CD8+ cytotoxic T lymphocytes in cancer immunotherapy: A review

CD8⁺ cytotoxic T lymphocytes (CTLs) are preferred immune cells for targeting cancer. During cancer progression, CTLs encounter dysfunction and exhaustion due to immunerelated tolerance and immunosuppression within the tumor microenvironment (TME), with all favor adaptive immune-resistance. Cancer-associated fibroblasts (CAFs), macrophage type 2 (M2) cells, and regulatory T cells (Tregs) could make immunologic barriers against CD8 ⁺ T cell-mediated antitumor immune responses. Thus, CD8 ⁺ T cells are needed to be primed and activated toward effector CTLs in a process called tumor immunity cycle for making durable and efficient antitumor immune responses. The CD8 ⁺ T cell priming is directed essentially as a corroboration work between cells of innate immunity including dendritic cells (DCs) and natural killer (NK) cells with CD4 ⁺ T cells in adoptive immunity. Upon activation, effector CTLs infiltrate to the core or invading site of the tumor (so-called infiltrated–inflamed [I–I] TME) and take essential roles for killing cancer cells. Exogenous reactivation and/or priming of CD8 ⁺ T cells can be possible using rational immunotherapy strategies. The increase of the ratio for costimulatory to coinhibitory mediators using immune checkpoint blockade (ICB) approach. Programmed death-1 receptor (PD-1)–ligand (PD-L1) and CTL-associated antigen 4 (CTLA-4) are checkpoint receptors that can be targeted for relieving exhaustion of CD8 ⁺ T cells and renewing their priming, respectively, and thereby eliminating antigen-expressing cancer cells. Due to a diverse relation between CTLs with Tregs, the Treg activity could be dampened for increasing the number and rescuing the functional potential of CTLs to induce immunosensitivity of cancer cells.     

Activated mouse CD4+Foxp3? T cells facilitate melanoma metastasis via Qa-1-dependent suppression of NK-cell cytotoxicity

The regulatory activities of mouse CD4⁺Foxp3⁺ T cells on various immune cells, including NK cells, have been well documented. Under some conditions, conventional CD4⁺Foxp3⁻ T cells in the periphery are able to acquire inhibitory function on other T cells, but their roles in controlling innate immune cells are poorly defined. As a potential cellular therapy for cancer, ex vivo activated CD4⁺Foxp3⁻ effector T cells are often infused back in vivo to suppress tumor growth and metastasis. Whether such activated T cells could affect NK-cell control of tumorigenesis is unclear. In the present study, we found that mitogen-activated CD4⁺Foxp3⁻ T cells exhibited potent suppressor function on NK-cell proliferation and cytotoxicity in vitro, and notably facilitated B16 melanoma metastasis in vivo. Suppression of NK cells by activated CD4⁺Foxp3⁻ T cells is cell-cell contact dependent and is mediated by Qa-1:NKG2A interaction, as administration of antibodies blocking either Qa-1 or NKG2A could completely reverse this suppression, and significantly inhibited otherwise facilitated melanoma metastasis. Moreover, activated CD4⁺Foxp3⁻ cells from Qa-1 knockout mice completely lost the suppressor activity on NK cells, and failed to facilitate melanoma metastasis when transferred in vivo. Taken together, our findings indicate that innate anti-tumor response is counter regulated by the activation of adaptive immunity, a phenomenon we term as “activation-induced inhibition”. Thus, the regulatory role of activated CD4⁺Foxp3⁻ T cells in NK-cell activity must be taken into consideration in the future design of cancer therapies.     

Identification and characterization of a tumor infiltrating CD56+/CD16− NK cell subset with specificity for pancreatic and prostate cancer cell lines

In a recent clinical trial, a patient exhibited regression of several pancreatic cancer metastases following the administration of the immune modulator Ipilimumab (anti-CTLA-4 antibody). We sought to characterize the immune cells responsible for this regression. Tumor infiltrating lymphocytes (TIL-2742) and an autologous tumor line (TC-2742) were expanded from a regressing metastatic lesion excised from this patient. Natural killer (NK) cells predominated in the TIL (92% CD56⁺) with few T cells (12% CD3⁺). A majority (88%) of the NK cells were CD56^brightCD16⁻. TIL-2742 secreted IFN-γ and GM-CSF following co-culture with TC-2742 and major histocompatibility complex mismatched pancreatic tumor lines. After sorting TIL-2742, the purified CD56⁺CD16⁻CD3⁻ subset showed reactivity similar to TIL-2742 while the CD56⁻CD16⁻CD3⁺ cells exhibited no tumor recognition. In co-culture assays, TIL-2742 and the NK subset expressed high reactivity to several pancreatic and prostate cancer cell lines and could lyse the autologous tumor as well as pancreas and prostate cancer lines. Reactivity was partially abrogated by blockade of TRAIL. We thus identified a unique subset of NK cells (CD56^brightCD16^dim) isolated from a regressing metastatic pancreatic cancer in a patient responding to Ipilimumab. This represents the first report of CD56⁺CD16⁻ NK cells with apparent specificity for pancreatic and prostate cancer cell lines and associated with tumor regression following the treatment with an immune modulating agent.     

