**2. Classification of immunocytes**

### **2.1 Patient-associated immunocytes**

## *2.1.1 Lymphokine-activated killer cells (LAKs)*

LAKs are effector cells with significant cytotoxic activity, which are induced by culturing with recombinant IL-2 and have been proven effective against NK cell-resistant allogeneic and autologous tumor cells or cell lines [28]. Generally, LAKs after regional intra-arterial perfusion mainly accumulate at tumor sites, whereas the intravenously infused LAKs first accumulate in lung tissues before migrating to the liver regions [29].

LAKs-mediated adoptive immunotherapy has been confirmed with limited efficacy in vivo ranging from 20–30% including the metastatic or advanced tumor models and clinical studies upon patients (e.g., hepatic carcinoma, renal cell carcinoma, melanoma, leukemia) [30–32]. For example, Rosenberg et al. reported the treatment of 157 patients with advanced cancer using LAKs and IL-2, and confirmed the marked tumor regression and even remission by the immunotherapeutic approach. Nevertheless, the ultimate role of LAKs-based cancer immunotherapy still awaits further improvement including the efficacy and the decrease of toxicity and complexity [32].

## *2.1.2 Tumor-infiltrating lymphocytes (TILs)*

TILs are infiltrating lymphocytes in cancer tissues and play a critical role in mediating response to chemotherapy [33]. To date, of the tumor-infiltrating immune cells such as mast cells, macrophages, dendritic cells and leucocytes, TILs have been

#### *Cancer Immunotherapy and Cytotoxicity: Current Advances and Challenges DOI: http://dx.doi.org/10.5772/intechopen.105184*

considered as selected heterogeneous populations of T lymphocytes with a higher and specific immunological reactivity against cancer cells than the non-infiltrating subset [34]. Despite the accumulation of TILs has been recognized as prognosis for elevated survival and clinical outcomes, yet tumors with high level of TILs also with increased PD-1 immune checkpoint expression [35]. Thus, the feasibility of TILs as biomarker for reflecting the immune responses and predicting the clinical outcomes of cancer immunotherapy still need systematic formulations and detailed explorations [34]. Generally, although ineffective for in vivo tumor elimination, yet TILs are adequate to functionate proliferation and effector capacity when separated from immunosuppressive tumor microenvironment [36]. Taken together, identification of specific subpopulations and the corresponding molecular mechanism of TILs in cancer might be of great importance for guiding prognosis and developing appropriate sequencing of immunotherapy [33].

#### *2.1.3 Cytokine-induced killer cells (CIKs)*

CIKs are a heterogeneous population of CD3<sup>+</sup> CD56<sup>+</sup> natural killer T (NKT) cells and recognized as pharmacological tools for tumor immunotherapy, and in particular, in refractory to conventional radiotherapy and chemotherapy [37, 38]. Generally, CIKs contain two main subsets including the CD3 + CD56+ and CD3 + CD56- subpopulations endowed with higher anti-tumor activity and higher proliferation ability, respectively. Mature CIKs express active receptors of NK cells including NKG2D and DNAM-1, whereas with minimal expression of NKG2A, NKp44, NKp46 or CD94 [39].

For decades, numerous clinical trials of CIKs-based tumor immunotherapy have been registered attribute to the distinctive properties including intense MHCindependent antitumor activity, low toxicity effects and high safety on healthy cells [39, 40]. For instance, Yu et al. verified that CIKs-based immunotherapy was an effective adjuvant strategy in the early stage of hepatocellular carcinoma (HCC) rather than advanced HCC, and suggested that targeting myeloid-derived suppressor cells (MDSCs) would provide additional therapeutic benefits alongside CIKs-based therapy [23]. Moreover, CIKs can be expanded to match the clinical relevant rates cost-effectively and conveniently by a simple protocol, which is also the key issue for clinical application of adoptive immunotherapy [39].

Of the multiple modalities for cancer immunotherapy, vaccination with DC-CIKs has revealed limited therapeutic success in the administration of advanced solid tumors [41]. Therefore, considering the shortcomings of CIKs-based cellular immunotherapy alone, integrated therapy or combined modality therapy such as CIKs, cytokines, gene editing, immune checkpoints, radiotherapy and chemotherapy have better potentiality in relieving the major side effects and improving the clinical outcomes of standard treatment options [40].

#### **2.2 Innate immunocytes**

#### *2.2.1 Natural killer (NK) cells*

NK cells are heterogeneous cell population with unique characteristics of and belong to the innate lymphoid cells (ILCs), which can be divided into the cytotoxic CD56dimCD16high and IFN-γ-producing CD56brightCD16low/neg subsets and play an important role in both innate and adoptive immune responses dispense

with preliminary antigen presentation via receptor-ligand mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), release of perforin and granzyme as well as cytokine-based paracrine effects (e.g., IFN-γ, GM-CSF) [21, 42].

