Galectin-9, a pro-survival factor inducing immunosuppression, leukemic cell transformation and expansion.

Leukemia is a malignancy of the bone marrow and blood originating from self-renewing cancerous immature blast cells or transformed leukocytes. Despite improvements in treatments, leukemia remains still a serious disease with poor prognosis because of disease heterogeneity, drug resistance and relapse. There is emerging evidence that differentially expression of co-signaling molecules play a critical role in tumor immune evasion. Galectin-9 (Gal-9) is one of the key proteins that leukemic cells express, secrete, and use to proliferate, self-renew, and survive. It also suppresses host immune responses controlled by T and NK cells, enabling leukemic cells to evade immune surveillance. The present review provides the molecular mechanisms of Gal-9-induced immune evasion in leukemia. Understanding the complex immune evasion machinery driven by Gal-9 expressing leukemic cells will enable the identification of novel therapeutic strategies for efficient immunotherapy in leukemic patients. Combined treatment approaches targeting T-cell immunoglobulin and mucin domain-3 (Tim-3)/Gal-9 and other immune checkpoint pathways can be considered, which may enhance the efficacy of host effector cells to attack leukemic cells.

TET2 guards against unchecked BATF3-induced CAR T cell expansion.

Further advances in cell engineering are needed to increase the efficacy of chimeric antigen receptor (CAR) and other T cell-based therapies1-5. As T cell differentiation and functional states are associated with distinct epigenetic profiles6,7, we hypothesized that epigenetic programming may provide a means to improve CAR T cell performance. Targeting the gene that encodes the epigenetic regulator ten-eleven translocation 2 (TET2)8 presents an interesting opportunity as its loss may enhance T cell memory9,10, albeit not cause malignancy9,11,12. Here we show that disruption of TET2 enhances T cell-mediated tumour rejection in leukaemia and prostate cancer models. However, loss of TET2 also enables antigen-independent CAR T cell clonal expansions that may eventually result in prominent systemic tissue infiltration. These clonal proliferations require biallelic TET2 disruption and sustained expression of the AP-1 factor BATF3 to drive a MYC-dependent proliferative program. This proliferative state is associated with reduced effector function that differs from both canonical T cell memory13,14 and exhaustion15,16 states, and is prone to the acquisition of secondary somatic mutations, establishing TET2 as a guardian against BATF3-induced CAR T cell proliferation and ensuing genomic instability. Our findings illustrate the potential of epigenetic programming to enhance T cell immunity but highlight the risk of unleashing unchecked proliferative responses.

RASA2 ablation in T cells boosts antigen sensitivity and long-term function.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells.

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.

The metabolism of cells regulates their sensitivity to NK cells depending on p53 status.

Leukemic cells proliferate faster than non-transformed counterparts. This requires them to change their metabolism to adapt to their high growth. This change can stress cells and facilitate recognition by immune cells such as cytotoxic lymphocytes, which express the activating receptor Natural Killer G2-D (NKG2D). The tumor suppressor gene p53 regulates cell metabolism, but its role in the expression of metabolism-induced ligands, and subsequent recognition by cytotoxic lymphocytes, is unknown. We show here that dichloroacetate (DCA), which induces oxidative phosphorylation (OXPHOS) in tumor cells, induces the expression of such ligands, e.g. MICA/B, ULBP1 and ICAM-I, by a wtp53-dependent mechanism. Mutant or null p53 have the opposite effect. Conversely, DCA sensitizes only wtp53-expressing cells to cytotoxic lymphocytes, i.e. cytotoxic T lymphocytes and NK cells. In xenograft in vivo models, DCA slows down the growth of tumors with low proliferation. Treatment with DCA, monoclonal antibodies and NK cells also decreased tumors with high proliferation. Treatment of patients with DCA, or a biosimilar drug, could be a clinical option to increase the effectiveness of CAR T cell or allogeneic NK cell therapies.

Lymphoma versus Carcinoma and Other Collaborations.

David Mason started his research career at a time when lymphoma diagnosis was based primarily on cellular morphology, clinical symptoms and special cytochemical stains using formalin fixed tissue sections. There were occasions, however, where the morphology was unhelpful, such as in the case of anaplastic or poorly differentiated tumours, where a distinction between lymphoma and a non-haematopoietic tumour was often problematical. Accurate diagnosis became even more important with the developments in the clinical staging of lymphoma and the availability of more effective treatments. One way forward to improve diagnosis was to use immunohistochemistry to study the antigens expressed by the tumor cells.