Human T Cell Crosstalk Is Induced by Tumor Membrane Transfer

Trogocytosis is a contact-dependent unidirectional transfer of membrane fragments between immune effector cells and their targets, initially detected in T cells following interaction with professional antigen presenting cells (APC). Previously, we have demonstrated that trogocytosis also takes place between melanoma-specific cytotoxic T lymphocytes (CTLs) and their cognate tumors. In the present study, we took this finding a step further, focusing on the ability of melanoma membrane-imprinted CD8⁺ T cells to act as APCs (CD8⁺T-APCs). We demonstrate that, following trogocytosis, CD8⁺T-APCs directly present a variety of melanoma derived peptides to fraternal T cells with the same TCR specificity or to T cells with different TCRs. The resulting T cell-T cell immune synapse leads to (1) Activation of effector CTLs, as determined by proliferation, cytokine secretion and degranulation; (2) Fratricide (killing) of CD8⁺T-APCs by the activated CTLs. Thus, trogocytosis enables cross-reactivity among CD8⁺ T cells with interchanging roles of effectors and APCs. This dual function of tumor-reactive CTLs may hint at their ability to amplify or restrict reactivity against the tumor and participate in modulation of the anti-cancer immune response.     

Immunotherapy with dendritic cells pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8+ cytotoxic T lymphocytes and natural killer cells

Background and purpose Immunization with heat shock proteins, gp96, elicits specific protective immunity against parent tumors. However, it is marginally effective as a therapeutic tool against established tumors. In the present study, we evaluated the efficacy and mechanism of immunotherapy with bone marrow-derived dendritic cells (DCs) pulsed with tumor-derived gp96 against murine lung cancer. Methods Mice were transplanted subcutaneously with ovalbumin (OVA)-transfected Lewis Lung Cancer (LLC-OVA) cells and immunized with gp96 derived from LLC-OVA, DCs, or DCs pulsed with gp96 derived from LLC-OVA. Results The antitumor effect was significantly enhanced in the mice immunized with DCs pulsed with gp96 derived from LLC-OVA, compared to mice immunized with gp96 or DCs (P < 0.05). The antitumor effect was significantly dependent on natural killer (NK) cells and CD8⁺ cells and partially dependent on CD4⁺ cells. Analysis by laser confocal microscopy demonstrated that gp96 was shown on the cell surface at 15 min, and after 30 min internalized in the endosomes and not in the endoplasmic reticulum or lysosomes. OVA-specific⁺ CD8⁺ cells were more readily recruited into the draining lymph nodes and higher CD8⁺ cytotoxic T cell activity against LLC-OVA was observed in splenocytes from mice immunized with DCs pulsed with gp96 derived from LLC-OVA. Re-challenge of the surviving mice with LLC-OVA tumors after the initial tumor inoculation showed dramatic retardation in tumor growth. Conclusion In conclusion, immunotherapy of DCs pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8⁺ cytotoxic T lymphocytes and NK cells.     

Neuropilin-1 Expression Is Induced on Tolerant Self-Reactive CD8+ T Cells but Is Dispensable for the Tolerant Phenotype

Establishing peripheral CD8⁺ T cell tolerance is vital to avoid immune mediated destruction of healthy self-tissues. However, it also poses a major impediment to tumor immunity since tumors are derived from self-tissue and often induce T cell tolerance and dysfunction. Thus, understanding the mechanisms that regulate T cell tolerance versus immunity has important implications for human health. Signals received from the tissue environment largely dictate whether responding T cells become activated or tolerant. For example, induced expression and subsequent ligation of negative regulatory receptors on the surface of self-reactive CD8⁺ T cells are integral in the induction of tolerance. We utilized a murine model of T cell tolerance to more completely define the molecules involved in this process. We discovered that, in addition to other known regulatory receptors, tolerant self-reactive CD8⁺ T cells distinctly expressed the surface receptor neuropilin-1 (Nrp1). Nrp1 was highly induced in response to self-antigen, but only modestly when the same antigen was encountered under immune conditions, suggesting a possible mechanistic link to T cell tolerance. We also observed a similar Nrp1 expression profile on human tumor infiltrating CD4⁺ and CD8⁺ T cells. Despite high expression on tolerant CD8⁺ T cells, our studies revealed that Nrp1 had no detectable role in the tolerant phenotype. Specifically, Nrp1-deficient T cells displayed the same functional defects as wild-type self-reactive T cells, lacking in vivo cytolytic potential, IFNγ production, and antitumor responses. While reporting mostly negative data, our findings have therapeutic implications, as Nrp1 is now being targeted for human cancer therapy in clinical trials, but the precise molecular pathways and immune cells being engaged during treatment remain incompletely defined.     

