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PD-1 blockade therapy has revolutionized melanoma treatment, but still not all patients benefit and pre-treatment identification of those patients is difficult. Increased expression of inflammatory markers such as interleukin (IL)-6 in blood of patients correlates with poor treatment response. We set out to study the effect of inflammatory cytokines on PD-1 blockade in vitro. For this, we studied the effect of IL-6 and type I interferon (IFN) in vitro on human T cells in a mixed leukocyte reaction (MLR) in the absence or presence of PD-1 blockade. While IL-6 reduced IFN-γ secretion by T cells in both the presence and absence of PD-1 blockade, IFN-α specifically reduced the IFN-γ secretion only in the presence of PD-1 blockade. IFN-α reduced T cell proliferation independent of PD-1 blockade and reduced the percentage of cells producing IFN-γ only in the presence of PD-1 blockade. Next we determined the type I IFN score in a cohort of 22 melanoma patients treated with nivolumab. In this cohort, we did not find a correlation between clinical response and type I IFN score, nor between clinical response and IFN-γ secretion in vitro in a MLR in the presence of PD-1 blockade. We conclude that IFN-α reduces the effectiveness of PD-1 blockade in vitro, but that in this cohort, type I IFN score in vivo, nor IFN-γ secretion in vitro in a MLR in the presence of PD-1 blockade correlated to decreased therapy responses in patients. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors (ICI) have revolutionized the treatment landscape of advanced malignancies, but come with a diverse spectrum of immune-related adverse events (irAEs). Mechanistic studies can aid the transition from expert-opinion to evidence-based irAE treatment strategies. We aimed to longitudinally characterize peripheral blood T and B cell dynamics in ICI-treated patients by multicolor flow cytometry and serum multiplex immunoassay at baseline, ± 3 weeks and ± 6 weeks or upon clinically relevant irAEs. We analyzed samples from 44 ICI-treated patients (24 anti-PD-1 monotherapy, 20 combined anti-PD-1/anti-CTLA-4; cICI), of whom 21 developed irAEs, and 10 healthy donors. IrAEs after cICI were characterized by significantly enhanced proliferation of Th1-associated, mainly (CD4+) CD27− effector memory T cells, as well as Th17-associated immune responses and germinal center activation (reflected by CXCL13 and IL-21 increases). We observed no changes in CD21lo, memory, class-switched or newly activated B cell subsets. Particularly double-positive PD-1+LAG-3+ CD8+ T cells showed enhanced cytotoxic capacity in patients with irAEs after cICI. Within anti-PD-1 monotherapy, irAEs were associated with modestly enhanced Th1-associated responses reflected by increased serum CXCL9 and CXCL10. In conclusion, ICI-induced toxicity is dominated by enhanced Th1-associated responses, but in cICI we also found evidence for Th17-associated responses and germinal center activation. Together, our data add to the growing body of evidence that irAEs may be driven by newly activated CD4+ helper T cells, specifically after cICI. This study also supports tailored irAE treatment, based on ICI regimen, and to deploy specific strategies such as Th17 inhibition especially in cICI-associated irAEs. [ABSTRACT FROM AUTHOR]

γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background1–5. γδ T cells can sense conserved cell stress signals prevalent in transformed cells2,3, although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells—the most abundant subset of human γδ T cells4—recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6–8), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1–BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.A combination of genome-wide CRISPR screens in target cancer cells identifies pathways that regulate γδ T cell killing and BTN3A cell surface expression. [ABSTRACT FROM AUTHOR]

We designed a study to describe the incidence of intracranial hemorrhage according to severity and duration of thrombocytopenia and to quantify the associations of platelet transfusions with intracranial hemorrhage in patients with acute leukemia. In this case-control study nested in a cohort of 859 leukemia patients, cases (n = 17) were patients diagnosed with intracranial hemorrhage who were matched with control patients (n = 55). We documented platelet counts and transfusions for seven days before the intracranial hemorrhage in cases and in a "matched" week for control patients. Three measures of platelet count exposure were assessed in four potentially important time periods before hemorrhage. Among these leukemia patients, we observed the cumulative incidence of intracranial hemorrhage of 3.5%. Low platelet counts were, especially in the three to seven days preceding intracranial hemorrhage, associated with the incidence of intracranial hemorrhage, although with wide confidence intervals. Platelet transfusions during the week preceding the hemorrhage were associated with higher incidences of intracranial hemorrhage; rate ratios (95% confidence interval) for one or two platelet transfusions and for more than two transfusions compared with none were 4.04 (0.73 to 22.27) and 8.91 (1.53 to 51.73) respectively. Thus, among acute leukemia patients, the risk of intracranial hemorrhage was higher among patients with low platelet counts and after receiving more platelet transfusions. Especially, the latter is likely due to clinical factors leading to increased transfusion needs. [ABSTRACT FROM AUTHOR]

