**4. Immunomodulation and immunotherapy**

There are many potential therapeutic strategies for circumventing mechanisms of tumor immune evasion, including reversal of the inhibition of adaptive immunity, blocking the Tcell checkpoint pathways such as CTLA4, PD-1, TIM-3, adenosine A2A receptor and LAG-3 checkpoint molecule with agents such as IMP321, BMS-986016, pembrolizumab, nivolumab, pidilizumab, AMP-224, ipilimumab, tremelimumab, etc.

Another therapeutic strategy consists of improving the function of innate immune cells by manipulating the activation of natural killer (NK)-cell inhibitory receptors (KIP) and by stimulating dendritic cells and macrophages with therapeutic agents in clinical development, such as Lirilumab, and Toll-like receptors including TLR 2/4, TLR7, TLR 7/8, and TLR9 agonists, such as Hiltonol, Imiquimod, Resiquimod, CpG7909, and Bacillus Calmette–Guerin.

An additional mechanism consists of switching on adaptive immunity by promoting T-cell costimulatory receptor signaling, using agonist antibodies for the promotion of CD137 signaling with Urelumab, enhancement of CD27 signaling with CDX-1127, activation of CD40 with CP-870,893, and ChiLob 7/4 promotion of GITP signaling with TRX518, enhancement of OX-40 signaling with MEDI 6469, and administration of systemic recombinant IL-7, IL-15, IL-21 with Denenicokin, rhIL-7, and rhIL-15 for enhancing immune cell function including Tcell development.

The final therapeutic strategy consists of the activation of the immune system by potentiating immune-cell effector function with IDO inhibition with Indoximab or INCB024360, various vaccine-based therapeutic strategies, inhibition of TGF-b signaling with IMC-TRI, TEW-7197, LY2157299, or GC1008, and systemic IFN-a or IL-2 administration [67].

There are many immunomodulatory effects of targeted therapies, which can circumvent tumor-mediated immunosuppression, improving the effector T-cell function that enhances eradication of targeted tumors. Tumor and immune system effects of approved and experi‐ mental targeted agents include Sunitinib, which by inhibiting multiple tumor-associated tyrosine kinases, such as PDGFR and VEGFR, downregulates STAT3 and VEGF signaling pathways, reducing the population and effectiveness of T-reg cells and MDSCs. By blocking tumor-associated tyrosine kinases, such as KIT and ABL, imatinib inhibits IDO, reduces the population and effectiveness of T-reg cells, enhances the population of B-1 B cells and the Other tumor microenvironment (TME)-induced immunosuppressive factors, which we must target with cancer immunotherapy not only in solid tumors but also in hematologic malig‐ nancies, include tumor intrinsic immunosuppressing ectoenzyme CD37, which is a disulfidelinked homodimer that regulates negatively the proinflammatory effects of extracellular ATP; activates P2X7R, which is a coactivator of the NLRP3 inflammasome-releasing proinflamma‐ tory cytokines such as IL-18 and IL-1b; and blocks antitumor T-cell immunity via upregulation of the adenosine receptor (AR) signaling, promoting tumor angiogenesis, growth, and

There are many potential therapeutic strategies for circumventing mechanisms of tumor immune evasion, including reversal of the inhibition of adaptive immunity, blocking the Tcell checkpoint pathways such as CTLA4, PD-1, TIM-3, adenosine A2A receptor and LAG-3 checkpoint molecule with agents such as IMP321, BMS-986016, pembrolizumab, nivolumab,

Another therapeutic strategy consists of improving the function of innate immune cells by manipulating the activation of natural killer (NK)-cell inhibitory receptors (KIP) and by stimulating dendritic cells and macrophages with therapeutic agents in clinical development, such as Lirilumab, and Toll-like receptors including TLR 2/4, TLR7, TLR 7/8, and TLR9 agonists, such as Hiltonol, Imiquimod, Resiquimod, CpG7909, and Bacillus Calmette–Guerin.