Downregulation of HLA class II is associated with relapse after allogeneic stem cell transplantation and alters recognition by antigen-specific T cells.

Genomic deletion of donor-patient-mismatched HLA alleles in leukemic cells is a major cause of relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Mismatched HLA is frequently lost as an individual allele or a whole region in HLA-class I, however, it is downregulated in HLA-class II. We hypothesized that there might be a difference in T cell recognition capacity against epitopes associated with HLA-class I and HLA-class II and consequently such allogeneic immune pressure induced HLA alterations in leukemic cells. To investigate this, we conducted in vitro experiments with T cell receptor-transduced T (TCR-T) cells. The cytotoxic activity of NY-ESO-1-specific TCR-T cells exhibited similarly against K562 cells with low HLA-A*02:01 expression. However, we demonstrated that the cytokine production against low HLA-DPB1*05:01 expression line decreased gradually from the HLA expression level approximately 2-log lower than normal expressors. Using sort-purified leukemia cells before and after HSCT, we applied the next-generation sequencing, and revealed that there were several marked downregulations of HLA-class II alleles which demonstrated consistently low expression from pre-transplantation. The marked downregulation of HLA-class II may lead to decreased antigen recognition ability of antigen-specific T cells and may be one of immune evasion mechanism associated with HLA-class II downregulation.

Key findings from the UKCCMP cohort of 877 patients with haematological malignancy and COVID-19: disease control as an important factor relative to recent chemotherapy or anti-CD20 therapy.

Patients with haematological malignancies have a high risk of severe infection and death from SARS-CoV-2. In this prospective observational study, we investigated the impact of cancer type, disease activity, and treatment in 877 unvaccinated UK patients with SARS-CoV-2 infection and active haematological cancer. The primary end-point was all-cause mortality. In a multivariate analysis adjusted for age, sex and comorbidities, the highest mortality was in patients with acute leukaemia [odds ratio (OR) = 1·73, 95% confidence interval (CI) 1·1-2·72, P = 0·017] and myeloma (OR 1·3, 95% CI 0·96-1·76, P = 0·08). Having uncontrolled cancer (newly diagnosed awaiting treatment as well as relapsed or progressive disease) was associated with increased mortality risk (OR = 2·45, 95% CI 1·09-5·5, P = 0·03), as was receiving second or beyond line of treatment (OR = 1·7, 95% CI 1·08-2·67, P = 0·023). We found no association between recent cytotoxic chemotherapy or anti-CD19/anti-CD20 treatment and increased risk of death within the limitations of the cohort size. Therefore, disease control is an important factor predicting mortality in the context of SARS-CoV-2 infection alongside the possible risks of therapies such as cytotoxic treatment or anti-CD19/anti-CD20 treatments.

Gene therapy in a murine model of chronic eosinophilic leukemia-not otherwise specified (CEL-NOS).

Chronic eosinophilic leukemia-not otherwise specified (CEL-NOS) is a rare, aggressive, fatal disease characterized by blood eosinophilia and dysfunction of organs infiltrated with eosinophils. Clinically, the disease manifests with weight loss, cough, weakness, diarrhea, and multi-organ dysfunction that is unresponsive to therapy. We developed a one-time gene therapy for CEL-NOS using an adeno-associated virus (AAV) expressing an anti-eosinophil monoclonal antibody (AAVrh.10mAnti-Eos) to provide sustained suppression of eosinophil numbers in blood, thus reducing eosinophil tissue invasion and organ dysfunction. A novel CEL-NOS model was developed in NOD-scid IL2rγnull (NSG) mice by administration of AAV expressing the cytokine IL5 (AAVrh.10mIL5), resulting in marked peripheral and tissue eosinophilia of the heart, lung, liver, and spleen, and eventually death. Mice were administered AAVrh.10mAnti-Eos (1011 genome copies) 4 wk after administration of AAVrh.10mIL5 and evaluated for anti-eosinophil antibody expression, blood eosinophil counts, organ eosinophil invasion, and survival. AAVrh.10mAnti-Eos expressed persistent levels of the anti-eosinophil antibody for >24 wk. Strikingly, CEL-NOS treated mice had markedly lower blood eosinophil levels and reduced mortality when compared with control treated mice. These results suggest that a single treatment with AAVrh.10mAnti-Eos has the potential to provide substantial therapeutic benefit to patients with CEL-NOS, a fatal malignant disorder.

