Block of T cell development in P53-deficient mice accelerates development of lymphomas with characteristic RAG-dependent cytogenetic alterations

SummaryMice deficient in the DNA damage sensor P53 display normal T cell development but eventually succumb to thymic lymphomas. Here, we show that inactivation of the TCR β gene enhancer (Eβ) results in a block of T cell development at stages where recombination-activating genes (RAG) are expressed. Introduction of the Eβ mutation into p53−/− mice dramatically accelerates the onset of lethal thymic lymphomas that harbor RAG-dependent aberrant rearrangements, chromosome 14 and 12 translocations, and amplification of the chromosomal region 9A1–A5.3. Phenotypic and genetic analyses suggest that lymphomas emerge through a normal thymocyte development pathway. These findings provide genetic evidence that block of lymphocyte development at stages with RAG endonuclease activity can provoke lymphomagenesis on a background with deficient DNA damage responses

First-in-human study of TK-positive oncolytic vaccinia virus delivered by adipose stromal vascular fraction cells

BackgroundACAM2000, a thymidine kinase (TK)-positive strain of vaccinia virus, is the current smallpox vaccine in the US. Preclinical testing demonstrated potent oncolytic activity of ACAM2000 against several tumor types. This Phase I clinical trial of ACAM2000 delivered by autologous adipose stromal vascular fraction (SVF) cells was conducted to determine the safety and feasibility of such a treatment in patients with advanced solid tumors or acute myeloid leukemia (AML).MethodsTwenty-four patients with solid tumors and two patients with AML participated in this open-label, non-randomized dose-escalation trial. All patients were treated with SVF derived from autologous fat and incubated for 15 min to 1 h with ACAM2000 before application. Six patients received systemic intravenous application only, one patient received intra-tumoral application only, 15 patients received combination intravenous with intra-tumoral deployment, 3 patients received intravenous and intra-peritoneal injection and 1 patient received intravenous, intra-tumoral and intra-peritoneal injections. Safety at each dose level of ACAM2000 (1.4 × 106 plaque-forming units (PFU) to 1.8 × 107 PFU) was evaluated. Blood samples for PK assessments, flow cytometry and cytokine analysis were collected at baseline and 1 min, 1 h, 1 day, 1 week, 1 month, 3 months and 6 months following treatment.ResultsNo serious toxicities (> grade 2) were reported. Seven patients reported an adverse event (AE) in this study: self-limiting skin rashes, lasting 7 to 18 days-an expected adverse reaction to ACAM2000. No AEs leading to study discontinuation were reported. Viral DNA was detected in all patients' blood samples immediately following treatment. Interestingly, in 8 patients viral DNA disappeared 1 day and re-appeared 1 week post treatment, suggesting active viral replication at tumor sites, and correlating with longer survival of these patients. No major increase in cytokine levels or correlation between cytokine levels and skin rashes was noted. We were able to assess some initial efficacy signals, especially when the ACAM2000/SVF treatment was combined with checkpoint inhibition.ConclusionsTreatment with ACAM2000/SVF in patients with advanced solid tumors or AML is safe and well tolerated, and several patients had signals of an anticancer effect. These promising initial clinical results merit further investigation of therapeutic utility. Trial registration Retrospectively registered (ISRCTN#10201650) on October 22, 2018

IAP inhibitors enhance co-stimulation to promote tumor immunity

The inhibitor of apoptosis proteins (IAPs) have recently been shown to modulate nuclear factor κB (NF-κB) signaling downstream of tumor necrosis factor (TNF) family receptors, positioning them as essential survival factors in several cancer cell lines, as indicated by the cytotoxic activity of several novel small molecule IAP antagonists. In addition to roles in cancer, increasing evidence suggests that IAPs have an important function in immunity; however, the impact of IAP antagonists on antitumor immune responses is unknown. In this study, we examine the consequences of IAP antagonism on T cell function in vitro and in the context of a tumor vaccine in vivo. We find that IAP antagonists can augment human and mouse T cell responses to physiologically relevant stimuli. The activity of IAP antagonists depends on the activation of NF-κB2 signaling, a mechanism paralleling that responsible for the cytotoxic activity in cancer cells. We further show that IAP antagonists can augment both prophylactic and therapeutic antitumor vaccines in vivo. These findings indicate an important role for the IAPs in regulating T cell–dependent responses and suggest that targeting IAPs using small molecule antagonists may be a strategy for developing novel immunomodulating therapies against cancer

Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host\u27s response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14-15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual\u27s recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4-5, 2019

Correction to: Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

Following publication of the original article [1], the author reported that an author name, Roberta Zappasodi, was missed in the authorship list

