A "Prime and Expand" strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy.

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming ("Prime" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 ("Expand" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting "Prime and Expand" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the "Prime and Expand" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

ACR Appropriateness Criteria® Urinary Tract Infection-Child: 2023 Update.

Urinary tract infection (UTI) is a frequent infection in childhood. The diagnosis is usually made by history and physical examination and confirmed by urine analysis. Cystitis is infection or inflammation confined to the bladder, whereas pyelonephritis is infection or inflammation of kidneys. Pyelonephritis can cause renal scarring, which is the most severe long-term sequela of UTI and can lead to accelerated nephrosclerosis, leading to hypertension and chronic renal failure. The role of imaging is to guide treatment by identifying patients who are at high risk to develop recurrent UTIs or renal scarring. This document provides initial imaging guidelines for children presenting with first febrile UTI with appropriate response to medical management, atypical or recurrent febrile UTI, and follow-up imaging for children with established vesicoureteral reflux. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision process support the systematic analysis of the medical literature from peer reviewed journals. Established methodology principles such as Grading of Recommendations Assessment, Development, and Evaluation or GRADE are adapted to evaluate the evidence. The RAND/UCLA Appropriateness Method User Manual provides the methodology to determine the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where peer reviewed literature is lacking or equivocal, experts may be the primary evidentiary source available to formulate a recommendation.

ACR Appropriateness Criteria® Soft Tissue Vascular Anomalies: Vascular Malformations and Infantile Vascular Tumors (Non-CNS)-Child.

Soft tissue vascular anomalies may be composed of arterial, venous, and/or lymphatic elements, and diagnosed prenatally or later in childhood or adulthood. They are divided into categories of vascular malformations and vascular tumors. Vascular malformations are further divided into low-flow and fast-flow lesions. A low-flow lesion is most common, with a prevalence of 70%. Vascular tumors may behave in a benign, locally aggressive, borderline, or malignant manner. Infantile hemangioma is a vascular tumor that presents in the neonatal period and then regresses. The presence or multiple skin lesions in an infant can signal underlying visceral vascular anomalies, and complex anomalies may be associated with overgrowth syndromes. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision process support the systematic analysis of the medical literature from peer reviewed journals. Established methodology principles such as Grading of Recommendations Assessment, Development, and Evaluation or GRADE are adapted to evaluate the evidence. The RAND/UCLA Appropriateness Method User Manual provides the methodology to determine the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where peer reviewed literature is lacking or equivocal, experts may be the primary evidentiary source available to formulate a recommendation.

An "off-the-shelf" CD2 universal CAR-T therapy for T-cell malignancies.

T-cell malignancies are associated with frequent relapse and high morbidity, which is partly due to the lack of effective or targeted treatment options. To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogeneic "universal" CD2-targeting CAR-T cell (UCART2), in which the CD2 antigen is deleted to prevent fratricide, and the T-cell receptor is removed to prevent GvHD. UCART2 demonstrated efficacy against T-ALL and CTCL and prolonged the survival of tumor-engrafted NSG mice in vivo. To evaluate the impact of CD2 on CAR-T function, we generated CD19 CAR-T cells (UCART19) with or without CD2 deletion, single-cell secretome analysis revealed that CD2 deletion in UCART19 reduced frequencies of the effector cytokines (Granzyme-B and IFN-γ). We also observed that UCART19ΔCD2 had reduced anti-tumor efficacy compared to UCART19 in a CD19+NALM6 xenograft model. Of note is that the reduced efficacy resulting from CD2 deletion was reversed when combined with rhIL-7-hyFc, a long-acting recombinant human interleukin-7. Treatment with rhIL-7-hyFc prolonged UCART2 persistence and increased survival in both the tumor re-challenge model and primary patient T-ALL model in vivo. Together, these data suggest that allogeneic fratricide-resistant UCART2, in combination with rhIL-7-hyFc, could be a suitable approach for treating T-cell malignancies.

Anti-myeloma efficacy of CAR-iNKT is enhanced with a long-acting IL-7, rhIL-7-hyFc.

