Anti-T-lymphocyte globulin exposure is associated with acute graft-versus-host disease and relapse in pediatric acute lymphoblastic leukemia patients undergoing hematopoietic stem cell transplantation: a multinational prospective study.

Anti-T-lymphocyte globulin (ATLG) is used in hematopoietic stem cell transplantation (HSCT) to prevent graft-versus-host disease (GVHD) and graft failure. To date, insight in ATLG pharmacokinetics and -dynamics (PK/PD) is limited, and population PK (POPPK) models are lacking. In this prospective study, we describe ATLG POPPK using NONMEM® and the impact of ATLG exposure on clinical outcome and immune reconstitution in a homogeneous cohort of pediatric acute lymphoblastic leukemia (ALL) patients transplanted with a matched unrelated donor and receiving uniform ATLG dosing. Based on 121 patients and 812 samples for POPPK analysis, a two-compartmental model with parallel linear and non-linear clearance and bodyweight as covariate, best described the ATLG concentration-time data. The level of ATLG exposure (day active ATLG <1 AU/mL, median 16 days post-HSCT) was strongly associated with aGVHD grade II-IV, with a lower incidence in patients with prolonged active ATLG exposure (≤day 16 50% vs. >day 16 8.2%; P<0.001). When stratified for remission state, patients transplanted in complete remission (CR) 2 or 3 with prolonged ATLG exposure had a higher relapse risk, while this effect was not seen in CR1 patients (P=0.010). High level ATLG exposure was associated with delayed CD4 T-cell recovery at 4 and 8 weeks post-HSCT, but not at 12 weeks, and overall and relapse-free survival were not influenced by CD4 recovery at 12 weeks post-HSCT. This study underlines the importance of individualized ATLG exposure with the use of model-informed precision dosing in order to optimize the HSCT outcome in pediatric ALL.

Population pharmacokinetics of subcutaneous alemtuzumab in kidney transplantation.

AIM: Alemtuzumab is a monoclonal antibody used as induction immunosuppressive therapy in kidney transplantation. It targets CD52 on lymphocytes, inducing profound immune cell depletion upon administration. Owing to its off-label status in kidney transplantation, its pharmacokinetic characteristics are largely unknown in this setting, and its current fixed dosing algorithm originates from other populations. We developed a population pharmacokinetic model for alemtuzumab in kidney transplant recipients and investigated the potential of personalized alemtuzumab therapy. METHODS: In total, 362 pharmacokinetic observations drawn 0-165 days after transplantation were available from 61 adult kidney transplant recipients who received two consecutive doses of 15 mg alemtuzumab subcutaneously. A population pharmacokinetic model was developed using nonlinear mixed-effects modelling and applied to simulate various dosing regimens. RESULTS: The alemtuzumab concentration-time data were best described by a two-compartmental model with first-order absorption and parallel first-order and time-varying concentration-dependent elimination, with between-subject variability on the first-order elimination (39.6%) and central distribution volume (39.6%). Alemtuzumab pharmacokinetics varied with body size, rendering lighter individuals exposed to lympholytic alemtuzumab concentrations (>0.1 mg/L) for prolonged durations as compared to their heavier peers. This between-subject variability could be reduced through lean bodyweight-adjusted dosing, showing a twofold to threefold reduction in the slope of the median alemtuzumab exposure over the bodyweight range. CONCLUSION: Alemtuzumab displays substantial pharmacokinetic variability in kidney transplant recipients, which may warrant a personalized treatment strategy. Lean bodyweight-adjusted dosing poses an option for individualized dosing, but further evaluation of its potential clinical benefit is warranted.

Development and Validation of an Efficient and Highly Sensitive Enzyme-Linked Immunosorbent Assay for Alemtuzumab Quantification in Human Serum and Plasma.

BACKGROUND: Alemtuzumab is a humanized monoclonal antibody that targets the CD52 glycoprotein expressed on most lymphocytes, subsequently inducing complement-mediated and antibody-mediated cytotoxicity. Owing to its ability to induce profound immune depletion, alemtuzumab is frequently used in patients before allogeneic hematopoietic stem cell transplantation to prevent graft rejection and acute graft-versus-host disease. In this clinical context, a stable immunoassay with high sensitivity and specificity to determine alemtuzumab levels is essential for performing pharmacokinetic and pharmacodynamic analyses; however, the available methods have several limitations. Here, we report the successful development and validation of an efficient and highly sensitive enzyme-linked immunosorbent assay technique based on commercially available reagents to quantify alemtuzumab in human serum or plasma. METHODS: This enzyme-linked immunosorbent assay technique was developed and validated in accordance with the European Medicines Agency guidelines on bioanalytical method validation. RESULTS: The assay sensitivity (lower limit of quantification) is 0.5 ng·mL -1 , and the dynamic range is 0.78-25 ng·mL -1 . To accommodate quantification of peak concentration and concentrations below the lympholytic level (<0.1 mcg·mL -1 ), patients' serum samples were prediluted 20-400 times according to the expected alemtuzumab concentration. The overall within-run accuracy was between 96% and 105%, whereas overall within-run precision (coefficient of variation) was between 3% and 9%. The between-run assessment provided an overall accuracy between 86% and 95% and an overall coefficient of variation between 5% and 14%. CONCLUSIONS: The developed assay provides accurate insight into alemtuzumab exposure and its effects on the clinical response to treatment, which is key to optimizing treatment strategies.

IL-7 and IL-15 Levels Reflect the Degree of T Cell Depletion during Lymphopenia and Are Associated with an Expansion of Effector Memory T Cells after Pediatric Hematopoietic Stem Cell Transplantation.

Differentially and functionally distinct T cell subsets are involved in the development of complications after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about factors regulating their recovery after HSCT. In this study, we investigated associations between immune-regulating cytokines, T cell differentiation, and clinical outcomes. We included 80 children undergoing allogeneic HSCT for acute leukemia using bone marrow or peripheral blood stem cells grafted from a matched sibling or unrelated donor. Cytokines (IL-7, IL-15, IL-18, SCF, IL-6, IL-2, and TNF-α) and active anti-thymocyte globulin (ATG) levels were longitudinally measured along with extended T cell phenotyping. The cytokine profiles showed a temporary rise in IL-7 and IL-15 during lymphopenia, which was strongly dependent on exposure to active ATG. High levels of IL-7 and IL-15 from graft infusion to day +30 were predictive of slower T cell recovery during the first 2 mo post-HSCT; however, because of a major expansion of memory T cell stages, only naive T cells remained decreased after 3 mo (p < 0.05). No differential effect was seen on polarization of CD4+ T cells into Th1, Th2, or Th17 cells or regulatory T cells. Low levels of IL-7 and IL-15 at day +14 were associated with acute graft-versus-host disease grades II-IV in ATG-treated patients (p = 0.0004 and p = 0.0002, respectively). Children with IL-7 levels comparable to healthy controls at day +14 post-HSCT were less likely to develop EBV reactivation posttransplant. These findings suggest that quantification of IL-7 and IL-15 may be useful as biomarkers in assessing the overall T cell depletion and suggest a potential for predicting complications after HSCT.

T and NK Cells in IL2RG-Deficient Patient 50 Years After Hematopoietic Stem Cell Transplantation.

The first successful European hematopoietic stem cell transplantation (HSCT) was performed in 1968 as treatment in a newborn with IL2RG deficiency using an HLA-identical sibling donor. Because of declining naive T and natural killer (NK) cells, and persistent human papilloma virus (HPV)-induced warts, the patient received a peripheral stem cell boost at the age of 37 years. NK and T cells were assessed before and up to 14 years after the boost by flow cytometry. The boost induced renewed reconstitution of functional NK cells that were 14 years later enriched for CD56dimCD27+ NK cells. T-cell phenotype and T-cell receptor (TCR) repertoire were simultaneously analyzed by including TCR Vß antibodies in the cytometry panel. Naive T-cell numbers with a diverse TCR Vß repertoire were increased by the boost. Before and after the boost, clonal expansions with a homogeneous TIGIT and PD-1 phenotype were identified in the CD27- and/or CD28- memory population in the patient, but not in the donor. TRB sequencing was applied on sorted T-cell subsets from blood and on T cells from skin biopsies. Abundant circulating CD8 memory clonotypes with a chronic virus-associated CD57+KLRG1+CX3CR1+ phenotype were also present in warts, but not in healthy skin of the patient, suggesting a link with HPV. In conclusion, we demonstrate in this IL2RG-deficient patient functional NK cells, a diverse and lasting naive T-cell compartment, supported by a stem cell boost, and an oligoclonal memory compartment half a century after HSCT.