MFG-E8 Blockade Enhances Tumor Immunity in a Murine Breast Cancer Model

Milk fat globule - epidermal growth factor - factor 8 protein (MFG-E8) is an important mediator of the tolerogenic functions of GM-CSF, and a dominant-negative RGE mutant augments the therapeutic potential of irradiated, GM-CSF-secreting tumor vaccines (GVAX) in the MFG-E8-negative B16 melanoma model. The frequent expression of MFG-E8 in various solid and hematological malignancies, however, prompted us to investigate the effect of the RGE mutant in a MFG-E8-positive transplantable breast tumor model. Here, we report that MFG-E8 blockade augmented anti-tumor humoral responses and modulated immune infiltrates at vaccination sites, which was associated with defective phagocytosis and clearance of apoptotic tumor cells. The RGE mutant enhanced the therapeutic potential of two irradiated, GM-CSF-secreting vaccines and improved protection correlated with augmented tumor-specific IgG1 and IgG2a antibody responses as well as increased ratios of T effectors to Tregs in TILs. These findings are consistent with the notion that MFG-E8 blockade potentiates anti-tumor responses through the preferential expansion of effector over regulatory T cells. Our data also validate the use of the RGE mutant to achieve therapeutically effective MFG-E8 blockade even in the context of tumors and vaccines that express high levels of endogenous MFG-E8

The ratio of ADSCs to HSC-progenitors in adipose tissue derived SVF may provide the key to predict the outcome of stem-cell therapy

Abstract Background Stromal vascular fraction (SVF) represents an attractive source of adult stem cells and progenitors, holding great promise for numerous cell therapy approaches. In 2017, it was reported that 1524 patients received autologous SVF following the enzymatic digestion of liposuction fat. The treatment was safe and effective and patients showed significant clinical improvement. In a collaborative study, we analyzed SVF obtained from 58 patients having degenerative, inflammatory, autoimmune diseases, and advanced stage cancer. Results Flow analysis showed that freshly isolated SVF was very heterogeneous and harbored four major subsets specific to adipose tissue; CD34high CD45− CD31− CD146− adipose-derived stromal/stem cells (ADSCs), CD34low CD45+ CD206+CD31− CD146− hematopoietic stem cell-progenitors (HSC-progenitors), CD34high CD45− CD31+CD146+ adipose tissue-endothelial cells and CD45−CD34−CD31−CD146+ pericytes. Culturing and expanding of SVF revealed a homogenous population lacking hematopoietic lineage markers CD45 and CD34, but were positive for CD90, CD73, CD105, and CD44. Flow cytometry sorting of viable individual subpopulations revealed that ADSCs had the capacity to grow in adherent culture. The identity of the expanded cells as mesenchymal stem cells (MSCs) was further confirmed based on their differentiation into adipogenic and osteogenic lineages. To identify the potential factors, which may determine the beneficial outcome of treatment, we followed 44 patients post-SVF treatment. The gender, age, clinical condition, certain SVF-dose and route of injection, did not play a role on the clinical outcome. Interestingly, SVF yield seemed to be affected by patient’s characteristic to various extents. Furthermore, the therapy with adipose-derived and expanded-mesenchymal stem cells (ADE-MSCs) on a limited number of patients, did not suggest increased efficacies compared to SVF treatment. Therefore, we tested the hypothesis that a certain combination, rather than individual subset of cells may play a role in determining the treatment efficacy and found that the combination of ADSCs to HSC-progenitor cells can be correlated with overall treatment efficacy. Conclusions We found that a 2:1 ratio of ADSCs to HSC-progenitors seems to be the key for a successful cell therapy. These findings open the way to future rational design of new treatment regimens for individuals by adjusting the cell ratio before the treatment

The T-Cell Receptor β Variable Gene Promoter Is Required for Efficient Vβ Rearrangement but Not Allelic Exclusion

To investigate the role of promoters in regulating variable gene rearrangement and allelic exclusion, we constructed mutant mice in which a 1.2-kb region of the Vβ13 promoter was either deleted (P13(−/−)) or replaced with the simian virus 40 minimal promoter plus five copies of Gal4 DNA sequences (P13(R/R)). In P13(−/−) mice, cleavage, rearrangement, and transcription of Vβ13, but not the flanking Vβ gene segments, were significantly inhibited. In P13(R/R) mice, inhibition of Vβ13 rearrangement was less severe and was not associated with any apparent reduction in Vβ13 cleavage. Expression of a T-cell receptor (TCR) transgene blocked cleavages at the normal Vβ13-recombination signal sequence junction and Vβ13 coding joint formation of both wild-type and mutant Vβ13 alleles. However, a low level of aberrant Vβ13 cleavage was consistently detected, especially in TCR transgenic P13(R/R) mice. These findings suggest that the variable gene promoter is required for promoting local recombination accessibility of the associated Vβ gene segment. Although the promoter is dispensable for allelic exclusion, it appears to suppress aberrant Vβ cleavages during allelic exclusion