Multiple myeloma (MM), a malignancy of mature plasma cells, remains incurable. B-cell maturation antigen (BCMA) is the lead protein target for chimeric antigen receptor (CAR) therapy because of its high expression in most MM, with limited expression in other cell types, resulting in favorable on-target, off tumor toxicity. The response rate to autologous BCMA CAR-T therapy is high; however, it is not curative and is associated with risks of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome. Outcomes in patients treated with BCMA CAR-T cells (CAR-Ts) may improve with allogeneic CAR T-cell therapy, which offer higher cell fitness and reduced time to treatment. However, to prevent the risk of graft-versus-host disease (GVHD), allogenic BCMA CAR-Ts require genetic deletion of the T-cell receptor (TCR), which has potential for unexpected functional or phenotype changes. Invariant natural killer T cells (iNKTs) have an invariant TCR that does not cause GVHD and, as a result, can be used in an allogeneic setting without the need for TCR gene editing. We demonstrate significant anti-myeloma activity of BCMA CAR-iNKTs in a xenograft mouse model of myeloma. We found that a long-acting interleukin-7 (IL-7), rhIL-7-hyFc, significantly prolonged survival and reduced tumor burden in BCMA CAR-iNKT-treated mice in both primary and re-challenge settings. Furthermore, in CRS in vitro assays, CAR-iNKTs induced less IL-6 than CAR-Ts, suggesting a reduced likelihood of CAR-iNKT therapy to induce CRS in patients. These data suggest that BCMA CAR-iNKTs are potentially a safer, effective alternative to BCMA CAR-Ts and that BCMA CAR-iNKT efficacy is further potentiated with rhIL-7-hyFc.

S100A9 upregulated by IFNGR signaling blockade functions as a novel GVHD suppressor without compromising GVL in mice.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for both malignant and nonmalignant hematologic disorders. However, graft-versus-host disease (GVHD) and malignant relapse limit its therapeutic success. We previously demonstrated that the blockade of interferon-gamma receptor (IFNGR) signaling in donor T cells resulted in a reduction in GVHD while preserving graft-versus-leukemia (GVL) effects. However, the underlying molecular mechanisms remain inconclusive. In this study, we found that S100A9 is a novel GVHD suppressor upregulated when IFNGR is blocked in T cells. Both Ifngr1-/- and S100a9-overexpressing T cells significantly reduced GVHD without compromising GVL, altering donor T-cell trafficking to GVHD target organs in our mouse model of allo-HSCT. In addition, in vivo administration of recombinant murine S100A9 proteins prolongs the overall survival of recipient mice. Furthermore, in vivo administration of anti-human IFNGRα neutralizing antibody (αhGR-Nab) significantly upregulates the expression of S100A9 in human T cells and improved GVHD in our mouse model of xenogeneic human peripheral blood mononuclear cell transplantation. Consistent with S100a9-overexpressing T cells in our allo-HSCT model, αhGR-Nab reduced human T-cell trafficking to the GVHD target organs. Taken together, S100A9, a downstream molecule suppressed by IFNGR signaling, functions as a novel GVHD suppressor without compromising GVL.

Chest CT in the evaluation of child abuse - When is it useful?

BACKGROUND: Indications for chest CT in evaluation of child abuse are unknown. OBJECTIVE: Determine which groups of children can best benefit from chest CT. PARTICIPANTS AND SETTING: 10-year (1/2010 to 12/2019) retrospective study of children <3 years who had chest CT within 3 days of the initial skeletal survey. METHODS: Demographic and clinical information were obtained from medical records. Two pediatric radiologists reviewed, independently and blinded to clinical information, anonymized rib X-rays (initial and follow up when available) and chest CT. Disagreements were resolved by a third pediatric radiologist. Agreement was evaluated using kappa statistics. Number and percentage of fractures were analyzed by negative binomial models and chi-square tests, respectively. RESULTS: 50 children (21 females) with average age of 9.7 months, 27 of whom had follow-up radiography. Agreement on initial and follow-up X-rays was substantial (k = 0.72) and perfect (k = 1.00), respectively, and almost perfect (k = 0.82) for CT scans. Chest CT demonstrated more fractures than X-ray, both initially (112 vs. 42, p < 0.0001) and at follow-up (93 vs. 49, p < 0.0001). Significantly more additional fractures were found at CT (11/13, 84.6 %) in patients with positive than in those with negative initial surveys (7/37, 18.9 %, p < 0.001). Ten initial surveys had only indeterminate fractures; four of them had fractures and six had no fractures on CT. Chest CT missed one patient (1/27, 3.7 %) with acute nondisplaced anterior rib fractures. CONCLUSION: Chest CT can be considered in children with negative skeletal survey and high clinical suspicion for child abuse, and when the diagnosis of rib fractures is indeterminate.

