IL-23 stabilizes an effector Treg cell program in the tumor microenvironment.

Interleukin-23 (IL-23) is a proinflammatory cytokine mainly produced by myeloid cells that promotes tumor growth in various preclinical cancer models and correlates with adverse outcomes. However, as to how IL-23 fuels tumor growth is unclear. Here, we found tumor-associated macrophages to be the main source of IL-23 in mouse and human tumor microenvironments. Among IL-23-sensing cells, we identified a subset of tumor-infiltrating regulatory T (Treg) cells that display a highly suppressive phenotype across mouse and human tumors. The use of three preclinical models of solid cancer in combination with genetic ablation of Il23r in Treg cells revealed that they are responsible for the tumor-promoting effect of IL-23. Mechanistically, we found that IL-23 sensing represents a crucial signal driving the maintenance and stabilization of effector Treg cells involving the transcription factor Foxp3. Our data support that targeting the IL-23/IL-23R axis in cancer may represent a means of eliciting antitumor immunity.

High-dimensional analysis of 16 SARS-CoV-2 vaccine combinations reveals lymphocyte signatures correlating with immunogenicity.

The range of vaccines developed against severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) provides a unique opportunity to study immunization across different platforms. In a single-center cohort, we analyzed the humoral and cellular immune compartments following five coronavirus disease 2019 (COVID-19) vaccines spanning three technologies (adenoviral, mRNA and inactivated virus) administered in 16 combinations. For adenoviral and inactivated-virus vaccines, heterologous combinations were generally more immunogenic compared to homologous regimens. The mRNA vaccine as the second dose resulted in the strongest antibody response and induced the highest frequency of spike-binding memory B cells irrespective of the priming vaccine. Priming with the inactivated-virus vaccine increased the SARS-CoV-2-specific T cell response, whereas boosting did not. Distinct immune signatures were elicited by the different vaccine combinations, demonstrating that the immune response is shaped by the type of vaccines applied and the order in which they are delivered. These data provide a framework for improving future vaccine strategies against pathogens and cancer.

Targeting MDM2 enhances antileukemia immunity after allogeneic transplantation via MHC-II and TRAIL-R1/2 upregulation.

Patients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell-mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2 inhibition.

Abatacept as salvage therapy in chronic graft-versus-host disease-a retrospective analysis.

The immunomodulatory fusion protein abatacept has recently been investigated for the treatment of steroid-refractory chronic graft-versus-host disease (cGvHD) in a phase 1 clinical trial. We analyzed the safety and efficacy of abatacept for cGvHD therapy in a retrospective study with 15 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) and received abatacept for cGvHD with a median age of 49 years. Grading was performed as part of the clinical routine according to the National Institute of Health's (NIH) consensus criteria at initiation of abatacept and 1, 3, 6, 9 and 12 months thereafter. The median time of follow-up was 191 days (range 55-393 days). Best overall response rate (ORR) was 40%. In particular, patients with bronchiolitis obliterans syndrome showed significant clinical improvement and durable responses following abatacept treatment with a response rate of 89% based on improvement in lung severity score (n = 6) or stabilized lung function (n = 4) or both (n = 3). Infectious complications CTCAE °III or higher were observed in 3/15 patients. None of the patients relapsed from the underlying malignancy. Thus, abatacept appears to be a promising treatment option for cGvHD, in particular for patients with lung involvement. However, further evaluation within a phase 2 clinical trial is required.

Cyclophosphamide for salvage therapy of chronic graft-versus-host disease: a retrospective analysis.

We retrospectively analyzed the safety and efficacy of cyclophosphamide (cyclo) for salvage treatment of chronic graft-versus-host disease (cGvHD) and cGvHD-associated (glomerulo-)nephritis at our center between 01/2010 and 11/2019. We identified 13 patients (pts) receiving cyclo for treatment of moderate (3/13) and severe (6/13) steroid-refractory cGvHD, cGvHD-associated (glomerulo-)nephritis (3/13), or vasculitis-like CNS manifestation of cGvHD (1/13). Cyclo was started on median day 509 (range 42-8193) after cGvHD onset; the median duration of application was 153 days (range 14-486) with 2/13 currently continuing treatment. The National Institute of Health organ grading and the intensity of immunosuppression (IS) were assessed at cyclo start and repeated after 3, 6, and 12 months. Response assessment was stopped at the start of any additional new IS. The median time of follow up was 407 days (range 86-1534). Best response was 1/13 CR, 6/13 PR, 4/13 SD, 1/13 MR, and 1/13 PD (ORR 54%). Significant and durable response was observed especially in cGvHD-associated (glomerulo-)nephritis (3/3). Infectious complications > CTCAE grade III were observed in 3/12 pts. During cyclo therapy, none of the pts suffered from recurrence of underlying malignancy. Overall, cyclo was relatively well tolerated and showed responses in heavily pretreated patients but requires further evaluation within clinical trials.