A Comprehensive Workflow for Applying Single-Cell Clustering and Pseudotime Analysis to Flow Cytometry Data.

The introduction of single-cell platforms inspired the development of high-dimensional single-cell analysis tools to comprehensively characterize the underlying cellular heterogeneity. Flow cytometry data are traditionally analyzed by (subjective) gating of subpopulations on two-dimensional plots. However, the increasing number of parameters measured by conventional and spectral flow cytometry reinforces the need to apply many of the recently developed tools for single-cell analysis on flow cytometry data, as well. However, the myriads of analysis options offered by the continuously released novel packages can be overwhelming to the immunologist with limited computational background. In this article, we explain the main concepts of such analyses and provide a detailed workflow to illustrate their implications and additional prerequisites when applied on flow cytometry data. Moreover, we provide readily applicable R code covering transformation, normalization, dimensionality reduction, clustering, and pseudotime analysis that can serve as a template for future analyses. We demonstrate the merit of our workflow by reanalyzing a public human dataset. Compared with standard gating, the results of our workflow provide new insights in cellular subsets, alternative classifications, and hypothetical trajectories. Taken together, we present a well-documented workflow, which utilizes existing high-dimensional single-cell analysis tools to reveal cellular heterogeneity and intercellular relationships in flow cytometry data.

NK Cell-Dependent Antibody-Mediated Immunotherapy Is Improved In Vitro and In Vivo When Combined with Agonists for Toll-like Receptor 2 in Head and Neck Cancer Models.

The immunosuppressive character of head and neck cancers may explain the relatively low response rates to antibody therapy targeting a tumor antigen, such as cetuximab, and anti-PD-1 checkpoint inhibition. Immunostimulatory agents that overcome tumor-derived inhibitory signals could augment therapeutic efficacy, thereby enhancing tumor elimination and improving patient survival. Here, we demonstrate that cetuximab treatment combined with immunostimulatory agonists for Toll-like receptor (TLR) 2 induces profound immune responses. Natural killer (NK) cells, isolated from healthy individuals or patients with head and neck cancer, harbored enhanced cytotoxic capacity and increased tumor-killing potential in vitro. Additionally, combination treatment increased the release of several pro-inflammatory cytokines and chemokines by NK cells. Tumor-bearing mice that received cetuximab and the TLR2 ligand Pam3CSK4 showed increased infiltration of immune cells into the tumors compared to mice that received cetuximab monotherapy, resulting in a significant delay in tumor growth or even complete tumor regression. Moreover, combination treatment resulted in improved overall survival in vivo. In conclusion, combining tumor-targeting antibody-based immunotherapy with TLR stimulation represents a promising treatment strategy to improve the clinical outcomes of cancer patients. This treatment could well be applied together with other therapeutic strategies such as anti-PD-(L)1 checkpoint inhibition to further overcome immunosuppression.

MRP8/14 and neutrophil elastase for predicting treatment response and occurrence of flare in patients with juvenile idiopathic arthritis.