A long-acting interleukin-7, rhIL-7-hyFc, enhances CAR T cell expansion, persistence, and anti-tumor activity.

Chimeric antigen receptor (CAR) T cell therapy is routinely used to treat patients with refractory hematologic malignancies. However, a significant proportion of patients experience suboptimal CAR T cell cytotoxicity and persistence that can permit tumor cell escape and disease relapse. Here we show that a prototype pro-lymphoid growth factor is able to enhance CAR T cell efficacy. We demonstrate that a long-acting form of recombinant human interleukin-7 (IL-7) fused with hybrid Fc (rhIL-7-hyFc) promotes proliferation, persistence and cytotoxicity of human CAR T cells in xenogeneic mouse models, and murine CAR T cells in syngeneic mouse models, resulting in long-term tumor-free survival. Thus, rhIL-7-hyFc represents a tunable clinic-ready adjuvant for improving suboptimal CAR T cell activity.

ACR Appropriateness Criteria® Osteomyelitis or Septic Arthritis-Child (Excluding Axial Skeleton).

Imaging plays an integral role in the evaluation of suspected musculoskeletal infections in children, not only in the accurate identification of infection such as osteomyelitis or septic arthritis, but also in guiding management. Various diagnostic modalities serve different purposes in the assessment of suspected pediatric musculoskeletal infections. The purpose of this document is to provide imaging guidance in the most frequently encountered clinical scenarios in which osteomyelitis and/or septic arthritis are suspected, outside of the axial skeleton. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion.

ACR Appropriateness Criteria® Crohn Disease-Child.

Crohn disease is an inflammatory condition of the gastrointestinal tract with episodes of exacerbation and remission occurring in children, adolescents, and adults. Crohn disease diagnosis and treatment depend upon a combination of clinical, laboratory, endoscopic, histological, and imaging findings. Appropriate use of imaging provides critical information in the settings of diagnosis, assessment of acute symptoms, disease surveillance, and therapy monitoring. Four variants are discussed. The first variant discusses the initial imaging for suspected Crohn disease before established diagnosis. The second variant pertains to appropriateness of imaging modalities during suspected acute exacerbation. The third variant is a substantial discussion of recommendations related to disease surveillance and monitoring of Crohn disease. Finally, panel recommendations and discussion of perianal fistulizing disease imaging completes the document. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment.

CS1 CAR-T targeting the distal domain of CS1 (SLAMF7) shows efficacy in high tumor burden myeloma model despite fratricide of CD8+CS1 expressing CAR-T cells.

Despite improvement in treatment options for myeloma patients, including targeted immunotherapies, multiple myeloma remains a mostly incurable malignancy. High CS1 (SLAMF7) expression on myeloma cells and limited expression on normal cells makes it a promising target for CAR-T therapy. The CS1 protein has two extracellular domains - the distal Variable (V) domain and the proximal Constant 2 (C2) domain. We generated and tested CS1-CAR-T targeting the V domain of CS1 (Luc90-CS1-CAR-T) and demonstrated anti-myeloma killing in vitro and in vivo using two mouse models. Since fratricide of CD8 + cells occurred during production, we generated fratricide resistant CS1 deficient Luc90- CS1- CAR-T (ΔCS1-Luc90- CS1- CAR-T). This led to protection of CD8 + cells in the CAR-T cultures, but had no impact on efficacy. Our data demonstrate targeting the distal V domain of CS1 could be an effective CAR-T treatment for myeloma patients and deletion of CS1 in clinical production did not provide an added benefit using in vivo immunodeficient NSG preclinical models.

