Repurposing CD19-directed immunotherapies for pediatric t(8;21) acute myeloid leukemia.

Not available.

Salivary polyreactive antibodies and Haemophilus influenzae are associated with respiratory infection severity in young children with recurrent respiratory infections.

BACKGROUND: Recurrent respiratory tract infections (rRTIs) are a common reason for immunodiagnostic testing in children, which relies on serum antibody level measurements. However, because RTIs predominantly affect the respiratory mucosa, serum antibodies may inaccurately reflect local immune defences. We investigated antibody responses in saliva and their interplay with the respiratory microbiota in relation to RTI severity and burden in young children with rRTIs. METHODS: We conducted a prospective cohort study including 100 children aged <10 years with rRTIs, their family members, and healthy healthcare professionals. Total and polyreactive antibody concentrations were determined in serum and saliva (ELISA); respiratory microbiota composition (16S-rRNA-sequencing) and respiratory viruses (qPCR) were characterised in nasopharyngeal swabs. Proteomic analysis (Olink®) was performed on saliva and serum samples. RTI symptoms were monitored with a daily cell phone application and assessed using latent class analysis and negative binomial mixed models. RESULTS: Serum antibody levels were not associated with RTI severity. Strikingly, 28% of salivary antibodies and only 2% of serum antibodies displayed polyreactivity (p<0.001). Salivary polyreactive immunoglobulin A (IgA) was negatively associated with recurrent lower RTIs (aOR 0.80 [95% CI 0.67-0.94]) and detection of multiple respiratory viruses (aOR 0.76 [95% CI 0.61-0.96]). Haemophilus influenzae abundance was positively associated with RTI symptom burden (regression coefficient 0.07 [95% CI 0.02-0.12]). CONCLUSION: These results highlight the importance of mucosal immunity in RTI severity and burden and suggest that the level of salivary polyreactive IgA and H. influenzae abundance may serve as indicators of infection risk and severity in young children with rRTIs.

Oncogenic and immunological targets for matched therapy of pediatric blood cancer patients: Dutch iTHER study experience.

Over the past 10 years, institutional and national molecular tumor boards have been implemented for relapsed or refractory pediatric cancer to prioritize targeted drugs for individualized treatment based on actionable oncogenic lesions, including the Dutch iTHER platform. Hematological malignancies form a minority in precision medicine studies. Here, we report on 56 iTHER leukemia/lymphoma patients for which we considered cell surface markers and oncogenic aberrations as actionable events, supplemented with ex vivo drug sensitivity for six patients. Prior to iTHER registration, 34% of the patients had received allogeneic hematopoietic cell transplantation (HCT) and 18% CAR-T therapy. For 51 patients (91%), a sample with sufficient tumor percentage (≥20%) required for comprehensive diagnostic testing was obtained. Up to 10 oncogenic actionable events were prioritized in 49/51 patients, and immunotherapy targets were identified in all profiled patients. Targeted treatment(s) based on the iTHER advice was given to 24 of 51 patients (47%), including immunotherapy in 17 patients, a targeted drug matching an oncogenic aberration in 12 patients, and a drug based on ex vivo drug sensitivity in one patient, resulting in objective responses and a bridge to HCT in the majority of the patients. In conclusion, comprehensive profiling of relapsed/refractory hematological malignancies showed multiple oncogenic and immunotherapy targets for a precision medicine approach, which requires multidisciplinary expertise to prioritize the best treatment options for this rare, heavily pretreated pediatric population.

Leukoencephalopathy with calcifications, developmental brain abnormalities and skeletal dysplasia due to homozygosity for a hypomorphic CSF1R variant: A report of three siblings.

We report three siblings homozygous for CSF1R variant c.1969 + 115_1969 + 116del to expand the phenotype of "brain abnormalities, neurodegeneration, and dysosteosclerosis" (BANDDOS) and discuss its link with "adult leukoencephalopathy with axonal spheroids and pigmented glia" (ALSP), caused by heterozygous CSF1R variants. We evaluated medical, radiological, and laboratory findings and reviewed the literature. Patients presented with developmental delay, therapy-resistant epilepsy, dysmorphic features, and skeletal abnormalities. Secondary neurological decline occurred from 23 years in sibling one and from 20 years in sibling two. Brain imaging revealed multifocal white matter abnormalities and calcifications during initial disease in siblings two and three. Developmental brain anomalies, seen in all three, were most severe in sibling two. During neurological decline in siblings one and two, the leukoencephalopathy was progressive and had the MRI appearance of ALSP. Skeletal survey revealed osteosclerosis, most severe in sibling three. Blood markers, monocytes, dendritic cell subsets, and T-cell proliferation capacity were normal. Literature review revealed variable initial disease and secondary neurological decline. BANDDOS presents with variable dysmorphic features, skeletal dysplasia, developmental delay, and epilepsy with on neuro-imaging developmental brain anomalies, multifocal white matter abnormalities, and calcifications. Secondary neurological decline occurs with a progressive leukoencephalopathy, in line with early onset ALSP. Despite the role of CSF1R signaling in myeloid development, immune deficiency is absent. Phenotype varies within families; skeletal and neurological manifestations may be disparate.

