Cells in Pediatric Patients with Primary Immune Thrombocytopenia

PB0267 High-Dose Dexamethasone Corrects the Elevated TCRαβ+ Double-Negative T Cells in Pediatric Patients with Primary Immune Thrombocytopenia

P‐TS‐91 | Transfusion of Red Cells in Pediatric Patients During ECMO Runs: A Single Center Audit

P‐TS‐91 | Transfusion of Red Cells in Pediatric Patients During ECMO Runs: A Single Center Audit

The Effect of Iron Overload on the Mobilization of Peripheral Blood Hematopoietic Stem Cells in Pediatric Patients with Thalassemia Major

Introduction: The purpose of this study was to examine the effect of iron overload on the mobilization of peripheral blood stem cells (PBSCs) in pediatric patients with β-thalassemia major (TM). Methods: We retrospectively reviewed the records of 226 patients with TM from whom PBSCs were collected. Iron overload was based on serum ferritin level, and liver and cardiac iron overload was measured by magnetic resonance imaging (MRI) T2*. Results: The mean age of the TM patients was 7.35 ± 3.41 years. Of the patients, only 171 received MRI. Of the 171 patients, 35 had normal liver iron levels, 39 mild liver iron overload, 90 intermediate liver iron overload, and 7 severe liver iron overload. The intermediate + severe group was associated with significantly higher age and BMI and lower leukapheresis product white blood cell count and CD34⁺ cell levels (all, p < 0.05). Conclusion: Leukapheresis indices were similar between patients with different degrees of iron overload according to the ferritin level and cardiac iron overload, in which the later might be due to the small number of patients with cardiac overload. In patients with TM, the intermediate and severe liver iron overload was associated with poorer mobilization of PBSCs.

Elevated TCR-αβ+ double-negative T cells in pediatric patients with acquired aplastic anemia

Background and aimsThe pathophysiology of acquired aplastic anemia (aAA) is most associated with T cell mediated immune dysfunction, but the role of CD4- CD8- double negative T cells (DNTs) in pediatric patients with aAA is unclear. In this study, we aimed to investigate the proportion of TCR-αβ+ DNTs in pediatric patients with aAA and correlation with the response to immunosuppressive therapy (IST). Materials and methodsAssessment of DNTs from peripheral blood was done by sensitive multi-color flow cytometry. The potential clinical value of TCR-αβ+ DNTs was then assessed by the receiver operating characteristic (ROC) curves. ResultsThe retrospective study evaluated 164 pediatric patients with aAA and 105 healthy donors (HD). Our data showed higher proportion of TCR-αβ+ DNTs in total lymphocytes [1.04% (0.79%-1.40%) vs 0.69% (0.47%-0.87%), p < 0.001] and CD3+ T cells [1.52% (1.10%-1.96%) vs 1.10% (0.70%-1.40%), p < 0.001] in aAA compared to HD. Patients with SAA/VSAA achieving complete response (CR) after IST had a higher proportion of TCR-αβ+ DNTs at initial diagnosis, than those not achieving CR for total (1.21%±0.39 vs 0.78%±0.38, p < 0.05) and CD3+ T cells (1.74%±0.53 vs 1.15%±0.59, p < 0.05). The ROC analysis showed areas under the curves (AUCs) for TCR-αβ+ DNT proportion in lymphocytes and CD3+ T cells were 0.756 (cutoff value 1.33, p < 0.05) and 0.758 (cutoff value 1.38, p < 0.05), respectively. And the complete response rate was higher in TCR-αβ+ DNT proportion high group than in TCR-αβ+ DNT proportion low group at baseline (p < 0.001). ConclusionOur observations suggest that elevated TCR-αβ+ DNTs seems to play a role in the pathogenesis of aAA, and it was involve in immune response to IST.

Decreased regulatory B cells in pediatric patients with asthma

Decreased regulatory B cells in pediatric patients with asthma

Bifidobacteria modulate immune response in pediatric patients with cow's milk protein allergy.