Multi-modality imaging characteristics of costochondral fractures, a highly specific rib fracture for child abuse.

BACKGROUND: Rib fractures in young children are strongly associated with nonaccidental trauma (NAT). Costochondral junction (CCJ) fractures are unique with most being identified in the healing phase on radiographs. NAT-associated CCJ fractures, therefore, may be underdiagnosed. Improved diagnoses of CCJ fractures may lead to better identification of NAT. OBJECTIVE: To document the association of CCJ fractures with NAT, and improve CCJ fracture recognition by documenting the imaging features with multiple radiologic modalities. MATERIALS AND METHODS: Children, ages 0-4 years, with CCJ fractures on radiologic reports were identified over a 10-year period. All available radiographic skeletal surveys, chest radiographs, computed tomography (CT), magnetic resonance imaging (MRI) and ultrasound (US) studies were reviewed. We chose CT as the radiologic gold standard. Imaging patterns of the primary fracture and healing changes were documented. The diagnosis of NAT by the child protective team was documented. RESULTS: One hundred and nine CCJ fractures were found in 22 patients, 21 of whom were diagnosed with NAT (95.5%). Radiographic skeletal survey identified 34.6% of CCJ fractures (P < 0.0001) with a sensitivity of 32.5% and specificity of 99.2%. MRI identified 50.0% of CCJ fractures with a sensitivity of 42.9% and specificity of 98.1%. CONCLUSION: CCJ fractures are highly specific for NAT. As sensitivity is low for radiographic skeletal survey in CCJ fracture diagnosis compared with CT, CT may have a role in confirming a clinical suspicion of NAT.

Antibody-drug conjugates plus Janus kinase inhibitors enable MHC-mismatched allogeneic hematopoietic stem cell transplantation.

Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.

Boosting and lassoing new prostate cancer SNP risk factors and their connection to selenium.

We begin by arguing that the often used algorithm for the discovery and use of disease risk factors, stepwise logistic regression, is unstable. We then argue that there are other algorithms available that are much more stable and reliable (e.g. the lasso and gradient boosting). We then propose a protocol for the discovery and use of risk factors using lasso or boosting variable selection. We then illustrate the use of the protocol with a set of prostate cancer data and show that it recovers known risk factors. Finally, we use the protocol to identify new and important SNP based risk factors for prostate cancer and further seek evidence for or against the hypothesis of an anticancer function for Selenium in prostate cancer. We find that the anticancer effect may depend on the SNP-SNP interaction and, in particular, which alleles are present.

CD7-deleted hematopoietic stem cells can restore immunity after CAR T cell therapy.

Targeting T cell malignancies with universal CD7-targeting chimeric antigen receptor T cells (UCART7) can lead to profound immune deficiency due to loss of normal T and NK cells. While a small population of endogenous CD7- T cells exists, these cells are unlikely to be able to repopulate the entire immune repertoire after UCART7 treatment, as they are limited in number and proliferative capacity. To rescue T and NK cells after UCART7, we created hematopoietic stem cells genetically deleted for CD7 (CD7-KO HSCs). CD7-KO HSCs were able to engraft immunodeficient mice and differentiate into T and NK cells lacking CD7 expression. CD7-KO T and NK cells could perform effector functions as robustly as control T and NK cells. Furthermore, CD7-KO T cells were phenotypically and functionally distinct from endogenous CD7- T cells, indicating that CD7-KO T cells can supplement immune functions lacking in CD7- T cells. Mice engrafted with CD7-KO HSCs maintained T and NK cell numbers after UCART7 treatment, while these were significantly decreased in control mice. These studies support the development of CD7-KO HSCs to augment host immunity in patients with T cell malignancies after UCART7 treatment.

Prospective evaluation of World Health Organization guidelines for diagnosis of pneumonia in children presenting to an emergency department in a resource-limited setting.