The IL-33/ST2 axis augments effector T-cell responses during acute GVHD.

Interleukin (IL)-33 binding to the receptor suppression of tumorigenicity 2 (ST2) produces pro-inflammatory and anti-inflammatory effects. Increased levels of soluble ST2 (sST2) are a biomarker for steroid-refractory graft-versus-host disease (GVHD) and mortality. However, whether sST2 has a role as an immune modulator or only as a biomarker during GVHD was unclear. We show increased IL-33 production by nonhematopoietic cells in the gastrointestinal (GI) tract in mice post-conditioning and patients during GVHD. Exogenous IL-33 administration during the peak inflammatory response worsened GVHD. Conversely, GVHD lethality and tumor necrosis factor-α production was significantly reduced in il33(-/-) recipients. ST2 was upregulated on murine and human alloreactive T cells and sST2 increased as experimental GVHD progressed. Concordantly, st2(-/-) vs wild-type (WT) donor T cells had a marked reduction in GVHD lethality and GI histopathology. Alloantigen-induced IL-18 receptor upregulation was lower in st2(-/-) T cells, and linked to reduced interferon-γ production by st2(-/-) vs WT T cells during GVHD. Blockade of IL-33/ST2 interactions during allogeneic-hematopoietic cell transplantation by exogenous ST2-Fc infusions had a marked reduction in GVHD lethality, indicating a role of ST2 as a decoy receptor modulating GVHD. Together, these studies point to the IL-33/ST2 axis as a novel and potent target for GVHD therapy.

Oncogene-induced TIM-3 ligand expression dictates susceptibility to anti-TIM-3 therapy in mice.

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting T cell (Tc) immunoglobulin and mucin-containing molecule 3 (TIM-3) for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ Tc enhanced Tc activation, proliferation, and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion, and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3 treatment-mediated GVL effects are Tc induced. In contrast to anti-programmed cell death protein 1 (anti-PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) treatment, anti-TIM-3-treatment did not enhance acute graft-versus-host disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post-allo-HCT relapse. We decipher the connections between oncogenic mutations found in AML and TIM-3 ligand expression and identify anti-TIM-3 treatment as a strategy for enhancing GVL effects via metabolic and transcriptional Tc reprogramming without exacerbation of aGVHD. Our findings support clinical testing of anti-TIM-3 Ab in patients with AML relapse after allo-HCT.

Antibodies Produced by CLL Phenotype B Cells in Patients With Myasthenia Gravis Are Not Directed Against Neuromuscular Endplates.

BACKGROUND AND OBJECTIVES: Myasthenia gravis (MG) can in rare cases be an autoimmune phenomenon associated with hematologic malignancies such as chronic lymphocytic leukemia (CLL). It is unclear whether in patients with MG and CLL, the leukemic B cells are the ones directly driving the autoimmune response against neuromuscular endplates. METHODS: We identified patients with acetylcholine receptor antibody-positive (AChR+) MG and CLL or monoclonal B-cell lymphocytosis (MBL), a precursor to CLL, and described their clinical features, including treatment responses. We generated recombinant monoclonal antibodies (mAbs) corresponding to the B-cell receptors of the CLL phenotype B cells and screened them for autoantigen binding. RESULTS: A computational immune cell screen revealed a subgroup of 5/38 patients with MG and 0/21 healthy controls who displayed a CLL-like B-cell phenotype. In follow-up hematologic flow cytometry, 2 of these 5 patients were diagnosed with an MBL. An additional patient with AChR+ MG as a complication of manifest CLL presented at our neuromuscular clinic and was successfully treated with the anti-CD20 therapy obinutuzumab plus chlorambucil. We investigated the specificities of expanding CLL-like B-cell clones to assess a direct causal link between the 2 diseases. However, we observed no reactivity of the clones against the AChR, antigens at the neuromuscular junction, or other common autoantigens. DISCUSSION: Our study suggests that AChR autoantibodies are produced by nonmalignant, polyclonal B cells The new anti-CD20 treatment obinutuzumab might be considered in effectively treating AChR+ MG. CLASSIFICATION OF EVIDENCE: This is a single case study and provides Class IV evidence that obinutuzumab is safe to use in patients with MG.

MDM2 Inhibition Enhances Immune Checkpoint Inhibitor Efficacy by Increasing IL15 and MHC Class II Production.