[Bispecific antibodies targeting CD3 in oncology and hematology]. / Anticorps bispécifiques ciblant CD3 en oncologie solide et onco-hématologie.

Bispecific therapies targeting CD3, so-called T-cell engagers (TCE), belong to the new spectrum of anti-tumor immunotherapies stimulating T-lymphocytes. TCE are unique constructs targeting the MHC-independent CD3 epsilon subunit (CD3e) and a tumor antigen. To date, only blinatumomab have reached market agreements in lymphoid malignancies with constructs targeting CD3exCD19. Other TCE are in advances development, with promising results targeting CD20 and BSMA in lymphoma and myeloma. These successes have relaunched the development of TCE in solid tumors, bringing mixed results so far (notably in terms of tolerance). Still, TCE pave the way to new immunotherapy in tumors considered to be refractory to inhibitors of immune checkpoints such as prostate cancer or colorectal cancer.

Born to survive: how cancer cells resist CAR T cell therapy.

Although chimeric antigen receptor T cells demonstrated remarkable efficacy in patients with chemo-resistant hematologic malignancies, a significant portion still resist or relapse. This immune evasion may be due to CAR T cells dysfunction, a hostile tumor microenvironment, or resistant cancer cells. Here, we review the intrinsic resistance mechanisms of cancer cells to CAR T cell therapy and potential strategies to circumvent them.

Single-cell proteo-genomic reference maps of the hematopoietic system enable the purification and massive profiling of precisely defined cell states.

Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

Mapping the evolution of T cell states during response and resistance to adoptive cellular therapy.

To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to "terminal" and "precursor" exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers.

γδ T Cells for Leukemia Immunotherapy: New and Expanding Trends.

Recently, many discoveries have elucidated the cellular and molecular diversity in the leukemic microenvironment and improved our knowledge regarding their complex nature. This has allowed the development of new therapeutic strategies against leukemia. Advances in biotechnology and the current understanding of T cell-engineering have led to new approaches in this fight, thus improving cell-mediated immune response against cancer. However, most of the investigations focus only on conventional cytotoxic cells, while ignoring the potential of unconventional T cells that until now have been little studied. γδ T cells are a unique lymphocyte subpopulation that has an extensive repertoire of tumor sensing and may have new immunotherapeutic applications in a wide range of tumors. The ability to respond regardless of human leukocyte antigen (HLA) expression, the secretion of antitumor mediators and high functional plasticity are hallmarks of γδ T cells, and are ones that make them a promising alternative in the field of cell therapy. Despite this situation, in particular cases, the leukemic microenvironment can adopt strategies to circumvent the antitumor response of these lymphocytes, causing their exhaustion or polarization to a tumor-promoting phenotype. Intervening in this crosstalk can improve their capabilities and clinical applications and can make them key components in new therapeutic antileukemic approaches. In this review, we highlight several characteristics of γδ T cells and their interactions in leukemia. Furthermore, we explore strategies for maximizing their antitumor functions, aiming to illustrate the findings destined for a better mobilization of γδ T cells against the tumor. Finally, we outline our perspectives on their therapeutic applicability and indicate outstanding issues for future basic and clinical leukemia research, in the hope of contributing to the advancement of studies on γδ T cells in cancer immunotherapy.

Position-Scanning Peptide Libraries as Particle Immunogens for Improving CD8+ T-Cell Responses.

Short peptides reflecting major histocompatibility complex (MHC) class I (MHC-I) epitopes frequently lack sufficient immunogenicity to induce robust antigen (Ag)-specific CD8+ T cell responses. In the current work, it is demonstrated that position-scanning peptide libraries themselves can serve as improved immunogens, inducing Ag-specific CD8+ T cells with greater frequency and function than the wild-type epitope. The approach involves displaying the entire position-scanning library onto immunogenic nanoliposomes. Each library contains the MHC-I epitope with a single randomized position. When a recently identified MHC-I epitope in the glycoprotein gp70 envelope protein of murine leukemia virus (MuLV) is assessed, only one of the eight positional libraries tested, randomized at amino acid position 5 (Pos5), shows enhanced induction of Ag-specific CD8+ T cells. A second MHC-I epitope from gp70 is assessed in the same manner and shows, in contrast, multiple positional libraries (Pos1, Pos3, Pos5, and Pos8) as well as the library mixture give rise to enhanced CD8+ T cell responses. The library mixture Pos1-3-5-8 induces a more diverse epitope-specific T-cell repertoire with superior antitumor efficacy compared to an established single mutation mimotope (AH1-A5). These data show that positional peptide libraries can serve as immunogens for improving CD8+ T-cell responses against endogenously expressed MHC-I epitopes.

Spatiotemporal proteomic profiling of the pro-inflammatory response to lipopolysaccharide in the THP-1 human leukaemia cell line.

Protein localisation and translocation between intracellular compartments underlie almost all physiological processes. The hyperLOPIT proteomics platform combines mass spectrometry with state-of-the-art machine learning to map the subcellular location of thousands of proteins simultaneously. We combine global proteome analysis with hyperLOPIT in a fully Bayesian framework to elucidate spatiotemporal proteomic changes during a lipopolysaccharide (LPS)-induced inflammatory response. We report a highly dynamic proteome in terms of both protein abundance and subcellular localisation, with alterations in the interferon response, endo-lysosomal system, plasma membrane reorganisation and cell migration. Proteins not previously associated with an LPS response were found to relocalise upon stimulation, the functional consequences of which are still unclear. By quantifying proteome-wide uncertainty through Bayesian modelling, a necessary role for protein relocalisation and the importance of taking a holistic overview of the LPS-driven immune response has been revealed. The data are showcased as an interactive application freely available for the scientific community.

The Association Between Single-Nucleotide Polymorphisms of Co-Stimulatory Genes Within Non-HLA Region and the Prognosis of Leukemia Patients With Hematopoietic Stem Cell Transplantation.

To avoid graft rejection, the hematopoietic stem cells with matched classical human leukocyte antigen (HLA) alleles are the primary choice for clinical allogeneic transplantation. However, even if the fully HLA-matched hematopoietic stem cells are used for transplantation, some patients still have poor prognosis after hematopoietic stem cell transplantation (HSCT), suggesting that the HLA system was not the only determinant of the outcomes of HSCT. In this study, we investigated whether the single-nucleotide polymorphisms (SNPs) of the co-stimulatory genes within non-HLA regions were related to the outcomes of HSCT. The genomic DNAs of 163 patients who had acute leukemia and received HSCT and their respective donors were collected for analysis. Thirty-four SNPs located in the four co-stimulatory genes including cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD28, tumor necrosis factor ligand superfamily 4 (TNFSF4), and programmed cell death protein 1 (PDCD1) were selected to explore their relationship with the adverse outcomes after transplantation, including mortality, cytomegalovirus infection, graft-versus-host disease, and relapse. Our results revealed that nine SNPs in the CTLA4 gene, five SNPs in the PDCD1 gene, two SNPs in the TNFSF4 gene, and four SNPs in the CD28 gene were significantly associated with the occurrence of adverse outcomes post-HSCT. These SNPs may play important roles in immune response to allografts post-HSCT and can be the targets for developing strategy to identify appropriate donors.

Granzyme B and perforin produced by SEC2 mutant-activated human CD4+ T cells and CD8+ T cells induce apoptosis of K562 leukemic cells by the mitochondrial apoptotic pathway.

Staphylococcal enterotoxin C2 (SEC2), a classical representative of superantigens, activates T cells that produce massive cytokines. This characteristic makes SEC2 a promising candidate drug for cancer immunotherapy. Previous study showed that ST-4, a SEC2 mutant, enhanced recognition of mouse T-cell receptor Vß regions, and activated the increased number of T cells that produced more cytokines. However, the underlying molecular mechanism for stimulation of human peripheral blood mononuclear cells (PBMCs) and antitumor effect on human tumor cells remains unknown. Herein, we showed that ST-4 significantly activated TCR Vß 12, 13A, 14, 15, 17, and 20 CD4+ and CD8+ T cells, which produced substantial amounts of granzyme B and perforin. These cytokines exhibited antitumor effect on K562 cells by promoting apoptosis and inducing S-phase cell cycle arrest. Conversely, the granzyme B inhibitor or perforin inhibitor significantly weakened antitumor effect of ST-4, accompanied by a decrease of cleaved proapoptotic BAX and cytochrome c, and an increase of antiapoptotic BCL2. Taken together, these data suggest that granzyme B and perforin produced by ST-4-activated CD4+ T cells and CD8+ T cells play a pivotal role in inducing K562 cell apoptosis by the mitochondrial apoptotic pathway, and support ST-4 as a potential candidate for cancer immunotherapy.