For decades, we and other investigators have reported the generation of NK cells from various sources such as cell lines (e.g., NK-92, NK-92MI, YT), perinatal blood (e.g., cord blood, placental blood), peripheral blood, hematopoietic stem cells (HSCs) and human pluripotent stem cells (e.g., human embryonic stem cells, human induced pluripotent stem cells) [21, 43–48]. Distinguish from the chimeric antigen receptortransduced T cells (CAR-T), NK cells with non-MHC-restricted recognition revealed reliable cytotoxicity against pathogenic microorganism and tumor cells without the significant disadvantages graft-versus-host disease (GvHD), cytokine release syndrome (CRS) and neurotoxicity [49–51]. Moreover, NK cells are supposed to eliminate non-proliferating or quiescent cancer stem cells (CSCs), which collectively highlights the possibility and preponderance of NK cell-based cytotherapy for cancer immunosurveillance and immunotherapy [52, 53].

#### *2.2.2 Dendritic cells (DCs)*

DCs are most potent antigen-presenting cells (APCs) arising from lymphomyeloid hematopoiesis and linking the innate and adaptive immunity, which are firstly identified in 1973 and capable of initiating and activating lymphocytes including T cells and B cells and thus play a unique function in cancer immunotherapy as well as the tumor microenvironment [54–57]. As recently reviewed by Stevens and the colleagues, the advent of DC-based immunotherapy and immune checkpoint inhibitors (e.g., PD-1/PD-L1, CTLA-4) has become a paradigm shift in the treatment of cancers including the small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [58].

Notably, DCs derived *in vitro* or naturally circulating DCs loaded with the tumor-associated antigens (TAAs) are safe and feasible for eliciting a robust and tumor-directed immune response [59]. Briefly, immature DCs are attracted by the damage-associated molecular patterns (DAMPs) and then transit to a mature phenotype with capabilities of phagocytic cargo processing and antigenic component engulfment [60]. Additionally, DCs have been reported with synergistic effect with CIKs or thoracic radiotherapy for the management of locally advanced or metastatic NSCLCs [61, 62].

#### *2.2.3 Macrophages (MΦ)*

Macrophages (MΦ), one type of innate immune cells including the M1 (inhibit growth and kill) and M2 (promote growth and repair) subtypes, possess superior antineoplastic effects due to the phenomena of engulfing pathogens and dead cells, which are pivotal modulators of tissue regeneration and hematopoietic stem cell renewal as well as cancer progression [63–66]. However, considering the purposiveness of identification of tumor-specific anticancer responses, the characteristics of MΦ in cancer as bidirectional executors have been largely overlooked for a long period [67, 68].

Current updates have indicated the biofunction of macrophages in the regression of cancers by modulating the polarization peculiarity of the predominate M2 repairtype MΦ (pro-tumor) to M1 kill-type (anti-tumor) [69]. Disturbances in MΦ function usually result in deficiency of anti-inflammatory MΦ, uncontrolled secretion

#### *Cancer Immunotherapy and Cytotoxicity: Current Advances and Challenges DOI: http://dx.doi.org/10.5772/intechopen.105184*

of inflammatory factors (e.g., TGF-β, VEGF, EGF, PGE2, IFN-γ) and production of nitric oxide (NO) as well as poor communication with functional cells (e.g., endothelial cells, epithelial cells, lymphocytes) and cancer cells [70, 71]. For instance, the tumor-associated MΦ (TAMs) are recognized as the prominent components in tumor microenvironment (TME), which thus considered as promising and advantaged targets for developing immunotherapeutic strategy [72, 73].

Thus, MΦ/innate immunology can be orchestrated to play a critical role in indirectly or directly combating numerous hematological malignancies and metastatic solid tumors via the activity of M1 kill-type and the stimulatory effect of M1-type MΦ to cytotoxic Th1 cells and relative effector cells, respectively [69, 74]. Moreover, state-of-the-art updates have suggested the feasibility of conversion of the dominating cancer growth-promoting MΦ into growth-inhibiting type and the resultant progression in cancer immunotherapy as well [69].

### **2.3 Adaptive immunocytes**

#### *2.3.1 T lymphocytes*

According to the tumor immunosurveillance theory, considerable attention has been caused on enhancing the effectiveness and cytotoxicity of antitumor immunity, and in particular, the T lymphocyte-based host immune response and the accompanied T cell signaling and metabolism as well [75]. T lymphocytes are CD3+ mononuclear cells and are essential for eradicating tumor cells, allergens and microorganisms as well as tissue repair, and thus people with the impairment or dysfunction of T cells are at high risk of cancers and infections and eventually poor prognosis or mortality [76, 77].

Immunotherapeutic resistance remains the substantial barrier of cancer immunotherapy, and the selection of appropriate standard-of-care treatments is critical for combating tumors in preclinical and clinical studies [78, 79]. Numerous investigations have indicated the feasibility of effectively eliminate immunotherapeutic resistance by promoting priming or activation of T lymphocytes, attracting or sustaining T cell-based immune response as well as favoring the immune-promoting the aforementioned TME, which is critical for cancer immunoediting and the component phases including elimination, equilibrium and escape [80, 81]. However, the developing of an effective T cell-based cytotherapy against cancer are extremely difficult largely due to the correspondence of tumor antigens to self MHC-associated fragments of self-proteins as well as the tumor heterogeneity [75, 82]. Encouragingly, functional cytolytic T lymphocyte (CTL)-mediated immune response has been reported with self-tumor antigen expression and efficient efficacy upon some cancer models and patients (e.g., melanoma, pancreatic carcinoma, breast cancer) [83, 84]. Collectively, T lymphocytes orchestrate various aspects of adaptive immunity, while subset delineation (e.g., TCF1<sup>+</sup> progenitor subsets in exhausted CD8<sup>+</sup> T cells, memory T cells) and tissue localization as well as targeted strategies (e.g., genetically engineered CAR-T or TCR-T) are key determinants of T lymphocyte function in immune responses [75, 77, 82, 85].

#### *2.3.2 B lymphocytes*

B cells, including the fetal liver-originated B-1 and bone marrow-derived B-2 subsets, are heterogeneous lymphocytes with antibody production and cytokine release capacity, which also represent a pivotal cellular constituent of humoral

immunity and function critically in the maintenance of tolerance and immune regulation [86–89]. Generally, B-1 lymphocytes, consist of B-1a and B-1b subsets, are part of innate immune system and mainly function in providing immunity to various specific pathogens via producing immunoglobulins [90]. B-2 lymphocytes, including the follicular B cells (FOB) and marginal zone B cells (MZB), have been considered as mediators of adaptive immunity and are capable of differentiating into memory cells [87].

Current studies have indicated the excessive inflammatory responses of regulatory B cells (Bregs) during autoimmune diseases, unresolved infections or chronic metabolic diseases, which also contribute to the dynamic balance of equilibrium required for tolerance by simultaneously reestablishing immune homeostasis and limiting ongoing immune responses [91–93]. Bregs encompassing all B cells for immune response suppression also play a promoting role in regulatory T cell (Treg) differentiation by accelerating anti-inflammatory cytokine (IL-10, IL-35, TGF-β) secretion and inhibiting proinflammatory cytokine production [91, 94]. Additionally, memory B cells (MBCs) have also been identified in humans and play an important role for the rapid development and response of protective immunity [95]. Collectively, with the aid of novel genetic and pharmacological technologies as well as the in-depth understanding of the phenotype, function and developmental processes, B cell-based cytotherapy has become a rapidly growing field in tumor immunotherapy [86, 94–96].

### **2.4 Engineered immunocytes**

#### *2.4.1 Chimeric antigen receptor-transduced T cells (CAR-Ts)*

T lymphocytes with the antibody-like CAR structure expression are adequate to recognize the unique structures on the surface of tumor-associated cells or tumor cells, and in particular, the remarkable efficacy of CAR-Ts for the management of hematological malignancies [27, 82, 97]. For instance, we and other investigators have reported the remission of various hematopoietic malignancies such as refractory or relapsed B acute lymphoblastic leukemia (r/rB-ALL) and B-cell non-Hodgkin's lymphoma (NHL) by utilizing the CAR-Ts targeting CD19, CD20, CD22 [98–102]. However, the adverse effects and the regulatory mechanisms of CAR-Ts-based tumor immunotherapy cannot be ignored [97, 103]. For example, Giavridis and the colleagues have reported the involvement of MΦ and IL-1 blockade in mediating and abating CAR-Ts-induced CRS [27, 104].

Despite the encouraging progress in hematologic malignancies, yet the applications of CAR-Ts-based cell therapy to solid tumors or as candidate pharmaceutical options have been challenging and suspicious [27, 68, 105–107]. In particular, the immunosuppressive TMEs of tumors represent the most important factor for limiting the efficacy of CAR-Ts-based immunotherapy [108]. It's noteworthy that pioneering investigators have suggested the synergistic effect of oncolytic virus with CAR-Ts in improving the therapeutic effect of solid tumors [109]. For instance, Watanabe and the colleagues took advantage of the Mesothelin-redirected CAR-Ts (meso-CAR-Ts) and combined with an OAd-TNFa-IL2 oncolytic adenovirus for TNF-α and IL-2 expression, which increased CAR-Ts and host T cell infiltration to TME and thus showed enhanced efficacy upon human- pancreatic ductal adenocarcinoma (PDA) xenograft immunodeficient mice [110].

*Cancer Immunotherapy and Cytotoxicity: Current Advances and Challenges DOI: http://dx.doi.org/10.5772/intechopen.105184*

### *2.4.2 T cell receptor-engineered T cells (TCR-Ts)*

Besides chimeric antigen receptor (CAR) transduction, T lymphocytes can also be genetically engineered with αβ T cell receptor expression, which is capable of recognizing MHC-restricted peptide antigens and therefore poised to turn into an important pillar of cellular cancer immunotherapy [82, 111]. Generally, TCRs are capable of recognizing a relatively broad range of human leucocyte antigen (HLA)/ specific peptide that can be expressed in the surface of patient's T cells [112–114]. Therefore, TCR-Ts hold the potential to redirect the recognition of tumor-associated surface antigens and the removal of cancer cells. In details, TCR-Ts-based cytotherapy are principally intended to redirect circulating CD8+ T lymphocytes to the targets of expressing class I epitopes of HLA compared to those CD4+ /CD8+ T cells with HLA Class-II epitopes. Collectively, TCR-transduced T cells hold promising prospective in targeting all intracellular proteins when peptide epitopes have been presented on the HLAs including over-expressed neoantigens and self -antigens, viral antigens [115]. Even though the characteristics of adaptive evolution in T cell immunity and the preferential expansion of T lymphocytes with high-affinity TCRs, yet whether the affinity maturation of T cells by clonal selection continues during the course of tumor development remains unresolved [114, 116].

#### *2.4.3 CAR-transduced NK cells (CAR-NKs)*

Compared to CAR-Ts, the genetically modified CAR-NKs not only inherit the splendid properties of adoptive NK cells including the aforementioned biological effects but also hold promising prospects for solid tumor administration without causing adverse effects including CRS, GvHD or immune cell- associated neurotoxicity syndrome (ICANS) [49–51, 117]. Thus, the CAR-NKs-based immunotherapy represents a virgin ground of immunotherapy innovation [118–120].

As to CAR transduction, a series of methodology has been developed to fulfill the high-efficient delivery demands via retrovirus-, lentivirus-, nonviral-mediated transfection with the range from 27–70% [121, 122]. In particular, the DNA transposon system composed of the sleeping beauty (SB) and the PiggyBac (PB) subsets is competent for delivering CAR structure into the genomes of induced pluripotent stem cells (iPSCs) or primary NK cells with high-efficient and long-lasting transgene expression [27, 123]. As to the CAR structures, a group of preclinical and clinical studies have reported the successful design and delivery of vectors carrying the cassettes of CAR-conjunct targets (e.g., CD19, CD5, CD137) into NK cells with the second- or third- or fourth-generation constructs against diverse malignancies, respectively [124–128]. Of note, Li *et al* announced the high-efficient generation of NK cells from human induced pluripotent stem cells (NK-CAR-iPSC-NKs) and superiority over T-CAR-expressing iPSC-NKs (T-CAR-iPSC-NKs), which highlighted the feasibility of the standardized and targeted CAR-NKs-based cancer immunotherapy in future [118].

#### *2.4.4 CAR-transduced macrophages (CAR-ms)*

State-of-the-art updates have indicated the feasibility of CAR-Ms for solid tumor management and virion load reduction [129, 130]. Differ from the other CARtransduced immune cells, CAR-Ms are important sources of matrix metalloproteinase (MMPs), which can enter tumor tissue and degrade almost all extracellular matrix (ECM) components and thus destroy malignant tumor progression [130]. Strikingly, Klichinsky *et al* has reported the successful application of CAR-Ms in two solid tumor models and confirmed the prolonged overall survival and decreased tumor burden, which indicates the possibility of CAR-Ms-focused immunotherapeutic modalities [68, 131]. Notably, the therapeutic benefit of HER-2-targeting CAR-Ms was mediated by direct antigen-specific phagocytosis and indirect pro-inflammatory effects [132]. Recently, Zhang and the colleagues derived CAR-Ms from induced pluripotent stem cells (iPSC-CAR-Ms) with splendid properties such as cytokine secretion, polarization, enhanced phagocytosis and *in vivo* antitumor activity, which for the first time made iPSCs as unlimited source for "off-the-shelf" CAR-M generation [133].