An additional mechanism consists of switching on adaptive immunity by promoting T-cell costimulatory receptor signaling, using agonist antibodies for the promotion of CD137 signaling with Urelumab, enhancement of CD27 signaling with CDX-1127, activation of CD40 with CP-870,893, and ChiLob 7/4 promotion of GITP signaling with TRX518, enhancement of OX-40 signaling with MEDI 6469, and administration of systemic recombinant IL-7, IL-15, IL-21 with Denenicokin, rhIL-7, and rhIL-15 for enhancing immune cell function including T-

The final therapeutic strategy consists of the activation of the immune system by potentiating immune-cell effector function with IDO inhibition with Indoximab or INCB024360, various vaccine-based therapeutic strategies, inhibition of TGF-b signaling with IMC-TRI, TEW-7197,

There are many immunomodulatory effects of targeted therapies, which can circumvent tumor-mediated immunosuppression, improving the effector T-cell function that enhances eradication of targeted tumors. Tumor and immune system effects of approved and experi‐ mental targeted agents include Sunitinib, which by inhibiting multiple tumor-associated tyrosine kinases, such as PDGFR and VEGFR, downregulates STAT3 and VEGF signaling pathways, reducing the population and effectiveness of T-reg cells and MDSCs. By blocking tumor-associated tyrosine kinases, such as KIT and ABL, imatinib inhibits IDO, reduces the population and effectiveness of T-reg cells, enhances the population of B-1 B cells and the

LY2157299, or GC1008, and systemic IFN-a or IL-2 administration [67].

metastasis [62–66].

24 Immunopathology and Immunomodulation

cell development.

**4. Immunomodulation and immunotherapy**

pidilizumab, AMP-224, ipilimumab, tremelimumab, etc.