OBJECTIVE: To study two neutrophil activation markers, myeloid-related protein (MRP) 8/14 and neutrophil elastase (NE), for their ability to predict treatment response and flare in patients with JIA. METHODS: Using samples from two cohorts (I and II), we determined MRP8/14 and NE levels of 32 (I) and 81 (II) patients with new-onset, DMARD-naïve arthritis and compared patients who responded to treatment (defined as fulfilling ≥ adjusted ACRpedi50 response and/or inactive disease) with non-responders (defined as fulfilling < adjusted ACRpedi50 response and/or active disease) at 6 and 12 months. Secondly, we compared biomarker levels of 54 (I) and 34 (II) patients with clinically inactive disease who did or did not suffer from a flare of arthritis after 6 or 12 months. Receiver operating characteristic analyses were carried out to study the predictive value of MRP8/14 and NE for treatment response and flare. RESULTS: For both cohorts, baseline MRP8/14 and NE levels for patients who did or did not respond to treatment were not different. Also, MRP8/14 and NE levels were not different in patients who did or did not flare. Receiver operating characteristic analysis of MRP8/14 and NE demonstrated areas under the curve <0.7 in both cohorts. CONCLUSION: In our cohorts, MRP8/14 and NE could not predict treatment response. Also, when patients had inactive disease, neither marker could predict flares.

Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population.

Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity.

Increased PD-L1 and T-cell infiltration in the presence of HLA class I expression in metastatic high-grade osteosarcoma: a rationale for T-cell-based immunotherapy.

INTRODUCTION: Immunotherapy may be an excellent choice for treating osteosarcoma given its exceptionally high genomic instability, potentially generating neoantigens. In this study, we aim to investigate the HLA class I expression, PD-L1 and tumour-infiltrating lymphocytes in primary osteosarcomas and relapses/metastases, as well as their changes during disease progression. MATERIALS AND METHODS: Tumour samples from multiple stages of the disease (pretreatment biopsies, surgical resections of primary osteosarcomas, relapses and metastases) were collected and stained for HLA-A (HCA2), HLA-B/C (HC10), ß2-microglobulin and PD-L1 using immunohistochemistry on whole sections. Density and type of T-cell infiltrate were characterised by a triple immunofluorescent staining CD3-CD8-FOXP3. RESULTS: Overall, 85 formalin-fixed, paraffin-embedded blocks from 25 osteosarcoma patients were included. HLA class I expression was detected in 94% of osteosarcomas (strongly positive in 56%, heterogeneous in 38%) and negative or weakly positive in 6%, without differences between the stages of the disease. HLA-A expression was more frequently negative than HLA-B/C. Tumour-infiltrating lymphocytes were highly heterogeneous and mainly observed in tumour areas with expression of HLA class I. Density of T cells was significantly higher in metastases than in primary tumours and local relapses (p = 0.0003). Positive PD-L1 expression was found in 13% of primary tumours, 25% of relapses and 48% of metastases and correlated with a high T-cell infiltrate (p = 0.002). CONCLUSION: An increased number of tumour-infiltrating T cells and PD-L1 expression in metastases compared with primary tumours, suggesting accessibility for T cells, could imply that osteosarcoma patients with metastatic disease may benefit from T-cell-based immunotherapy.

Glioblastoma-derived extracellular vesicles modify the phenotype of monocytic cells.

Glioblastoma multiforme (GBM) is the most common primary brain tumor and is without exception lethal. GBMs modify the immune system, which contributes to the aggressive nature of the disease. Particularly, cells of the monocytic lineage, including monocytes, macrophages and microglia, are affected. We investigated the influence of GBM-derived extracellular vesicles (EVs) on the phenotype of monocytic cells. Proteomic profiling showed GBM EVs to be enriched with proteins functioning in extracellular matrix interaction and leukocyte migration. GBM EVs appeared to skew the differentiation of peripheral blood-derived monocytes to alternatively activated/M2-type macrophages. This was observed for EVs from an established cell line, as well as for EVs from primary cultures of GBM stem-like cells (GSCs). Unlike EVs of non-GBM origin, GBM EVs induced modified expression of cell surface proteins, modified cytokine secretion (e.g., an increase in vascular endothelial growth factor and IL-6) and increased phagocytic capacity of the macrophages. Most pronounced effects were observed upon incubation with EVs from mesenchymal GSCs. GSC EVs also affected primary human microglia, resulting in increased expression of Membrane type 1-matrix metalloproteinase, a marker for GBM microglia and functioning as tumor-supportive factor. In conclusion, GBM-derived EVs can modify cells of the monocytic lineage, which acquire characteristics that resemble the tumor-supportive phenotypes observed in patients.