Chemotherapy-induced intestinal epithelial damage directly promotes galectin-9-driven modulation of T cell behavior.

The intestine is vulnerable to chemotherapy-induced damage due to the high rate of intestinal epithelial cell (IEC) proliferation. We have developed a human intestinal organoid-based 3D model system to study the direct effect of chemotherapy-induced IEC damage on T cell behavior. Exposure of intestinal organoids to busulfan, fludarabine, and clofarabine induced damage-related responses affecting both the capacity to regenerate and transcriptional reprogramming. In ex vivo co-culture assays, prior intestinal organoid damage resulted in increased T cell activation, proliferation, and migration. We identified galectin-9 (Gal-9) as a key molecule released by damaged organoids. The use of anti-Gal-9 blocking antibodies or CRISPR/Cas9-mediated Gal-9 knock-out prevented intestinal organoid damage-induced T cell proliferation, interferon-gamma release, and migration. Increased levels of Gal-9 were found early after HSCT chemotherapeutic conditioning in the plasma of patients who later developed acute GVHD. Taken together, chemotherapy-induced intestinal damage can influence T cell behavior in a Gal-9-dependent manner which may provide novel strategies for therapeutic intervention.

Thymic NK-Cells and Their Potential in Cancer Immunotherapy.

Natural killer (NK)-cells are innate immune cells with potent anti-tumor capacity, capable of recognizing target cells without prior exposure. For this reason, NK-cells are recognized as a useful source of cell therapy. Although most NK-cells are derived from the bone marrow (BM), a separate developmental pathway in the thymus also exists, producing so-called thymic NK-cells. Unlike conventional NK-cells, thymic NK (tNK)-cells have a combined capacity for cytokine production and a natural ability to kill tumor cells in the presence of NK-cell receptor stimulatory ligands. Furthermore, tNK-cells are reported to express CD3 subunits intracellularly, without the presence of a rearranged T-cell receptor (TCR). This unique feature may enable harnessing of these cells with a TCR to combine NK- and T-cell effector properties in one cell type. The development, phenotype, and function of tNK-cells, and potential as a cell therapy is, however, poorly explored. In this review, we provide an overview of current literature on both murine and human tNK-cells in comparison to conventional BM-derived NK-cells, and discuss the potential applications of this cellular subset in the context of cancer immunotherapy.

Progressive demyelinating polyneuropathy after hematopoietic cell transplantation in metachromatic leukodystrophy: a case series.

Metachromatic leukodystrophy (MLD) is a neuro-metabolic disorder due to arylsulfatase A deficiency, causing demyelination of the central and peripheral nervous system. Hematopoietic cell transplantation (HCT) can provide a symptomatic and survival benefit for pre-symptomatic and early symptomatic patients by stabilizing CNS disease. This case series, however, illustrates the occurrence of severely progressive polyneuropathy shortly after HCT in two patients with late-infantile, one with late-juvenile, and one with adult MLD, leading to the inability to walk or sit without support. The patients had demyelinating polyneuropathy before HCT, performed at the ages of 2 years in the first two patients and at 14 and 23 years in the other two patients. The myeloablative conditioning regimen consisted of busulfan, fludarabine and, in one case, rituximab, with anti-thymocyte globulin, cyclosporine, steroids, and/or mycophenolate mofetil for GvHD prophylaxis. Polyneuropathy after HCT progressed parallel with tapering immunosuppression and paralleled bouts of infection and graft-versus-host disease (GvHD). Differential diagnoses included MLD progression, neurological GvHD or another (auto)inflammatory cause. Laboratory, electroneurography and pathology investigations were inconclusive. In two patients, treatment with immunomodulatory drugs led to temporary improvement, but not sustained stabilization of polyneuropathy. One patient showed recovery to pre-HCT functioning, except for a Holmes-like tremor, for which a peripheral origin cannot be excluded. One patient showed marginal response to immunosuppressive treatment and died ten months after HCT due to respiratory failure. The extensive diagnostic and therapeutic attempts highlight the challenge of characterizing and treating progressive polyneuropathy in patients with MLD shortly after HCT. We advise to consider repeat electro-neurography and possibly peripheral nerve biopsy in such patients. Nerve conduction blocks, evidence of the presence of T lymphocytes and macrophages in the neuronal and surrounding nerve tissue, and beneficial effects of immunomodulatory drugs may indicate a partially (auto)immune-mediated pathology. Polyneuropathy may cause major residual disease burden after HCT. MLD patients with progressive polyneuropathy could potentially benefit from a more intensified immunomodulatory drug regime following HCT, especially at times of immune activation.