In children with an allergy to cow's milk proteins (CMA), the altered composition of intestinal microbiota influences the immune tolerance to milk proteins (CMP). This study aims to investigate the effect of probiotics on the phenotype and activation status of peripheral basophils and lymphocytes in a pediatric CMA cohort. CMA children underwent 45 days of treatment with Bifidobacteria. The basophil degranulation and the immune phenotype of B cells, T helper cells, and regulatory T cells were analyzed in peripheral blood at diagnosis (T0), after a 45-day probiotic treatment (T1), and 45 days after the probiotic wash-out (T2). We observed in probiotic-treated CMA patients a decrease in naive T lymphocytes. Among the CD3+ cell subsets, both naive and activated CD4+ cells resulted markedly reduced after taking probiotics, with the lowest percentages at T2. A decreased basophil degranulation was observed in response to all analyzed CMP at T1 compared to T0. The probiotic treatment resulted in a decrease of circulating naive and activated CD4+ T cells, as well as degranulating basophils. These data suggest that the Bifidobacteria could have a beneficial effect in the modulation of oral tolerance to CMP. ISRCTN69069358. URL of registration: https://www.isrctn.com/ISRCTN69069358 . Probiotic treatment with Bifidobacteria induces a reduction of both naive and activated circulating CD4+ T cells in pediatric patients with cow's milk allergy (CMA). The probiotic supplementation induces a decreased basophil degranulation. The immunological tolerance persists even after 45 days of the probiotic wash-out. Bifidobacteria in vivo supplementation down-modulates the activation of innate and adaptive immunity in pediatric patients with cow's milk allergy. Bifidobacteria contribute to the development of immune tolerance in CMA patients.

115 - Preliminary Results from a Phase 1 Trial Showing Safety and Anti-Leukemic Activity of CD123-CAR T Cells in Pediatric Patients with AML

115 - Preliminary Results from a Phase 1 Trial Showing Safety and Anti-Leukemic Activity of CD123-CAR T Cells in Pediatric Patients with AML

Indication and selection of pediatric patients for genetically modified T cells

The use of CAR-T cells will completely change the treatment of many hematological diseases, including relapsed pediatric B-lineage acute lymphoblastic leukemia. This article is part of the Brazilian Society of Bone Marrow Transplantation (SBTMO)’s “Technical manual of cellular therapy” to guide physicians to select patients and indicate the use of the CAR-T cells in pediatric patients with acute lymphoblastic leukemia. Other aspects of CAR-T cell therapy and management of important toxicities are included on other articles of this volume.

Safety and Anti-Leukemic Activity of CD123-CAR T Cells in Pediatric Patients with AML: Preliminary Results from a Phase 1 Trial