Background: Fludarabine is often used as an important drug in reduced toxicity conditioning regimens prior to hematopoietic cell transplantation (HCT). As no definitive pharmacokinetic (PK) basis for HCT dosing for the wide age and weight range in HCT is available, linear body surface area (BSA)-based dosing is still used.Objective: We sought to describe the population PK of fludarabine in HCT recipients of all ages.Methods: From 258 HCT recipients aged 0.3-74 years, 2605 samples were acquired on days 1 (42%), 2 (17%), 3 (4%) and 4 (37%) of conditioning. Herein, the circulating metabolite of fludarabine was quantified, and derived concentration-time data were used to build a population PK model using non-linear mixed-effects modelling.Results: Variability was extensive where area under the curve ranged from 10 to 66 mg h/L. A three-compartment model with first-order kinetics best described the data. Actual body weight (BW) with standard allometric scaling was found to be the best body-size descriptor for all PK parameters. Estimated glomerular filtration rate (eGFR) was included as a descriptor of renal function. Thus, clearance was differentiated into a non-renal (3.24 ± 20% L/h/70 kg) and renal (eGFR × 0.782 ± 11% L/h/70 kg) component. The typical volumes of distribution of the central (V1), peripheral (V2), and second peripheral (V3) compartments were 39 ± 8%, 20 ± 11%, and 50 ± 9% L/70 kg respectively. Intercompartmental clearances between V1 and V2, and V1 and V3, were 8.6 ± 8% and 3.8 ± 13% L/h/70 kg, respectively.Conclusion: BW and eGFR are important predictors of fludarabine PK. Therefore, current linear BSA-based dosing leads to highly variable exposure, which may lead to variable treatment outcome. [ABSTRACT FROM AUTHOR]

Over half a century ago, the first allogeneic stem cell transplantation (allo-SCT) initiated cellular immunotherapy. For several decades, little progress was made, and toxicity of allo-SCT remained a major challenge. However, recent breakthroughs have opened new avenues to further develop this modality and to provide less toxic and equally efficient interventions for patients suffering from hematological or solid malignancies. Current novel cellular immune interventions include ex vivo expansion and adoptive transfer of tumor-infiltrating immune cells or administration of drugs which antagonize tolerizing mechanisms. Alternatively, transfer of immune cells engineered to express defined T cell receptors (TCRs) and chimeric antigen receptors (CARs) has shown its potential. A valuable addition to 'engineered' adaptive immunity has emerged recently through the improved understanding of how innate immune cells can attack cancer cells without substantial side effects. This has enabled the development of transplantation platforms with limited side effects allowing early immune interventions as well as the design of engineered immune cells expressing innate immune receptors. Here, we focus on innate immune interventions and their orchestration with TCR- and CAR-engineered immune cells. In addition, we discuss how the exploitation of the full potential of cellular immune interventions is influenced by regulatory frameworks. Finally, we highlight and discuss substantial differences in the current landscape of clinical trials in Europe as compared to the USA. The aim is to stimulate international efforts to support regulatory authorities and funding agencies, especially in Europe, to create an environment that will endorse the development of engineered immune cells for the benefit of patients. [ABSTRACT FROM AUTHOR]

T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8 T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8 T cells was observed. An increase in PADRE-specific CD4 T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4 T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration. [ABSTRACT FROM AUTHOR]

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8×A2.1/Kb mouse-derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81–88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81–88), we found that the heterodimeric CD8αβ coreceptor, but not normally expressed homodimeric CD8αα, is required for tetramer binding and functional redirection of TCR-transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 antibody and required more peptide than wild-type (WT) MDM2 TCR+ T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) αβ domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expresion, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease. [ABSTRACT FROM AUTHOR]

We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1?restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1?restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon expression of wild-type and partially humanized high-affinity T cell antigen receptor (TCR) genes derived from the transgenic mice. These results demonstrate that TCR gene transfer can be used to circumvent self-tolerance of autologous T lymphocytes to universal tumor antigens and thus provide the basis for a TCR gene transfer?based broad-spectrum immunotherapy of malignant disease. [ABSTRACT FROM AUTHOR]

Autologous haematopoietic stem cell transplantation (HSCT) is the only treatment that is able to induce long-term, drug-free and symptom-free remission in several refractory autoimmune rheumatic diseases. Over 3,000 HSCT procedures for rheumatic and nonrheumatic severe autoimmune diseases have been performed worldwide. Specific conditioning regimens are currently used to eradicate the autoreactive immunological memory of patients. Although in vivo immune cell depletion with antithymocyte globulin or anti-CD52 is the norm for many regimens, ex vivo selection of CD34+ stem cells from the graft is controversial. Following the extensive immune depletion associated with serotherapy and chemotherapy, HSCT effectively resets the immune system by renewing the CD4+ T cell compartment, especially the regulatory T cell population. The risk of transplant-related mortality (TRM) within the first 100 days should be weighed against the risk of disease-related mortality, and the careful selection and screening of patients before transplantation is essential. Systemic sclerosis is the first autoimmune disease for which HSCT has been shown, in a randomized, controlled trial, to be associated with increased TRM in the first year but a significant long-term, event-free survival benefit afterwards. In this Review, we discuss the immunological mechanisms of HSCT in various autoimmune diseases and current HSCT regimens. After carefully taking into consideration the risks and benefits of HSCT and alternative therapies, we also discuss the efficacy, complications and proposed indications of this procedure. [ABSTRACT FROM AUTHOR]