concentration of natural antitumor carbohydrate antibodies, and promotes the crosstalk between NK and DC cells. By sensitizing tumor cells to the induction of apoptosis or type I PCD, IAP inhibitors stimulate responses of T cells, NKT cells, and NK cells. GSK3b inhibitors facilitate differentiation toward stem cell memory T-cell population by blocking GSK3bmediated signaling of tumor cell growth, enhancing TLR4 signaling. By downregulating PI3K-AKT signaling in tumor cells, PI3K-AKT inhibitors enhance tumor susceptibility to perforin and granzyme-mediated lysis involving NK cells and CTLs, downregulating prosurvival signaling and reducing tumor promoting inflammation. By downregulating HSP-90, which enhances unfolded protein-associated stress in tumor cells, HSP-90 inhibitors exert immunos‐ timulatory action by enhancing the expression of NKG2D ligands and by stimulating the CTL recognition of tumor cells. JAK2 inhibitors increase the maturation of DCs, enhance DCmediated antigen presentation and T-cell priming, and downregulate immunosuppressive STAT3 signaling and expression of IAP and PDL1 of tumor cells by blocking JAK2 signaling in tumor cells. By downregulating BRAF-V600E, vemurafenib upregulates MART1, gp100, and other antigens, while it reduces tumor secretion of immunosuppressive cytokines. By inhibiting 26S subunit of the proteasome, bortezomib sensitizes tumor cells to lysis mediated by CTL and natural killer (NK) cells after the downregulation of the expression of MHC class I molecule, while it boosts antigen-specific T-cell response to vaccination. By inhibiting the mTOR pathway, rapamycin, temsirolimus, and other mTOR inhibitors exert immunostimu‐ latory actions, increasing CD8+T-cell activation and production of IFN-γ, enhancing CD8+ Tcell differentiation into memory T cells, impairing the homeostasis of T-reg cells, and downregulating IDO. Cetuximab as a neutralizing antibody against EGFR inhibits tumoral growth signals and activates the immune system by complement fixation, antibody-dependent cellular cytotoxicity, MHC class I and class II upregulation, and enhancement of DC priming of tumor-specific CTLs. Trastuzumab inhibits tumor growth signaling by the downregulation of HER2, which activates antitumor CTL activity, activates NK cells to secrete IFN-γ, and induces antibody-dependent cell-mediated cytotoxicity (ADCC). Bevacizumab, which is a neutralizing antibody against VEGF, inhibits angiogenesis and subsequent metastasis, while it enhances the maturation of dendritic cells (DCs) and the DC priming of T cells and shifts differentiation of DC toward mature DCs instead of MDSCs [68]. Thus, by interfering with these targeted pathways that drive tumor maintenance and growth, we exert immune therapeutic action by modulating the differentiation, activation, function, and development of the immune cells, which are responsible for inhibiting tumor growth and development, while tumor-induced immunosuppressive mechanisms are circumvented. These immunomodula‐ tory properties that activate the antitumor response include antagonism of tumor-mediated immunosuppressive mechanisms; increase of T-cell activation, differentiation, and effector function; and enhancement of T-cell priming and bolstering of presentation of tumor antigens, indicating a synergistic antitumor action between targeted therapies, which inhibit genomic pathways and anticancer immunomodulatory effects. These synergistic anticancer effects may become even much more effective with the use of combinatorial immunotherapies, which can be used in combination with other conventional anticancer treatment modalities, such as chemotherapy, radiotherapy, and surgery whose inflammatory and immunosuppressive actions may be circumvented with immunonutrition which can improve metabolomics, while it may circumvent the deadly risk of infection to cancer patients. More analytically, combina‐ torial immunotherapy may act synergistically by combining two different immunotherapeutic agents, such as inhibitors of immune checkpoints for preventing T-cell energy, and cancer vaccines for producing antitumor T cells. For instance, PD1 inhibitors or CTLA4 vaccines, such as autologous granulocyte macrophage colony-stimulating factor (GM-CSF) secreting tumor vaccine, may exert a significant synergistic antitumor action associated with higher overall survival rates by targeting multiple immunosuppressive pathways. Another combinatorial immune therapeutic approach consists of combining costimulatory receptors, which are overexpressed on activated T cells with agonistic antibodies, leading to enhancement of antitumor T-cell function, which eradicates tumors. Promising combinatorial immunothera‐ pies target synergistically the dual T-cell checkpoints, downregulating CTLA-4, PD-1, PD-L1, and LAG-3 with ipilimumab, tremelimumab, nivolumab, pembrolizumab, MEDI4736, and BMS-986016 against NSCLC, colon Ca, gastric Ca, SCLC, pancreatic Ca, melanoma, RCC, triple (-) breast Ca, and other solid tumors. Combinatorial immunotherapeutic regimens include Tcell inhibitors with costimulatory receptor agonists targeting CTLA-4 and CD40 with admin‐ istration of tremelimumab and CP-870,893 against metastatic melanoma. Another combinatorial regimen consists of T-cell inhibitors, and function enhancers of innate immune cells targeting CTLA-4, PD-1, and KIR with administration of lirilumab, ipilimumab, and nivolumab against solid tumors. Finally, T-cell inhibitors are combined with other activators of the immune system, such as vaccines and passive immunotherapeutics targeting CTLA-4, IL-21, PD-1, IDO with administration of denenicokin, ipilimumab, Nivolumab, INCB024360, indoximod, sipuleucel-T, nivolumab, gp100, NY-ESO-1, TriMix-DC, and adoptive cell transfer against melanoma, prostate Ca, and other solid tumors [67]. Currently, combinatorial thera‐ peutics may combine more than two agents, such as, immunotoxins, Fc-fusion proteins, and bispecific T-cell engagers (BiTEs) [69–72].

Another even more efficient antitumor strategy consists of combining targeted therapies with immunotherapies which exert many antitumor synergies. As we have observed previously antitumor targeted therapies by breaking oncogene addiction, they may optimize the action of immunotherapies by enhancing their sensitivity after circumvention of resistant immuno‐ suppressive mechanisms, leading to elimination of tumorigenic inflammation, enhancing long lived memory T-cell priming, activation, differentiation, function, and effective dendritic cell (DC) maturation, which trigger tumor cell senescence and eradication of tumor cells by induction of apoptosis or type I PCD leading to a bystander killing effect [73]. The derived apoptotic bodies release large quantities of multiple cancer-associated antigenic debris, which activate dendritic cell (DC) functioning as a vaccination in situ, leading to long-lasting remissions by combining the inhibition of oncogenic downstream signaling pathways, enhancing immunosensitivity after elimination of tumor-induced immunosuppressive mechanisms, which may lead to immunomodulatory effects, such as attenuation of the function of specific immunocomponents that block the action of cytotoxic T lymphocytes (CTLs), including myeloid-derived suppressor cells (MDSCs) and FOXP3+ regulatory T (Treg) cells. Other targeted antitumor agents may enhance the priming of tumor-specific CTLs and increase tumor antigen presentation by dendritic cells [74–76].