Background The prognosis of pediatric patients with relapsed or refractory (r/r) Acute Myeloid Leukemia (AML) remains dismal. Evaluation of novel therapies such as chimeric antigen receptor (CAR) T cells is therefore urgently needed. CD123 is overexpressed on AML blasts and leukemic stem cells (LSC), making it an attractive target for CAR T-cell directed therapy. Methods and Results We designed a 'bridge to allogeneic hematopoietic cell transplant (HCT)’ Phase 1 study to evaluate the safety and feasibility of treating pediatric patients with r/r AML with CD123-CAR T-cell on four dose levels (DL): DL1: 3x105/kg, DL2: 1x106/kg, DL3: 3x106/kg, DL4: 1x107/kg (NCT04318678). CD123-CAR T-cells were generated from CD4/CD8-selected autologous leukapheresis products using a lentiviral vector, which encoded a CD123-CAR with CD28.z signaling domain and a CD20 safety switch. Patients received lymphodepleting chemotherapy with Fludarabine and Cyclophosphamide followed by a single CAR T cell infusion. To date we have enrolled 12 patients. The median age of treated patients was 17 years (range 12-21years). Except for the first patient who had primary refractory disease, all other patients had relapsed following HCT (n=1-4 previous HCTs). Despite patients being heavily pre-treated, we successfully manufactured products on all patients. The infused products had a predominantly CD4+ effector memory immunophenotype, with a median CD123-CAR+/CD20+ expression of 60.2% (range 46.1-79.4%, N=6). All infused CAR T cell products exhibited potent antitumor activity in vitro when cultured against CD123+ targets (including autologous blasts for 2 patients). We infused 2 patients on DL1 and 3 patients on DL2. Two patients were infused on single patient protocols. All infusions were well tolerated, without any adverse infusion events. Post infusion, we observed isolated fevers that resolved within 24 hours (possible Grade 1 Cytokine Release Syndrome (CRS)). No Grade 2 ≥ CRS or neurotoxicity was seen. Multiplex cytokine analysis showed statistically significant increase of IL15 after lymphodepletion without significant increase in Th1 cytokines or IL6 after T cell infusion. While transient cytopenias were observed, patients did not develop persistent marrow aplasia. No dose limiting toxicities were noted. Disease evaluation was performed at 4-6 weeks following CAR T cell infusion. The 2 patients on DL1 showed no response. In the 3 patients on DL2, we observed: reduction in blast percentage without complete remission (CR) in 1 patient, no response in 1 patient and CR in 1 patient. The patient with CR had isolated extramedullary disease, showing complete resolution of all lesions by week 4 by PET imaging (Figure 1). She recurred 2 months following infusion, coinciding with loss of CAR T-cell detection by qPCR. She received a second infusion off study and again achieved a short-lived CR. One additional patient was infused on dose level 2 off protocol. She achieved morphological CR at day 28 with low level minimal residual disease (0.19%). Correlative studies revealed CD123-CAR T cell expansion in patients on DL2 (Figure 2), but not on DL1. However, peak expansion on DL2 was low in comparison to published reports on CAR T cells for Acute Lymphoblastic Leukemia. Detailed phenotypic analysis revealed that CD123-CAR T cell products were predominantly effector memory (CD45RO+CCR7-) with <2% naïve-like (CD45RO-CCR7+ or CD45RO-CD62L+) T cells. In addition, PD1, TIM3 and CD39 were expressed at a median of 43, 81 and 16% in CD4+ and 3, 72 and 45% in CD8+ T cells. This phenotype could be completely reversed by inhibiting CAR signaling with dasatinib during the manufacturing process, resulting in a CAR T cell product dominated by naïve-like T cells that did not express TIM3, PD1 or CD39, leading to enhanced T-cell effector function. Mechanistic analysis revealed that the observed phenotype could be explained by the transient, increased cell surface expression of CD123 on T cells upon activation during manufacturing, and not by tonic CAR signaling. Conclusions Our initial clinical experience with CD123-CAR T cells for pediatric patients with r/r AML demonstrates feasibility, safety, and evidence of anti-leukemic activity. Upcoming CD123-CAR T cell products will be manufactured in the presence of dasatinib to limit T-cell differentiation and exhaustion as we continue to explore our CD123-CAR T cell therapy approach for pediatric r/r AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Prognostic Value of Eight Immunohistochemical Markers Expressed in the Tumor Microenvironment and on Hodgkin Reed-Sternberg Cells in Pediatric Patients With Classical Hodgkin Lymphoma.

Immunohistochemical markers are associated with treatment outcome in adults with classical Hodgkin Lymphoma (cHL). Studies in children are scarce and inconsistent. We investigated in 67 children with cHL, whether the expression of CD15, CD30, PAX5, PD-1, PD-L1, CD68, CD163 and TARC at diagnosis is associated with disease free survival (DFS) and with interim remission status. Low CD15 and low TARC expression were associated with relapsed disease. Low expression of PD-L1 was associated with complete remission at interim PET-scan. Our data suggest a difference between pediatric and adult cHL. This underlines the importance of future research into specific prognostic factors in pediatric cHL, indispensable for improvement of treatment in this population.

Dynamics and analysis of a mathematical model of neuroblastoma treated with Celyvir

Celyvir viro-immunotherapy - a therapy using mesenchymal stem cells (MSCs) infected with the oncolytic adenovirus ICOVIR 5, an innovative oncolytic adenovirus - is a new therapeutic approach in cancer treatment. The purpose of this study is to create a mathematical model describing the relationships between immune, cancer and viral cells in pediatric patients with neuroblastoma. This was done by studying two different ways of applying the treatment: continuous and periodic therapy. Analysis of the first mathematical model identifies equilibrium points, their stability properties and bifurcation points. It can also identify bistability regimes in which therapy can induce either tumour-free equilibrium or tumour progression, depending only on the initial conditions. The second model reveals the existence of a threshold value of viral load, beyond which the patient’s recovery could be assured. The models make it clear that both the intensity of the viral load and the time over which treatment is given must be sufficient to ensure the success of the therapy. Low levels of treatment may fail to eliminate neuroblastoma, while stopping therapy early could lead to recurrence.