The effect of cidofovir on adenovirus plasma DNA levels in stem cell transplantation recipients without T cell reconstitution.

Cidofovir is frequently used to treat life-threatening human adenovirus (HAdV) infections in immunocompromised children after hematopoietic stem cell transplantation (HSCT). However, the antiviral effect irrespective of T cell reconstitution remains unresolved. Plasma HAdV DNA levels were monitored by real-time quantitative PCR during 42 cidofovir treatment episodes for HAdV viremia in 36 pediatric allogeneic HSCT recipients. HAdV load dynamics were related to T and natural killer (NK) cell reconstitution measured by flow cytometry. To evaluate the in vivo antiadenoviral effect of cidofovir, we focused on 20 cidofovir treatment episodes lacking concurrent T cell reconstitution. During 2 to 10 weeks of follow-up in the absence of T cells, HAdV load reduction (n = 7) or stabilization (n = 8) was observed in 15 of 20 treatments. Although HAdV load reduction was always accompanied by NK cell expansion, HAdV load stabilization was measured in 2 children lacking both T and NK cell reconstitution. In cases with T cell reconstitution, rapid HAdV load reduction (n = 14) or stabilization (n = 6) was observed in 20 of 22 treatments. In the absence of T cells, cidofovir treatment was associated with HAdV viremia control in the majority of cases. Although the contribution of NK cells cannot be excluded, cidofovir has the potential to mediate HAdV load stabilization in the time pending T cell reconstitution.

Role of NKG2D, DNAM-1 and natural cytotoxicity receptors in cytotoxicity toward rhabdomyosarcoma cell lines mediated by resting and IL-15-activated human natural killer cells.

Children with advanced stages (relapsed/refractory and stage IV) of rhabdomyosarcoma (RMS) have a poor prognosis despite intensive chemotherapy and autologous stem cell rescue, with 5-year survival rates ranging from 5 to 35 %. Development of new, additional treatment modalities is necessary to improve the survival rate. In this preclinical study, we investigated the potential of resting and cytokine-activated natural killer (NK) cells to lyse RMS cell lines, as well as the pathways involved, to explore the eventual clinical application of (activated) NK cell immunotherapy. RMS cell lines (n = 3 derived from embryonal RMS and n = 2 derived from alveolar RMS) were susceptible to cytolysis mediated by resting NK cells, and this susceptibility was significantly increased using IL-15-activated NK cells. Flow cytometry and cytolytic assays were used to define the activating and inhibitory pathways of NK cells involved in recognizing and lysing RMS cells. NKG2D and DNAM-1 receptor-ligand interactions were essential in cytolysis by resting NK cells, as simultaneous blocking of both pathways resulted in almost complete abrogation of the cytotoxicity. In contrast, combined blocking of DNAM-1 and NKG2D only led to partial reduction of the lytic activity of IL-15-activated NK cells. In this respect, residual lysis was, at least partly, mediated by pathways involving the natural cytotoxicity receptors NKp30 and NKp46. These findings support further exploration of NK cell-based immunotherapy as adjuvant modality in current treatment strategies of RMS.

Expression of the immune regulation antigen CD70 in osteosarcoma.