Targeting pediatric cancers via T-cell recognition of the monomorphic MHC class I-related protein MR1.

Human leukocyte antigen (HLA) restriction of conventional T-cell targeting introduces complexity in generating T-cell therapy strategies for patients with cancer with diverse HLA-backgrounds. A subpopulation of atypical, major histocompatibility complex-I related protein 1 (MR1)-restricted T-cells, distinctive from mucosal-associated invariant T-cells (MAITs), was recently identified recognizing currently unidentified MR1-presented cancer-specific metabolites. It is hypothesized that the MC.7.G5 MR1T-clone has potential as a pan-cancer, pan-population T-cell immunotherapy approach. These cells are irresponsive to healthy tissue while conferring T-cell receptor(TCR) dependent, HLA-independent cytotoxicity to a wide range of adult cancers. Studies so far are limited to adult malignancies. Here, we investigated the potential of MR1-targeting cellular therapy strategies in pediatric cancer. Bulk RNA sequencing data of primary pediatric tumors were analyzed to assess MR1 expression. In vitro pediatric tumor models were subsequently screened to evaluate their susceptibility to engineered MC.7.G5 TCR-expressing T-cells. Targeting capacity was correlated with qPCR-based MR1 mRNA and protein overexpression. RNA expression of MR1 in primary pediatric tumors varied widely within and between tumor entities. Notably, embryonal tumors exhibited significantly lower MR1 expression than other pediatric tumors. In line with this, most screened embryonal tumors displayed resistance to MR1T-targeting in vitro MR1T susceptibility was observed particularly in pediatric leukemia and diffuse midline glioma models. This study demonstrates potential of MC.7.G5 MR1T-cell immunotherapy in pediatric leukemias and diffuse midline glioma, while activity against embryonal tumors was limited. The dismal prognosis associated with relapsed/refractory leukemias and high-grade brain tumors highlights the promise to improve survival rates of children with these cancers.

BEHAV3D: a 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response.

Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond.

High-dose individualized antithymocyte globulin with therapeutic drug monitoring in high-risk cord blood transplant.

BACKGROUND: Graft-versus-host disease (GvHD) and rejection are main limitations of cord blood transplantation (CBT), more so in patients with severe inflammation or previous rejections. While rigorous T-cell depletion with antithymocyte globulin (ATG) is needed to prevent GvHD and rejection, overexposure to ATG leads to slow T-cell recovery after transplantation, especially in CBT. OBJECTIVE: To evaluate high-dose, upfront ATG with individualized dosing and therapeutic drug monitoring (TDM) in pediatric CBT for patients at high risk for GvHD and rejection. STUDY DESIGN: Heavily inflamed patients and patients with a recent history of rejection were eligible for individualized high-dose ATG with real-time TDM. The ATG dosing scheme was adjusted to target a post-CBT exposure of <10 AU*day/mL, while achieving a pre-CBT exposure of 60-120 AU*day/mL; exposure levels previously defined for optimal efficacy and safety in terms of reduced GvHD and rejection, respectively. Main outcomes of interest included efficacy (target exposure attainment) and safety (incidence of GvHD and rejection). Other outcomes of interest included T-cell recovery and survival. RESULTS: Twenty-one patients were included ranging from 2 months to 18 years old, receiving an actual median cumulative dose of ATG of 13.3 mg/kg (range 6-30 mg/kg) starting at a median 15 days (range 12-17) prior to CBT. Dosing was adjusted in 14 patients (increased in 3 and decreased in 11 patients). Eighteen (86%) and 19 (91%) patients reached the target pre-CBT and post-CBT exposure, respectively. Cumulative incidence for acute GvHD was 34% (95% CI 23-45) and 5% (95% CI 0-10%) for grade 2-4 and grade 3-4, respectively; cumulative incidence of rejection was 9% (95% CI 2-16%). Overall survival was 75% (95% CI 65-85%). CONCLUSION: Individualized high-dose ATG with TDM is feasible and safe for patients with hyperinflammation in a CBT setting. We observe high target ATG exposure attainment, good immune reconstitution (despite very high doses of ATG) and acceptable rates of GvHD and rejection.

B-cell precursor acute lymphoblastic leukemia elicits an interferon-α/ß response in bone marrow-derived mesenchymal stroma.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can hijack the normal bone marrow microenvironment to create a leukemic niche which facilitates blast cell survival and promotes drug resistance. Bone marrow-derived mesenchymal stromal cells (MSC) mimic this protective environment in ex vivo co-cultures with leukemic cells obtained from children with newly diagnosed BCP-ALL. We examined the potential mechanisms of this protection by RNA sequencing of flow-sorted MSC after co-culture with BCP-ALL cells. Leukemic cells induced an interferon (IFN)-related gene signature in MSC, which was partially dependent on direct cell-cell signaling. The signature was selectively induced by BCP-ALL cells, most profoundly by ETV6-RUNX1-positive ALL cells, as co-culture of MSC with healthy immune cells did not provoke a similar IFN signature. Leukemic cells and MSC both secreted IFNα and IFNß, but not IFNγ. In line, the IFN gene signature was sensitive to blockade of IFNα/ß signaling, but less to that of IFNγ. The viability of leukemic cells and level of resistance to three chemotherapeutic agents was not affected by interference with IFN signaling using selective IFNα/ß inhibitors or silencing of IFN-related genes. Taken together, our data suggest that the leukemia-induced expression of IFNα/ß-related genes by MSC does not support survival of BCP-ALL cells but may serve a different role in the pathobiology of BCP-ALL.

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Influence of ATLG serum levels on CD3/CD19-depleted hematopoietic grafts and on immune recovery in pediatric haplo-HSCT.

ABSTRACT: Anti-T lymphocyte globulin (ATLG) significantly reduces the risk of engraftment failure in allogeneic hematopoietic stem cell transplant (HSCT) but hampers posttransplant immune reconstitution. We hypothesized that in patients receiving haploidentical CD3/CD19-depleted grafts, these double-edged effects could be better balanced by attaining high ATLG serum concentrations before transplant but as low as possible on the day of transplant. Therefore, we moved the start of ATLG application to day -12 and determined serum concentrations of T-cell-specific ATLG in pediatric patients treated with 3 established dosing regimens (15, 30, or 60 mg/kg). Corresponding mean T-cell-specific ATLG serum concentrations at day 0 were 1.14, 2.99, or 12.10 µg/mL, respectively. Higher ATLG doses correlated with higher peak levels at days -8 and -7 and reduced graft rejection, whereas lower ATLG doses correlated with significantly faster posttransplant recovery of T and natural killer cells. The rate of graft-versus-host disease remained low, independent of ATLG doses. Moreover, in vitro assays showed that ATLG concentrations of 2.0 µg/mL and lower only slightly reduced the activity of natural killer cells, and therefore, the function of such effector cells might be preserved in the grafts. Pharmacokinetic analysis, compatible with linear first-order kinetics, revealed similar half-life values, independent of ATLG doses. Hence, the day on which a desired ATLG serum level is reached can be calculated before HSCT. Our retrospective study demonstrates the relevance of dosing and time of administration of ATLG on engraftment and immune recovery in ex vivo CD3/CD19-depleted haploidentical HSCT.

Daily intranasal palivizumab to prevent respiratory syncytial virus infection in healthy preterm infants: a phase 1/2b randomized placebo-controlled trial.

Background: Mucosal administration of monoclonal antibodies (mAbs) against respiratory pathogens is a promising alternative for systemic administration because lower doses are required for protection. Clinical development of mucosal mAbs is a highly active field yet clinical proof-of-concept is lacking. Methods: In this investigator-initiated, double-blind, randomized placebo-controlled trial, we evaluated intranasal palivizumab for the prevention of RSV infection in preterm infants (Dutch Trial Register NTR7378 and NTR7403). We randomized infants 1:1 to receive intranasal palivizumab (1 mg/mL) or placebo once daily during the RSV season. Any RSV infection was the primary outcome and RSV hospitalization was the key secondary outcome. The primary outcome was analyzed with a mixed effect logistic regression on the modified intention-to-treat population. Findings: We recruited 268 infants between Jan 14, 2019 and Jan 28, 2021, after which the trial was stopped for futility following the planned interim analysis. Adverse events were similar in both groups (22/134 (16.4%) palivizumab arm versus 26/134 (19.4%) placebo arm). There were 6 dropouts and 168 infants were excluded from the efficacy analyses due to absent RSV circulation during the SARS-CoV-2 pandemic. Any RSV infection was similar in infants in both groups (18/47 (38.3%) palivizumab arm versus 11/47 (23.4%) placebo arm; aOR 2.2, 95% CI 0.7-6.5). Interpretation: Daily intranasal palivizumab did not prevent RSV infection in late preterm infants. Our findings have important implications for the clinical development of mucosal mAbs, namely the necessity of timely interim analyses and further research to understand mucosal antibody half-life. Funding: Funded by the Department of Pediatrics, University Medical Centre Utrecht, the Netherlands.