Hypoxia-inducible factor regulation of NK cell dysfunction in lupus

Abstract Natural Killer (NK) cells are innate antiviral and antitumor effector cells that have emerged as potent regulators of adaptive immune responses. However, NK cells are known to be dysfunctional in many diseases including systemic lupus erythematosus (lupus), where inability to restrain autoreactive T and B cells leads to potentially irreversible organ damage. To determine mechanisms driving NK-cell dysfunction in autoimmunity, we performed transcriptomic analysis of these cells in pediatric patients with lupus. Analysis of the differentially expressed genes revealed a putative gene regulatory node controlled by the heterodimeric pairing of the transcription factors EPAS1 and ARNT that is active in NK cells from patients with lupus, but not controls. Cytokines linked to lupus promote up-regulation of the protein encoded by EPAS1 in NK cells. Small molecule inhibitors of EPAS1 (PT2385) enhanced the capacity of NK cells to kill K562 cells. These results suggest that a hypoxia-related transcriptional program contributes to functional restraint of NK cells in lupus. Supported by T32GM063483, AR073228, AR47363 (Flow Cytometry Core), DK78392 (Flow Cytometry Core), DK90971 (Flow Cytometry Core)

Immunophenotypically defined stem cell subsets in paediatric AML are highly heterogeneous and demonstrate differences in BCL-2 expression by cytogenetic subgroups.

In adult acute myeloid leukaemia (AML), immunophenotypic differences enable discrimination of leukaemic stem cells (LSCs) from healthy haematopoietic stem cells (HSCs). However, immunophenotypic stem cell characteristics are less explored in paediatric AML. Employing a 15-colour flow cytometry assay, we analysed the expression of eight aberrant surface markers together with BCL-2 on CD34+ CD38- bone marrow stem cells from 38 paediatric AML patients and seven non-leukaemic, age-matched controls. Furthermore, clonality was investigated by genetic analyses of sorted immunophenotypically abnormal stem cells from six patients. A total of 50 aberrant marker positive (non-HSC-like) subsets with 41 different immunophenotypic profiles were detected. CD123, CLEC12A, and IL1RAP were the most frequently expressed markers. IL1RAP, CD93, and CD25 expression were not restricted to stem cells harbouring leukaemia-associated mutations. Differential BCL-2 expression was found among defined cytogenetic subgroups. Interestingly, only immunophenotypically abnormal non-HSC-like subsets demonstrated BCL-2 overexpression. Collectively, we observed pronounced immunophenotypic heterogeneity within the stem cell compartment of paediatric AML patients. Additionally, certain aberrant markers used in adults seemed to be ineligible for detection of leukaemia-representing stem cells in paediatric patients implying that inference from adult studies must be done with caution.

The expression and function of programmed death-ligand 1 and related cytokines in neutrophilic asthma.

BackgroundProgrammed death-ligand 1 (PD-L1) is an important immune checkpoint inhibitor. Recent studies suggest that the PD-L1-mediated pathway may be a promising target in allergic asthma. However, the mechanism by which PD-L1 represses neutrophilic asthma (NA) remains unclear. In this study, we examined correlations between the expression of PD-L1 and the production of T helper cell type 1 (Th1), T helper cell type 2 (Th2), and T helper cell type 17 (Th17) cells in pediatric patients with NA and a mouse model.MethodsThe clinical samples of 26 children with asthma and 15 children with a bronchial foreign body were collected over a period of 12 months by the Children’s Hospital of Soochow University. An experimental mouse model of asthma was established to study NA. An enzyme-linked immunoassay (ELISA) was used to assess soluble PD-L1 (sPD-L1) and cytokines [e.g., interleukin (IL)-4, IL-6, interferon gamma (IFN-γ), IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF)] in bronchoalveolar lavage fluid (BALF).ResultsNA patients had significantly higher levels of sPD-L1, IL-6, IL-17, and GM-CSF in their BALF than non-NA and control patients (P<0.05). In a murine model of asthma, the positive rate and fluorescence intensity of PD-L1 in the NA group and the immunoglobulin G (IgG)-treated NA group were higher than in the PD-L1 antibody (Ab)-treated NA group and the phosphate-buffered saline (PBS) control group (P<0.05). In the plasma and the BALF of the NA group and the IgG-treatment NA group, the levels of IL-17, IL-4, tumor necrosis factor alpha (TNF-α), and granulocyte colony-stimulating were higher than those in the PBS control group (P<0.05). The histopathological examination of lung tissues from all mice groups showed that a large number of inflammatory cells were found around the airway in the NA group and the IgG-treatment group.ConclusionsPD-L1 may contribute to the Th17/IL-17 immune response, which is associated with neutrophilic inflammation and asthma. A PD-L1 blockade reduces pulmonary neutrophils and mucus production.