Osteosarcoma is the most frequent bone cancer in children and young adults. The outcome of patients with advanced disease is dismal. Exploitation of tumor-immune cell interactions may provide novel therapeutic approaches. CD70-CD27 interactions are important for the regulation of adaptive immunity. CD70 expression has been reported in some solid cancers and implicated in tumor escape from immunosurveillance. In this study, expression of CD70 and CD27 was analyzed in osteosarcoma cell lines and tumor specimens. CD70 protein was expressed on most osteosarcoma cell lines (5/7) and patient-derived primary osteosarcoma cultures (4/6) as measured by flow cytometry. In contrast, CD70 was detected on few Ewing sarcoma cell lines (5/15) and was virtually absent from neuroblastoma (1/7) and rhabdomyosarcoma cell lines (0/5). CD70(+) primary cultures were derived from CD70(+) osteosarcoma lesions. CD70 expression in osteosarcoma cryosections was heterogeneous, restricted to tumor cells and not attributed to infiltrating CD3(+) T cells as assessed by immunohistochemistry/immunofluorescence. CD70 was detected in primary (1/5) but also recurrent (2/4) and metastatic (1/3) tumors. CD27, the receptor for CD70, was neither detected on tumor cells nor on T cells in CD70(+) or CD70(-) tumors, suggesting that CD70 on tumor cells is not involved in CD27-dependent tumor-immune cell interactions in osteosarcoma. CD70 gene expression in diagnostic biopsies of osteosarcoma patients did not correlate with the occurrence of metastasis and survival (n = 70). Our data illustrate that CD70 is expressed in a subset of osteosarcoma patients. In patients with CD70(+) tumors, CD70 may represent a novel candidate for antibody-based targeted immunotherapy.

Communication between human mast cells and CD4(+) T cells through antigen-dependent interactions.

Mast cells (MCs) are immune cells residing in tissues where pathogens are first encountered. It has been indicated that MCs might also be involved in setting the outcome of T-cell responses. However, little is known about the capacity of human MCs to express MHC class II and/or to capture and present antigens to CD4(+) T cells. To study the T-cell stimulatory potential of human MCs, CD34(+) stem cell derived MCs were generated. These cells expressed HLA-DR when stimulated with IFN-γ, and, importantly, presented peptide and protein for activation of antigen-specific CD4(+) T cells. The interplay between MC and T cell led to increased HLA-DR expression on MCs. MCs were present in close proximity to T cells in tonsil and expressed HLA-DR and CD80, indicating their ability to present antigens to CD4(+) T cells in T-cell areas of human LNs. Our data show that MCs can present native antigens to human CD4(+) T cells and that HLA-DR expressing MCs are present in tonsil tissue, indicating that human MCs can directly activate T cells and provide a rationale to study the potential of MCs to prime and/or skew human T-cell responses.

Antibody-dependent cell lysis by NK cells is preserved after sarcoma-induced inhibition of NK cell cytotoxicity.

Osteosarcoma and Ewing's sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40 h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-ß. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.

DNAM-1 mediates epithelial cell-specific cytotoxicity of aberrant intraepithelial lymphocyte lines from refractory celiac disease type II patients.

In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3(-) intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3(-) IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8(+) T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.

Targeting FcαRI on polymorphonuclear cells induces tumor cell killing through autophagy.

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.

Predicting Patient Death after Allogeneic Stem Cell Transplantation for Inborn Errors Using Machine Learning (PREPAD): A European Society for Blood and Marrow Transplantation Inborn Errors Working Party Study.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many inborn errors of immunity, metabolism, and hematopoiesis. No predictive models are available for these disorders. We created a machine learning model using XGBoost to predict survival after HSCT using European Society for Blood and Marrow Transplant registry data of 10,888 patients who underwent HSCT for inborn errors between 2006 and 2018, and compared it to a simple linear Cox model, an elastic net Cox model, and a random forest model. The XGBoost model had a cross-validated area under the curve value of .73 at 1 year, which was significantly superior to the other models, and it accurately predicted for countries excluded while training. It predicted close to 0% and >30% mortality more often than other models at 1 year, while maintaining good calibration. The 5-year survival was 94.7% in the 25% of patients at lowest risk and 62.3% in the 25% at highest risk. Within disease and donor subgroups, XGBoost outperformed the best univariate predictor. We visualized the effect of the main predictors-diagnosis, performance score, patient age and donor type-using the SHAP ML explainer and developed a stand-alone application, which can predict using the model and visualize predictions. The risk of mortality after HSCT for inborn errors can be accurately predicted using an explainable machine learning model. This exceeds the performance of models described in the literature. Doing so can help detect deviations from expected survival and improve risk stratification in trials.