The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction-mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor-treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma. IMPLICATIONS: MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II-production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti-PD-1 immunotherapy for metastatic melanoma is planned.

Stereotactic Microwave Ablation of Hepatocellular Carcinoma: The Impact of Tumor Size and Minimal Ablative Margin on Therapeutic Success.

BACKGROUND: Microwave ablation (MWA) has gained relevance in the treatment of hepatic malignancies and especially in hepatocellular carcinoma (HCC), and it is an important alternative to surgery. The purpose of the study was to evaluate whether the minimal ablative margin (MAM) or the initial tumor size has a greater effect on the success of stereotactic MWA of HCC regarding the time to local tumor progression (LTP) and overall survival (OS). METHODS: 88 patients, who received stereotactic MWA of 127 tumor lesions with a curative intention were included in this single-center, retrospective study. The MAM was evaluated in a side-by-side comparison of pre- and post-ablative, contrast-enhanced slice imaging. A Cox proportional hazard model with a frailty term was computed to assess the influence of the MAM and the maximum tumor diameter on the time to LTP and the OS. RESULTS: The maximum tumor diameter was identified as a significant positive predictor for LTP (hazard ratio 1.04, 95% CI 1.00-1.08, p = 0.03), but it was not a significant positive predictor for the OS (p = 0.20). The MAM did not have a significant influence on LTP-free survival (p = 0.23) and OS (p = 0.67). CONCLUSION: For the successful stereotactic MWA of HCC, the MAM and maximum tumor diameter might not have an influence on the OS, but the maximum tumor diameter seems to be an independent predictor of the time to LTP.

Parametric Imaging of Contrast-Enhanced Ultrasound (CEUS) for the Evaluation of Acute Gastrointestinal Graft-Versus-Host Disease.

In recent years contrast-enhanced ultrasound (CEUS) has been an emerging diagnostic modality for the detection of acute gastrointestinal (GI) graft-versus-host disease (GvHD) in patients after allogeneic stem cell transplantation. However, broad clinical usage has been partially limited by its high dependence on the expertise of an experienced examiner. Thus, the aim of this study was to facilitate detection of acute GI GvHD by implementing false color-coded parametric imaging of CEUS. As such, two inexperienced examiners with basic knowledge in abdominal and vascular ultrasound analyzed parametric images obtained from patients with clinical suspicion for acute GvHD in a blinded fashion. As diagnostic gold standard, histopathological GvHD severity score on intestinal biopsies obtained from lower GI tract endoscopy was performed. The evaluation of parametric images by the two inexperienced ultrasound examiners in patients with histological confirmation of acute GI GvHD was successful in 17 out of 19 patients (89%) as opposed to analysis of combined B-mode ultrasound, strain elastography, and CEUS by an experienced examiner, which was successful in 18 out of 19 of the patients (95%). Therefore, CEUS with parametric imaging of the intestine was technically feasible and has the potential to become a valuable diagnostic tool for rapid and widely accessible detection of acute GvHD in clinical practice.

Corrigendum: Suppression of luteinizing hormone enhances HSC recovery after hematopoietic injury.

This corrects the article DOI: 10.1038/nm.4470.

Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration.

The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4) ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as Dll4, a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.

Suppression of luteinizing hormone enhances HSC recovery after hematopoietic injury.

There is a substantial unmet clinical need for new strategies to protect the hematopoietic stem cell (HSC) pool and regenerate hematopoiesis after radiation injury from either cancer therapy or accidental exposure. Increasing evidence suggests that sex hormones, beyond their role in promoting sexual dimorphism, regulate HSC self-renewal, differentiation, and proliferation. We and others have previously reported that sex-steroid ablation promotes bone marrow (BM) lymphopoiesis and HSC recovery in aged and immunodepleted mice. Here we found that a luteinizing hormone (LH)-releasing hormone antagonist (LHRH-Ant), currently in wide clinical use for sex-steroid inhibition, promoted hematopoietic recovery and mouse survival when administered 24 h after an otherwise-lethal dose of total-body irradiation (L-TBI). Unexpectedly, this protective effect was independent of sex steroids and instead relied on suppression of LH levels. Human and mouse long-term self-renewing HSCs (LT-HSCs) expressed high levels of the LH/choriogonadotropin receptor (LHCGR) and expanded ex vivo when stimulated with LH. In contrast, the suppression of LH after L-TBI inhibited entry of HSCs into the cell cycle, thus promoting HSC quiescence and protecting the cells from exhaustion. These findings reveal a role of LH in regulating HSC function and offer a new therapeutic approach for hematopoietic regeneration after hematopoietic injury.

Sex steroid blockade enhances thymopoiesis by modulating Notch signaling.

Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.