Thus, this complex interplay of targeted anticancer agents, and immunotherapy may sensitize tumor cells to immune-mediated eradication with long-lasting immunotherapeutic effects, which may inhibit induction of tumor dormancy [77–79].

it may circumvent the deadly risk of infection to cancer patients. More analytically, combina‐ torial immunotherapy may act synergistically by combining two different immunotherapeutic agents, such as inhibitors of immune checkpoints for preventing T-cell energy, and cancer vaccines for producing antitumor T cells. For instance, PD1 inhibitors or CTLA4 vaccines, such as autologous granulocyte macrophage colony-stimulating factor (GM-CSF) secreting tumor vaccine, may exert a significant synergistic antitumor action associated with higher overall survival rates by targeting multiple immunosuppressive pathways. Another combinatorial immune therapeutic approach consists of combining costimulatory receptors, which are overexpressed on activated T cells with agonistic antibodies, leading to enhancement of antitumor T-cell function, which eradicates tumors. Promising combinatorial immunothera‐ pies target synergistically the dual T-cell checkpoints, downregulating CTLA-4, PD-1, PD-L1, and LAG-3 with ipilimumab, tremelimumab, nivolumab, pembrolizumab, MEDI4736, and BMS-986016 against NSCLC, colon Ca, gastric Ca, SCLC, pancreatic Ca, melanoma, RCC, triple (-) breast Ca, and other solid tumors. Combinatorial immunotherapeutic regimens include Tcell inhibitors with costimulatory receptor agonists targeting CTLA-4 and CD40 with admin‐ istration of tremelimumab and CP-870,893 against metastatic melanoma. Another combinatorial regimen consists of T-cell inhibitors, and function enhancers of innate immune cells targeting CTLA-4, PD-1, and KIR with administration of lirilumab, ipilimumab, and nivolumab against solid tumors. Finally, T-cell inhibitors are combined with other activators of the immune system, such as vaccines and passive immunotherapeutics targeting CTLA-4, IL-21, PD-1, IDO with administration of denenicokin, ipilimumab, Nivolumab, INCB024360, indoximod, sipuleucel-T, nivolumab, gp100, NY-ESO-1, TriMix-DC, and adoptive cell transfer against melanoma, prostate Ca, and other solid tumors [67]. Currently, combinatorial thera‐ peutics may combine more than two agents, such as, immunotoxins, Fc-fusion proteins, and

Another even more efficient antitumor strategy consists of combining targeted therapies with immunotherapies which exert many antitumor synergies. As we have observed previously antitumor targeted therapies by breaking oncogene addiction, they may optimize the action of immunotherapies by enhancing their sensitivity after circumvention of resistant immuno‐ suppressive mechanisms, leading to elimination of tumorigenic inflammation, enhancing long lived memory T-cell priming, activation, differentiation, function, and effective dendritic cell (DC) maturation, which trigger tumor cell senescence and eradication of tumor cells by induction of apoptosis or type I PCD leading to a bystander killing effect [73]. The derived apoptotic bodies release large quantities of multiple cancer-associated antigenic debris, which activate dendritic cell (DC) functioning as a vaccination in situ, leading to long-lasting remissions by combining the inhibition of oncogenic downstream signaling pathways, enhancing immunosensitivity after elimination of tumor-induced immunosuppressive mechanisms, which may lead to immunomodulatory effects, such as attenuation of the function of specific immunocomponents that block the action of cytotoxic T lymphocytes (CTLs), including myeloid-derived suppressor cells (MDSCs) and FOXP3+ regulatory T (Treg) cells. Other targeted antitumor agents may enhance the priming of tumor-specific CTLs and

bispecific T-cell engagers (BiTEs) [69–72].

26 Immunopathology and Immunomodulation

increase tumor antigen presentation by dendritic cells [74–76].