CD30-Directed Chimeric Antigen Receptor (CAR)-T Cells for Treatment of Hodgkin Lymphoma and Non-Hodgkin Lymphoma in Pediatric Patients

Background:Chimeric antigen receptor (CAR)-T cell therapy targeting the CD19 antigen has been effective in treating B-cell acute lymphoblastic leukemia. As CAR-T cells targeting new antigens are being explored for the treatment of other cancers in adults, parallel studies are warranted for pediatric cohorts. We have previously shown the safety and efficacy in adults of CAR-T cells targeting CD30, which is expressed in classical Hodgkin Lymphoma (HL) and in some Non-Hodgkin Lymphoma (NHL). We have therefore sought to study the feasibility and the safety of CD30.CAR-T cells in pediatric patients with relapsed/refractory CD30-expressing HL and Anaplastic Large Cell Lymphoma (ALCL).Design/Methods:Six pediatric patients (ages 9 to 17 years) with CD30+ HL (4) and NHL (2) were enrolled on two trials at the University of North Carolina. One NHL patient with ALK+ ALCL was enrolled on both trials. Two patients, one HL and one NHL, were enrolled on a phase I study and received 2x10 7 CD30.CAR-T cells/m 2 as consolidation for high-risk of relapse after autologous stem cell transplant (ASCT, NCT02663297). Five patients, 3 HL and 2 NHL, were enrolled on a phase Ib/II study and received 1x10 8 CD30.CAR-T cells/m 2, as treatment for relapsed disease, after lymphodepletion with bendamustine and fludarabine (NCT02690545). HL patients had failed multiple lines of therapies (5-6), including 2 with prior pembrolizumab, 2 with prior ASCT, and all 3 with prior brentuximab vedotin (BV) and radiation therapy. The two NHL patients both had ALCL, one was ALK positive and one was ALK negative. Both had been treated with prior BV. The ALK negative patient had been treated with 3 prior lines of therapy and the ALK positive patient had been treated with 6 lines of prior therapy including ASCT and two ALK inhibitors, crizotinib and brigatinib. The brigatinib was stopped 3 weeks prior to starting lymphodepletion.Results:CD30.CAR-T cells were successfully manufactured for all 7 patients and no differences were observed as compared to products manufactured for adults, based on cell number, transduction, potency or immunophenotype. For all patients, infusions were well-tolerated and no neurotoxicity experienced.On the post-ASCT study, 1 patient with HL and 1 with ALCL were treated. All adverse events (AE) were less than grade 4. The patient with HL remains in complete remission (CR) 41 months following therapy, while the patient with ALCL progressed.Five patients with relapsed/refractory disease (3 HL and 2 ALCL) were treated on the post-lymphodepletion study. Most grade 3 or higher AEs were anticipated hematologic toxicity secondary to lymphodepletion. The youngest HL patient on the study developed symptoms consistent with grade 2 cytokine release syndrome (CRS) and a concomitant pneumonia based on imaging, accompanied by a marked inflammatory response based on labs with maximum ferritin 9,920 ng/mL and CRP 150.7 mg/L. He responded to antibacterial agents and two doses of tocilizumab, as well as brief supplemental oxygen by nasal cannula. He did not require vasopressors. One patient with ALK+ ALCL had symptoms compatible with CRS and macrophage activation syndrome (maximum ferritin >100,000 ng/mL and CRP 39.7 mg/L), which were ultimately determined to be secondary to progressive disease, confirmed at autopsy 5 weeks post therapy. The other four patients (3 HL and 1 ALCL) achieved CR and remain in CR 4 to 27 months post CAR-T cell infusion. Of note, 2 of the HL patients chose to come off study while in CR to receive treatment with checkpoint inhibitor therapy.Conclusion:Our studies show that CD30.CAR-T cells are well tolerated in pediatric patients. CRs were observed in all heavily pre-treated and refractory HL patients, highlighting the potential of this strategy. All patients treated on both studies had previously received BV, which suggests CD30.CAR-T cells are effective even post BV progression. We continue to investigate how to better tailor CD30.CAR-T cells in NHL given the need for better therapies in ALCL, which is often aggressive at relapse. One patient with ALK negative ALCL remains in CR while the patient with ALK+ ALCL had rapid relapse.After study initiation, UNC entered into a research collaboration with Tessa Therapeutics. DisclosuresSavoldo: Tessa: Patents & Royalties: Approach for CD30.CAR-T Cells for Hodgkin Lymphoma. Dotti: Tessa: Patents & Royalties: Approach for CD30.CAR-T Cells for Hodgkin Lymphoma. Grover: Tessa: Consultancy; Kite: Other: Advisory Board; Novartis: Consultancy; Genentech: Research Funding; ADC: Other: Advisory Board. Morrison: Vesselon: Consultancy. Riches: Jazz Pharmaceuticals: Other: Payment; ATARA Biotherapeutics: Other: Payment; BioIntelect: Membership on an entity's Board of Directors or advisory committees. Serody: Tessa: Patents & Royalties: Approach for CD30.CAR-T Cells for Hodgkin Lymphoma.

NK Cell Subpopulation Is Altered and the Expression of TLR1 and TLR9 Is Decreased in Patients with Acute Lymphoblastic Leukemia.

NK cells represent a heterogeneous subpopulation of lymphocytes of the innate immune system, which possess powerful antitumor activity. NK cells exhibit their function through a complex collection of receptors that act synergistically to recognize, regulate, or amplify the immune response. TLRs allow cells to detect PAMPs, MAMPs, or DAMPs, which are essential for the initiation of the immune response. Studies on the different subpopulations of NK cells and their expression profile of innate immune receptors in hematological cancers are limited. In this study, the specific subpopulations of NK cells in pediatric patients with acute lymphoblastic leukemia (ALL) and the repertoire and level of expression of TLRs in cytotoxic NK cells were assessed. The results suggested that pediatric patients with ALL exhibited a significant decrease in NK cells in peripheral blood and bone marrow, in addition to alterations in the distribution of the subpopulations of cells. Regulatory and cytotoxic NK cells were diminished, whereas dysfunctional phenotype was considerably increased. Cytotoxic NK cells from children with ALL expressed all 10 TLRs, and expression of TLR1 and TLR9 was decreased compared with the controls. Interestingly, cytotoxic NK cells exhibited a higher expression of TLR1 in the bone marrow than in the peripheral blood of patients with ALL. The present study is the first to show that TLR10 was expressed in the cytotoxic NK cells and the first to assess the profile and levels of the 10 known TLRs in cytotoxic NK cells from patients with ALL. The alterations in expression levels and cellular distribution may be involved in the immune response.

Expression of YY1 in pro-B and T phenotypes correlation with poor survival in pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, constituting 80% of all acute leukemias in minors. Despite the increase in the success of therapies, disease-free survival is over 80% in most cases. For the remaining 20% of patients, new strategies are needed to allow us to know and select those at greatest risk of relapse. We evaluated by immunohistochemistry the expression of the transcription factor YY1 and found that it is overexpressed in peripheral blood leukemia cells of pediatric patients with ALL with Pro-B and T phenotype compared to control samples. Over expression of YY1 was associated with a significantly lower chance of survival. We also evaluated by RT-PCR in bone marrow samples from ALL pediatric patients the association of YY1 expression with the percentage of blasts. High levels of YY1 were present in samples with higher percent of blasts in these patients. In addition, ALL pediatric patients with a poor response to therapy had higher levels at the nuclear level of YY1 than those who responded well to chemotherapy. In conclusion, our data suggest that YY1 could serve in pediatric ALL as markers of evolution and response for this disease, mainly in patients with pro-B and T immunophenotype. It is also suggested that YY1 is implicated in the expanse of blast in these patients.