The Interplay Between Epigenetic Regulation and CD8+ T Cell Differentiation/Exhaustion for T Cell Immunotherapy

Effective immunotherapy treats cancers by eradicating tumourigenic cells by activated tumour antigen-specific and bystander CD8+ T-cells. However, T-cells can gradually lose cytotoxicity in the tumour microenvironment, known as exhaustion. Recently, DNA methylation, histone modification, and chromatin architecture have provided novel insights into epigenetic regulations of T-cell differentiation/exhaustion, thereby controlling the translational potential of the T-cells. Thus, developing strategies to govern epigenetic switches of T-cells dynamically is critical to maintaining the effector function of antigen-specific T-cells. In this mini-review, we 1) describe the correlation between epigenetic states and T cell phenotypes; 2) discuss the enzymatic factors and intracellular/extracellular microRNA imprinting T-cell epigenomes that drive T-cell exhaustion; 3) highlight recent advances in epigenetic interventions to rescue CD8+ T-cell functions from exhaustion. Finally, we express our perspective that regulating the interplay between epigenetic changes and transcriptional programs provides translational implications of current immunotherapy for cancer treatments.

T Cell Immune Profiles of Blood and Tumor in Dogs Diagnosed With Malignant Melanoma

Investigation of canine T cell immunophenotypes in canine melanomas as prognostic biomarkers for disease progression or predictive biomarkers for targeted immunotherapeutics remains in preliminary stages. We aimed to examine T cell phenotypes and function in peripheral blood mononuclear cells (PBMC) and baseline tumor samples by flow cytometry, and to compare patient (n = 11–20) T cell phenotypes with healthy controls dogs (n = 10–20). CD3, CD4, CD8, CD25, FoxP3, Ki67, granzyme B, and interferon-γ (IFN-γ) were used to classify T cell subsets in resting and mitogen stimulated PBMCs. In a separate patient cohort (n = 11), T cells were classified using CD3, CD4, CD8, FoxP3, and granzyme B in paired PBMC and single cell suspensions of tumor samples. Analysis of flow cytometric data of individual T cell phenotypes in PBMC revealed specific T cell phenotypes including FoxP3+ and CD25+FoxP3- populations that distinguished patients from healthy controls. Frequencies of IFN-γ+ cells after ConA stimulation identified two different patient phenotypic responses, including a normal/exaggerated IFN-γ response and a lower response suggesting dysfunction. Principle component analysis of selected T cell immunophenotypes also distinguished patients and controls for T cell phenotype and revealed a clustering of patients based on metastasis detected at diagnosis. Findings supported the overall hypothesis that canine melanoma patients display a T cell immunophenotype profile that is unique from healthy pet dogs and will guide future studies designed with larger patient cohorts necessary to further characterize prognostic T cell immunophenotypes.

Heterocellular OSM-OSMR signalling reprograms fibroblasts to promote pancreatic cancer growth and metastasis

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with a complex microenvironment. Dichotomous tumour-promoting and -restrictive roles have been ascribed to the tumour microenvironment, however the effects of individual stromal subsets remain incompletely characterised. Here, we describe how heterocellular Oncostatin M (OSM) - Oncostatin M Receptor (OSMR) signalling reprograms fibroblasts, regulates tumour growth and metastasis. Macrophage-secreted OSM stimulates inflammatory gene expression in cancer-associated fibroblasts (CAFs), which in turn induce a pro-tumourigenic environment and engage tumour cell survival and migratory signalling pathways. Tumour cells implanted in Osm-deficient (Osm−/−) mice display an epithelial-dominated morphology, reduced tumour growth and do not metastasise. Moreover, the tumour microenvironment of Osm−/− animals exhibit increased abundance of α smooth muscle actin positive myofibroblasts and a shift in myeloid and T cell phenotypes, consistent with a more immunogenic environment. Taken together, these data demonstrate how OSM-OSMR signalling coordinates heterocellular interactions to drive a pro-tumourigenic environment in PDA.