Thus, we can combine targeted therapies with combinatorial immunotherapies, which consist of conventional immunotherapy, including administration of cytokines and/or chemokines, such as IL-7, IL-15, IL-21, adoptive T-cell transfusion with effector T cells, APC vaccination with dendritic cells (DCs), and tumor-associated antigens with tumor peptides combined with novel tumor immunotherapies, which target tumor-induced immunosuppressive molecules, circumventing tumor immunoresistance by inhibition of soluble suppressive molecules, such as TGFb, COX2, VEGF, and IL-10; suppressive molecules, such as PD1, and CTLA4 on T cells; and suppressive molecules, such as arginase, B7-H1, B7-H4, and IDO on APCs. They also target immunoresistant regulatory T cells by inhibition of trafficking with CCL22-specific antibody differentiation and signaling, such as FOXP3 signal, and depletion of T-reg cells with deni‐ leukin diftitox, cyclophosphamide, and CD25-specific antibody [80].

These combinatorial immunotherapies with targeted therapies can be used as neoadjuvants and adjuvant treatments with conventional anticancer strategies, such as surgical debulking, radiation therapy, and chemotherapy. For instance, immunotherapies such as indoximod, Denecikocin, CP-870,893, PF-05082566, urelumab, IMP321, pidilizumab, MEDI14763, MPDL3280A, pembrolizumab, tremelimumab, and nivolumab [67], which target CTLA-4, PD-1, PD-L1, LAG-3, CD137, CD40, IL-21, and IDO, have been combined with chemothera‐ peutic regimens or agents, such as FOLFOX, paclitaxel, cyclophosphamide, carboplatin, docetaxel, gemcitabine, etc., and molecular targeting agents, such as gefitinib, dasatinib, bevacizumab, erlotinib, sunitinib, pazopanid, lenalidomide, vemurafenib, trametinib, rituxi‐ mab, sorafenib, etc., against liquid tumors, such as CML, NHL, etc., and solid tumors including NSCLC, RCC, multiple myeloma, melanoma, pancreatic Ca, CRC, prostate Ca, breast Ca, etc.

In conventional anticancer treatment, the chemotherapeutic-induced immunosuppression inhibits the anticancer efficiency of cell therapies, which are based on activated lymphocytes for eradication of tumor cells enhancing susceptibility to infections [81].The majority of conventional chemotherapeutic agents interfere with hematopoiesis and subsequently with the immune system affecting the surveillance of cancer cells promoting tumor development and growth [82].

Generally, cancer surgery causes tremendous alterations in the neuroendocrine, metabolic, and immune systems constituting the stress response, which may lead to infection, and cancer recurrence due to release of catecholamines, cortisol, and cytokines that interfere with the adaptive or specific immunity, which is composed of humoral immunity that consists of B cells, and cellular immunity containing T-cytotoxic cells, T-suppressor cells, and T-helper cells, and the innate or nonspecific immunity. During the postoperative stage, there is balance between pro-inflammatory and anti-inflammatory cytokines. Deficient responses may cause immunosuppression leading to infections. Excessive responses may cause the systemic inflammatory response syndrome (SIRS), which has been associated with the clinical syn‐ drome of sepsis and multiorgan failure (MOF) or multiple organ dysfunction syndrome (MODS) [83].

