CTLA-4 is a crucial immune checkpoint receptor involved in the maintenance of immune homeostasis, tolerance, and tumour control. Antibodies targeting CTLA-4 have been promising treatments for numerous cancers, but the mechanistic basis of their anti-tumoral immune boosting effects are poorly understood. Although the ctla4 gene also encodes an alternatively-spliced soluble variant (sCTLA-4), preclinical/clinical evaluation of anti-CTLA-4-based immunotherapies have not considered the contribution of this isoform. Here, we explore the functional properties of sCTLA-4 and evaluate the efficacy of isoform-specific anti-sCTLA-4 antibody targeting in murine cancer model. We show that expression of sCTLA-4 by tumour cells suppresses CD8⁺ T-cells in vitro, and accelerates growth and experimental metastasis of murine tumours in vivo. These effects were accompanied by modification of the immune infiltrate, notably restraining CD8⁺ T-cells in a non-cytotoxic state. sCTLA-4 blockade with isoform-specific antibody reversed this restraint, enhancing intratumoural CD8⁺ T-cell activation and cytolytic potential, correlating with therapeutic efficacy and tumour control. This previously unappreciated role of sCTLA-4 suggests that better understanding of the biology and function of multi-gene products of immune checkpoint receptors needs to be fully elucidated for improved mechanistic understanding of cancer immunotherapies.

Immune profiling of tissue through multiplex immunohistochemistry is important for the investigation of immune cell dynamics, and it can contribute to disease prognosis and evaluation of treatment response in cancer patients. However, protocols for mouse formalin-fixed, paraffin-embedded tissue have been less successful. Given that formalin fixation and paraffin embedding remains the most common preparation method for processing mouse tissue, this has limited the options to study the immune system and the impact of novel therapeutics in preclinical models. In an attempt to address this, we developed an improved immunohistochemistry protocol with a more effective Ag-retrieval buffer. We also validated 22 Abs specific for mouse immune cell markers to distinguish B cells, T cells, NK cells, macrophages, dendritic cells, and neutrophils. In addition, we designed and tested novel strategies to identify immune cells for which unique Abs are currently not available. Last, in the 4T1 model of breast cancer, we demonstrate the utility of our protocol and Ab panels in the quantitation and spatial distribution of immune cells.

Neuroblastoma is the most frequent extracranial childhood tumour but effective treatment with current immunotherapies is challenging due to its immunosuppressive microenvironment. Efforts to date have focused on using immunotherapy to increase tumour immunogenicity and enhance anticancer immune responses, including anti-GD2 antibodies; immune checkpoint inhibitors; drugs which enhance macrophage and natural killer T (NKT) cell function; modulation of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway; and engineering neuroblastoma-targeting chimeric-antigen receptor-T cells. Some of these strategies have strong preclinical foundation and are being tested clinically, although none have demonstrated notable success in treating paediatric neuroblastoma to date. Recently, approaches to overcome heterogeneity of neuroblastoma tumours and treatment resistance are being explored. These include rational combination strategies with the aim of achieving synergy, such as dual targeting of GD2 and tumour-associated macrophages or natural killer cells; GD2 and the B7-H3 immune checkpoint; GD2 and enhancer of zeste-2 methyltransferase inhibitors. Such combination strategies provide opportunities to overcome primary resistance to and maximize the benefits of immunotherapy in neuroblastoma.

The inhibitory CTLA-4 molecule is a crucial regulator of immune responses and a target for therapeutic intervention in both autoimmunity and cancer. In particular, CTLA-4 is important in controlling antigen-specific immunity, including responses to autoantigens associated with autoimmune disease. Here, we investigate cytokine responses to a range of lupus-associated autoantigens and assess whether the alternatively spliced isoform of CTLA-4, soluble CTLA-4 (sCTLA-4), contributes to immune regulation of autoantigen-specific immunity in systemic lupus erythematosus (SLE). The cell culture supernatant production of sCTLA-4 as well as the cytokines IL-10, IFN-γ, and IL-17 from peripheral blood mononuclear cells (PBMC) from lupus patients and age- and sex-matched healthy volunteer donors were measured in response to previously identified histone and small nuclear ribonucleoprotein (snRNP) autoantigen-derived peptides (H391-105, H471-93, and U170K131-151) by ELISA. We also examined the functional contribution of sCTLA-4 to immune regulation in the context of these autoantigenic peptides following blockade of sCTLA-4 with a selective anti-sCTLA-4 monoclonal antibody, JMW-3B3. We identified responses to autoantigenic peptides, which revealed qualitative differences in cytokine (IL-10, IL-17, and IFN-γ) profiles between SLE patients and healthy donors. PBMC from healthy donors responded to each of the lupus peptides by secreting IFN-γ and IL-17, but PBMC from SLE patients produced IL-10. Although we did not observe differences in the levels of serum or PBMC culture supernatant sCTLA-4 in either cohort, blockade of sCTLA-4 in PBMC cultures responding to antigen enhanced the cytokine profiles associated with each group. The results show that lupus autoantigen-derived peptides display varied immunogenicity in lupus versus healthy volunteer donors, while sCTLA-4 acts to regulate the T-cell activity independently of response profile.

Background: The immunomodulatory enzyme, indoleamine 2,3-dioxygenase (IDO) facilitates tryptophan catabolism at the rate-limiting step of the kynurenine (Kyn) pathway. IDO expression and elevations in Kyn metabolites are associated with immunosuppressive tumor microenvironment including T cell proliferative arrest and generation of regulatory T cells (Tregs) which can favor tumor progression. However, the extent of the role of IDO in acute myeloid leukemia (AML) is currently ill-defined. This study reviews the role of IDO-driven Treg function in AML and evaluates the current body of evidence implicating IDO in AML pathogenesis.Method: Studies related to IDO in AML were identified through a systematic review of PubMed and Scopus. Data extracted described sample analysis, IDO expression, IDO in prognosis, techniques used in Treg phenotypic studies, and the effect of IDO inhibitors.Results: Twenty studies were included in the systematic review. Expression of IDO was identified in a range of cells in AML, both inducible and constitutive. Seven studies indicated an association between elevated expression and poor clinical prognosis. Six studies suggested a positive correlation between IDO expression and Treg induction, with FoxP3 being the prominent Treg phenotypic marker. Of eight studies investigating IDO inhibition, some reported reductions in Treg frequency and enhanced effector T cell proliferation.Conclusion: This review highlights that IDO expression in AML is associated with poor prognosis and measurement of IDO and its Kyn metabolites may offer utility as prospective prognostic markers. Pharmacological inhibition of IDO using novel drugs may hold promise for the treatment of AML.

Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches.