Chimeric antigen receptor (CAR)-engineered T-cell (CAR-T) therapy has demonstrated impressive therapeutic efficacy against hematological malignancies, but multiple challenges have hindered its application, particularly for the eradication of solid tumors. Innate killer cells (IKCs), particularly NK cells, NKT cells, and γδ T cells, employ specific antigen-independent innate tumor recognition and cytotoxic mechanisms that simultaneously display high antitumor efficacy and prevent tumor escape caused by antigen loss or modulation. IKCs are associated with a low risk of developing GVHD, thus offering new opportunities for allogeneic "off-the-shelf" cellular therapeutic products. The unique innate features, wide tumor recognition range, and potent antitumor functions of IKCs make them potentially excellent candidates for cancer immunotherapy, particularly serving as platforms for CAR development. In this review, we first provide a brief summary of the challenges hampering CAR-T-cell therapy applications and then discuss the latest CAR-NK-cell research, covering the advantages, applications, and clinical translation of CAR- and NK-cell receptor (NKR)-engineered IKCs. Advances in synthetic biology and the development of novel genetic engineering techniques, such as gene-editing and cellular reprogramming, will enable the further optimization of IKC-based anticancer therapies.

Chimeric antigen receptor- and natural killer cell receptor-engineered innate killer cells in cancer immunotherapy.

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.

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

A growing number of studies have shown that γδ T cells play a pivotal role in mediating the clearance of tumors and pathogen-infected cells with their potent cytotoxic, cytolytic, and unique immune-modulating functions. Unlike the more abundant αβ T cells, γδ T cells can recognize a broad range of tumors and infected cells without the requirement of antigen presentation via major histocompatibility complex (MHC) molecules. Our group has recently demonstrated parts of the mechanisms of T-cell receptor (TCR)-dependent activation of Vγ9Vδ2+ T cells by tumors following the presentation of phosphoantigens, intermediates of the mevalonate pathway. This process is mediated through the B7 immunoglobulin family-like butyrophilin 2A1 (BTN2A1) and BTN3A1 complexes. Such recognition results in activation, a robust immunosurveillance process, and elicits rapid γδ T-cell immune responses. These include targeted cell killing, and the ability to produce copious quantities of cytokines and chemokines to exert immune-modulating properties and to interact with other immune cells. This immune cell network includes αβ T cells, B cells, dendritic cells, macrophages, monocytes, natural killer cells, and neutrophils, hence heavily influencing the outcome of immune responses. This key role in orchestrating immune cells and their natural tropism for tumor microenvironment makes γδ T cells an attractive target for cancer immunotherapy. Here, we review the current understanding of these important interactions and highlight the implications of the crosstalk between γδ T cells and other immune cells in the context of anti-tumor immunity.

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γδ T Cells in the Tumor Microenvironment—Interactions With Other Immune Cells

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia among adults. It is the clonal expansion of B cells expressing CD19 and CD5. Despite significant progress in treatment, CLL is still incurable. γδ T cells comprise an important subset of the cytotoxic T cells. Although γδ T cells in CLL are dysfunctional, they still can possibly be used for immunotherapy. The current paper reviews our understanding of γδ T lymphocytes in CLL.

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The Mysterious Actor—γδ T Lymphocytes in Chronic Lymphocytic Leukaemia (CLL)

Background Heterogeneous T cells in acute myeloid leukemia (AML) have the combinatorial variety generated by different T cell receptors (TCRs). γδ T cells are a distinct subgroup of T cells containing TCRγ (TRGV) and TCRδ (TRDV) subfamilies with diverse structural and functional heterogeneity. Our previous study showed that clonally expanded TRDV T cells might benefit the immune response directed against AML. However, the features of the TRGV repertoire in AML remain unknown. To fully characterize the features of γδ T cells, we analyzed the distribution and clonality of TRGV I-III subfamilies (TRGV II is also termed TRVG 9), the proportions of γδ T cell subsets, and their effects on the overall survival (OS) of patients with AML. Methods In this study, the complementarity-determining region 3 (CDR3) size of TRGV subfamilies in γδ T cells of peripheral blood (PB) from de novo AML patients were analyzed by Genescan analysis. Expression levels of TRGV subfamilies were performed by real-time quantitative PCR. The proportions of total γδ T cells and their Vγ9+ Vδ2+ T cells subsets were detected by multicolor flow cytometry assay. We further compared the correlation among the TRGV gene expression levels, the proportion of Vγ9+ Vδ2+ T cells, and OS in AML. Results We first found that the distribution pattern and clonality of TRGV subfamilies were changed. The expression frequencies and gene expression levels of three TRGV subfamilies in AML samples were significantly lower than those in healthy individuals (HIs). Compared with HIs, the proportions of total γδ T cells and Vγ9+ Vδ2+ T cells were also significantly decreased in patients with AML. In addition, patients with AML who had higher expression levels of the TRGV gene and higher proportion of Vγ9+ Vδ2+ T cells showed better OS than their counterparts. Furthermore, high expression levels of TRGV 9 and proportion of Vγ9+ Vδ2+ T cells were identified as independent protective factors for complete remission in patients with AML. Conclusions The restriction of TRGV usage might be related to the preference of usage of γδ T cells. Higher expression of TRGV subfamilies might be associated with better OS in AML. Higher TRGV 9 expression and increased Vγ9+ Vδ2+ T cells subfamilies might indicate a better prognosis in patients with AML.

High TRGV 9 Subfamily Expression Marks an Improved Overall Survival in Patients With Acute Myeloid Leukemia

Recently, cell-mediated immune response in malignant neoplasms has become the focus in immunotherapy against cancer. However, in leukemia, most studies on the cytotoxic potential of T cells have concentrated only on T cells that recognize peptide antigens (Ag) presented by polymorphic molecules of the major histocompatibility complex (MHC). This ignores the great potential of unconventional T cell populations, which include gamma-delta T cells (γδ), natural killer T cells (NKT), and mucosal-associated invariant T cells (MAIT). Collectively, these T cell populations can recognize lipid antigens, specially modified peptides and small molecule metabolites, in addition to having several other advantages, which can provide more effective applications in cancer immunotherapy. In recent years, these cell populations have been associated with a repertoire of anti- or protumor responses and play important roles in the dynamics of solid tumors and hematological malignancies, thus, encouraging the development of new investigations in the area. This review focuses on the current knowledge regarding the role of unconventional T cell populations in the antitumor immune response in leukemia and discusses why further studies on the immunotherapeutic potential of these cells are needed.

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Translating Unconventional T Cells and Their Roles in Leukemia Antitumor Immunity.

The leukemic microenvironment has a high diversity of immune cells that are phenotypically and functionally distinct. However, our understanding of the biology, immunology, and clinical implications underlying these cells remains poorly investigated. Among the resident immune cells that can infiltrate the leukemic microenvironment are myeloid cells, which correspond to a heterogeneous cell group of the innate immune system. They encompass populations of neutrophils, macrophages, and myeloid-derived suppressor cells (MDSCs). These cells can be abundant in different tissues and, in the leukemic microenvironment, are associated with the clinical outcome of the patient, acting dichotomously to contribute to leukemic progression or stimulate antitumor immune responses. In this review, we detail the current evidence and the many mechanisms that indicate that the activation of different myeloid cell populations may contribute to immunosuppression, survival, or metastatic dissemination, as well as in immunosurveillance and stimulation of specific cytotoxic responses. Furthermore, we broadly discuss the interactions of tumor-associated neutrophils and macrophages (TANs and TAMs, respectively) and MDSCs in the leukemic microenvironment. Finally, we provide new perspectives on the potential of myeloid cell subpopulations as predictive biomarkers of therapeutical response, as well as potential targets in the chemoimmunotherapy of leukemias due to their dual Yin-Yang roles in leukemia.

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Mateus de Souza Barros

Papers

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

Translating Unconventional T Cells and Their Roles in Leukemia Antitumor Immunity.

The Yin-Yang of myeloid cells in the leukemic microenvironment: Immunological role and clinical implications