The postoperative immune response is multifactorial with the release of inflammatory Th1 cytokines, such as IL-6 and TNF-a, and corticosteroids immediately after cancer surgery. Subsequently, even after 2 h from the surgical procedure, there is a reduction of the Th1 cytokines, while the Th2 cytokines, such as TGF-b, and IL-10 rise rapidly increasing the accumulation of immunosuppressive myeloid-derived suppressor cells, and immuneinhibitory cytokines [84]. This shift toward the Th2 immune response deregulates the cellular immunity, enhancing susceptibility of the cancer patient to infection, sepsis, and MOF [85– 87]. Furthermore, there is a quantitative reduction of T lymphocytes, which depends on the volume of blood loss during surgery. Also, there is a reduction in the number of white blood cells (WBCs) called leucopenia, which causes immunosuppression that combined with reduced cytokine secretion and suppression of T-lymphocyte responses, and reduced levels of macrophages may cause postoperative sepsis that may lead to morbidity. However, sepsis may be inhibited by postoperative release of anti-inflammatory cytokines, prostaglandins, and nitric oxide, which requires arginine as a substrate for its production by nitric oxide synthase [88]. Since plasma levels of arginine are reduced in septic patients, we need to establish a positive nitrogen balance by supplementation of arginine as an immunonutrition approach. This amino acid regulates blood flow by producing nitric oxide (NO), and it functions as an immunomodulator by enhancing the antitumor cytotoxicity of neutrophils and macrophages [89–92]. Furthermore, the proper antitumor function of T cells requires arginine. The tumor microenvironment contains nitric oxide synthase (NOS) and arginase I, which are upregulated by tumor-induced MDSC, acting as an immunosuppressive mechanism that leads to a deficiency of arginine, which subsequently suppresses the antigen-specific T-cell responses by downregulating the T-cell receptor [93,94]. Within a few hours after cancer surgery, there is an evident reduction of arginine in the circulation of the cancer patient [95,96] because arginine is metabolized by arginase-I, which may be downregulated by omega-3 fatty acids that are metabolized to PGE3, inhibiting production of immunosuppressive Th2 cytokine, and increasing the production of protectins and resolvins, which promote tissue repair [97]. Immunonutrition in the surgical cancer patient with arginine may improve trauma healing, enhance macrophage function, and lymphocyte immune responses enhancing resistance to infection at the postoperative stage [98]. A functional immune system is required for protecting the surgical cancer patient from the high risk of postoperative infections, which can be achieved by perioperative immunomodulating formulations that can circumvent postoperative immunoparesis and prevent sepsis by activating the immune cell responses, and modulating inflammation.

Other protective perioperative practices include minimally invasive surgical procedures, circumvention of immunosuppressive drugs, and reduction of blood transfusions [99]. Radical surgery combined with old-age neuroendocrine response and administration of analgesics may suppress the activity of the innate immunity and specifically NK cells, which leads to tumor progression since tumor cells circumvent tumor immunosurveillance and subsequent cytolysis [100–104]. In addition, operative anesthetics, such as halothane, thiopental, and ketamine, may suppress even further the activity of NK cells promoting metastasis. Thus, immunonutrition may stimulate the immunity, while other factors such as hypothermia, alcohol, and mainly stress may enhance tumor progression [105].

Supplementation with polysaccharides or glutamine may increase natural killer (NK) cell activity [106]. A requirement for a functional anticancer immunity includes a balanced Th1/Th2 ratio because after surgery, a dominant Th2-type immune response, especially in tumors of the gastrointestinal (GI) tract, may suppress tumor surveillance and cellular immunity [107]. Other immunosuppressive and inflammatory factors such as IL-6 and immunosuppressive acidic protein (IAP) may reduce the antitumor activity of cellular immunity leading to tumor progression.

Other immunosuppressive factors include IL-10, TGF-b, and angiogenic VEGF, which is regulated by CD47 signaling that suppresses activity of T cells promoting tumor growth [108]. Thus, after oncological surgery, we must help the patient to maintain homeostasis against the consequences of cancer, tissular attrition, hormonal and metabolic changes, and mainly inflammatory reaction, which induces metastases by a cascade of genomic signaling pathways that may lead to angiogenesis, which is associated to a potent immunosuppression [109,110]. In addition to surgery, other conventional cancer treatments, such as chemotherapy and radiation therapy, may suppress the immune system of the cancer patient by causing a tremendous reduction in the production of all the cells of the bone marrow leading to leuco‐ penia and anemia, which may lead to severe infections. Specifically, if a neutrophil count is below 1000, the risk of infection by bacteria, germs, and fungi is increased, which becomes worse if the count is below 500 where we have neutropenia. This may be treated with admin‐ istration of colony stimulating factors (CSF) or white blood cell (Leukocyte) growth factors. With bone radiation for metastatic tumors, leucopenia and even neutropenia may be caused by chemotherapy. Furthermore, local radiation therapy can irritate the skin causing small breaks from which germs and bacteria may enter causing infections. Also when lymph nodes are irradiated, infection may occur, which leads to lymphedema. Moreover, there are many tumor-induced immunosuppressive mechanisms, which have been described previously that act synergistically.