IMMU-34. CAMKK2 PROMOTES AN IMMUNOSUPPRESSIVE PROGRAM AND CHECKPOINT BLOCKADE RESISTANCE IN THE GLIOBLASTOMA TUMOR MICROENVIRONMENT

BACKGROUND Immunotherapy has demonstrated efficacy in several cancers but has shown only modest effects in Glioblastoma (GBM). This is linked to the anti-inflammatory nature of the tumor microenvironment (TME) and the pro-tumor functions of brain native cells. Targeting stromal cells, such as tumor associated macrophages (TAMs) and neurons, is a promising approach. Re-analysis of human and murine brain single cell-RNAseq (scRNAseq) datasets shows Calmodulin Dependent Kinase Kinase 2 (CaMKK2) is highly expressed in both neurons and TAMs. Loss of CaMKK2 polarizes TAMs to an immunostimulatory phenotype and reduces pro-tumor neuronal functions. Thus, we hypothesize that CaMKK2 promotes the pro-tumor nature of the GBM TME and immunotherapy resistance. RESULTS Murine GBM was orthotopically implanted into wild-type and CaMKK2-/- mice. CaMKK2-/- mice exhibited significantly prolonged survival. To determine if anti-tumor immune function was enhanced, we probed the TME using flow cytometry and scRNAseq. CaMKK2-/- mice showed increased abundance of precursor exhausted, potentially immune checkpoint blockade (ICB) responsive, CD8 T cells. Furthermore, T cell depletion abrogated the survival benefit observed in CaMKK2-/- mice. Considering these T cell phenotypes, we treated CaMKK2-/- mice with ICB, and indeed they were sensitive. To determine if the CaMKK2-/- survival phenotype and ICB response depended on CaMKK2 expression in hematopoietic or in non-hematopoietic cells, we utilized a reciprocal chimera model. Loss of CaMKK2 in the non-hematopoietic cells was more vital for survival and ICB response than in hematopoietic cells, suggesting a potential novel and unique role for CaMKK2 in brain native cells - potentially neurons - in coordinating ICB resistance. CONCLUSIONS We find that CaMKK2 exacerbates mortality and drives ICB resistance by limiting the anti-tumor response in GBM via both hematopoietic and brain native cells. Our findings identify a novel therapeutic target for GBM, and a unique role for CaMKK2 in the TME.

Single cell T cell landscape and T cell receptor repertoire profiling of AML in context of PD-1 blockade therapy

In contrast to the curative effect of allogenic stem cell transplantation in acute myeloid leukemia via T cell activity, only modest responses are achieved with checkpoint-blockade therapy, which might be explained by T cell phenotypes and T cell receptor (TCR) repertoires. Here, we show by paired single-cell RNA analysis and TCR repertoire profiling of bone marrow cells in relapsed/refractory acute myeloid leukemia patients pre/post azacytidine+nivolumab treatment that the disease-related T cell subsets are highly heterogeneous, and their abundance changes following PD-1 blockade-based treatment. TCR repertoires expand and primarily emerge from CD8+ cells in patients responding to treatment or having a stable disease, while TCR repertoires contract in therapy-resistant patients. Trajectory analysis reveals a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B and a bone marrow-residing memory CD8+ T cell subset, in which a population with stem-like properties expressing granzyme K is enriched in responders. Chromosome 7/7q loss, on the other hand, is a cancer-intrinsic genomic marker of PD-1 blockade resistance in AML. In summary, our study reveals that adaptive T cell plasticity and genomic alterations determine responses to PD-1 blockade in acute myeloid leukemia.

Pan-Cancer Analysis of Immune Complement Signature C3/C5/C3AR1/C5AR1 in Association with Tumor Immune Evasion and Therapy Resistance

Despite the advances in our understanding of the genetic and immunological basis of cancer, cancer remains a major public health burden with an ever-increasing incidence rate globally. Nevertheless, increasing evidence suggests that the components of the complement system could regulate the tumor microenvironment (TME) to promote cancer progression, recurrence, and metastasis. In the present study, we used an integrative multi-omics analysis of clinical data to explore the relationships between the expression levels of and genetic and epigenetic alterations in C3, C5, C3AR1, and C5AR1 and tumor immune evasion, therapy response, and patient prognosis in various cancer types. We found that the complements C3, C5, C3AR1, and C5AR1 have deregulated expression in human malignancies and are associated with activation of immune-related oncogenic processes and poor prognosis of cancer patients. Furthermore, we found that the increased expression levels of C3, C5, C3AR1, and C5AR1 were primarily predicted by copy number variation and gene methylation and were associated with dysfunctional T-cell phenotypes. Single nucleotide variation in the gene signature co-occurred with multiple oncogenic mutations and is associated with the progression of onco-immune-related diseases. Further correlation analysis revealed that C3, C5, C3AR1, and C5AR1 were associated with tumor immune evasion via dysfunctional T-cell phenotypes with a lesser contribution of T-cell exclusion. Lastly, we also demonstrated that the expression levels of C3, C5, C3AR1, and C5AR1 were associated with context-dependent chemotherapy, lymphocyte-mediated tumor killing, and immunotherapy outcomes in different cancer types. In conclusion, the complement components C3, C5, C3AR1, and C5AR1 serve as attractive targets for strategizing cancer immunotherapy and response follow-up.