BACKGROUND: Clinicians in resource-limited settings commonly use the World Health Organization criteria to diagnose pneumonia in children. AIM: The aim of this study was to prospectively evaluate the diagnostic accuracy of the WHO criteria compared with chest radiograph for the diagnosis of pneumonia in children under 5 years of age presenting to an emergency department (ED) in Nepal. METHODS: A prospective cross-sectional study of children presenting to an ED with respiratory complaints in Nepal was conducted. It included all children under 5 years of age with cough or difficulty breathing who received a chest radiograph. Paediatric pneumonia was diagnosed according to WHO criteria when a child presented with a cough or difficulty breathing and met the age-related WHO-defined respiratory rate for tachypnoea. The criterion standard was radiographic pneumonia. The primary outcome was the sensitivity and specificity of the WHO criteria for diagnosis of pneumonia. RESULTS: Of 324 patients enrolled, 72 had radiographic pneumonia. The median (IQR) age was 17 (23) months. Overall, WHO criteria had a sensitivity of 71% (95% CI 59-81) and specificity of 57% (95% CI 50-63). Respiratory rate had poor diagnostic accuracy for pneumonia with an area under the curve of 0.65. CONCLUSION: The WHO criteria had poor sensitivity and specificity for the diagnosis of pneumonia in children presenting to the ED in a resource-limited setting.

Multidimensional Analyses of Donor Memory-Like NK Cells Reveal New Associations with Response after Adoptive Immunotherapy for Leukemia.

Natural killer (NK) cells are an emerging cancer cellular therapy and potent mediators of antitumor immunity. Cytokine-induced memory-like (ML) NK cellular therapy is safe and induces remissions in patients with acute myeloid leukemia (AML). However, the dynamic changes in phenotype that occur after NK-cell transfer that affect patient outcomes remain unclear. Here, we report comprehensive multidimensional correlates from ML NK cell-treated patients with AML using mass cytometry. These data identify a unique in vivo differentiated ML NK-cell phenotype distinct from conventional NK cells. Moreover, the inhibitory receptor NKG2A is a dominant, transcriptionally induced checkpoint important for ML, but not conventional NK-cell responses to cancer. The frequency of CD8α+ donor NK cells is negatively associated with AML patient outcomes after ML NK therapy. Thus, elucidating the multidimensional dynamics of donor ML NK cells in vivo revealed critical factors important for clinical response, and new avenues to enhance NK-cell therapeutics. SIGNIFICANCE: Mass cytometry reveals an in vivo memory-like NK-cell phenotype, where NKG2A is a dominant checkpoint, and CD8α is associated with treatment failure after ML NK-cell therapy. These findings identify multiple avenues for optimizing ML NK-cell immunotherapy for cancer and define mechanisms important for ML NK-cell function.This article is highlighted in the In This Issue feature, p. 1775.

CAR-modified memory-like NK cells exhibit potent responses to NK-resistant lymphomas.

Natural killer (NK) cells are a promising cellular immunotherapy for cancer. Cytokine-induced memory-like (ML) NK cells differentiate after activation with interleukin-12 (IL-12), IL-15, and IL-18, exhibit potent antitumor responses, and safely induce complete remissions in patients with leukemia. However, many cancers are not fully recognized via NK cell receptors. Chimeric antigen receptors (CARs) have been used to enhance tumor-specific recognition by effector lymphocytes. We hypothesized that ML differentiation and CAR engineering would result in complementary improvements in NK cell responses against NK-resistant cancers. To test this idea, peripheral blood ML NK cells were modified to express an anti-CD19 CAR (19-CAR-ML), which displayed significantly increased interferon γ production, degranulation, and specific killing against NK-resistant lymphoma lines and primary targets compared with nonspecific control CAR-ML NK cells or conventional CAR NK cells. The 19-CAR and ML responses were synergistic and CAR specific and required immunoreceptor tyrosine-based activation motif signaling. Furthermore, 19-CAR-ML NK cells generated from lymphoma patients exhibited improved responses against their autologous lymphomas. 19-CAR-ML NK cells controlled lymphoma burden in vivo and improved survival in human xenograft models. Thus, CAR engineering of ML NK cells enhanced responses against resistant cancers and warrants further investigation, with the potential to broaden ML NK cell recognition against a variety of NK cell-resistant tumors.