Manganese is critical for antitumor immune responses via cGAS-STING and improves the efficacy of clinical immunotherapy

CD8⁺ T cell-mediated cancer clearance is often suppressed by the interaction between inhibitory molecules like PD-1 and PD-L1, an interaction acts like brakes to prevent T cell overreaction under normal conditions but is exploited by tumor cells to escape the immune surveillance. Immune checkpoint inhibitors have revolutionized cancer therapeutics by removing such brakes. Unfortunately, only a minority of cancer patients respond to immunotherapies presumably due to inadequate immunity. Antitumor immunity depends on the activation of the cGAS-STING pathway, as STING-deficient mice fail to stimulate tumor-infiltrating dendritic cells (DCs) to activate CD8⁺ T cells. STING agonists also enhance natural killer (NK) cells to mediate the clearance of CD8⁺ T cell-resistant tumors. Therefore STING agonists have been intensively sought after. We previously discovered that manganese (Mn) is indispensable for the host defense against cytosolic dsDNA by activating cGAS-STING. Here we report that Mn is also essential in innate immune sensing of tumors and enhances adaptive immune responses against tumors. Mn-insufficient mice had significantly enhanced tumor growth and metastasis, with greatly reduced tumor-infiltrating CD8⁺ T cells. Mechanically, Mn²⁺ promoted DC and macrophage maturation and tumor-specific antigen presentation, augmented CD8⁺ T cell differentiation, activation and NK cell activation, and increased memory CD8⁺ T cells. Combining Mn²⁺ with immune checkpoint inhibition synergistically boosted antitumor efficacies and reduced the anti-PD-1 antibody dosage required in mice. Importantly, a completed phase 1 clinical trial with the combined regimen of Mn²⁺ and anti-PD-1 antibody showed promising efficacy, exhibiting type I IFN induction, manageable safety and revived responses to immunotherapy in most patients with advanced metastatic solid tumors. We propose that this combination strategy warrants further clinical translation.Subject terms: Pattern recognition receptors, Immunosurveillance     

Expansion of CD11b+Ly6G+Ly6Cint cells driven by medroxyprogesterone acetate in mice bearing breast tumors restrains NK cell effector functions

The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b⁺Gr-1⁺, mostly CD11b⁺Ly6G⁺Ly6C^int and CD11b⁺Ly6G^?Ly6C^high cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b⁺Ly6G⁺Ly6C^int cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b⁺Ly6G⁺Ly6C^int cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b⁺Gr-1⁺ cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b⁺Gr-1⁺ cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b⁺Ly6G⁺Ly6C^int cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.     

Prostate Tumor-Derived Exosomes Down-Regulate NKG2D Expression on Natural Killer Cells and CD8+ T Cells: Mechanism of Immune Evasion

Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC) progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK) and CD8⁺ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8⁺ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8⁺ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape.     

Active hexose correlated compound enhances tumor surveillance through regulating both innate and adaptive immune responses

Active hexose correlated compound (AHCC) is a mixture of polysaccharides, amino acids, lipids and minerals derived from cocultured mycelia of several species of Basidiomycete mushrooms. AHCC has been implicated to modulate immune functions and plays a protective role against infection. However, the potential role of AHCC in tumor immune surveillance is unknown. In this study, C57BL/6 mice were orally administered AHCC or water, followed by tumor cell inoculation. We showed that compared to pure water-treated mice, AHCC treatment significantly delayed tumor development after inoculation of either melanoma cell line B16F0 or lymphoma cell line EL4. Treatment with AHCC enhanced both Ag-specific activation and proliferation of CD4⁺ and CD8⁺ T cells, increased the number of tumor Ag-specific CD8⁺ T cells, and more importantly, increased the frequency of tumor Ag-specific IFN-γ producing CD8⁺ T cells. Interestingly, AHCC treatment also showed increased cell number of NK and γδ T cells, indicating the role of AHCC in activating these innate-like lymphocytes. In summary, our results demonstrate that AHCC can enhance tumor immune surveillance through regulating both innate and adaptive immune responses.     

in vitro generation of IFN-γ-producing Listeria-specific T cells is dependent on IFN-γ production by non-NK cells

In vitro 5-day cultures of naive spleen cells with viable Listeria monocytogenes (VLM), but not heat-killed L. monocytogenes, induced CD4⁺ T cells that produced IFN-γ upon secondary antigen stimulation. The VLM-induced Listeria-specific T cells produced IFN-γ but lacked expression of IL-2 and IL-4. To study the role of IFN-γ in the induction of the IFN-γ-producing T cells, we added anti-IFN-γ mAb to the primary culture and analyzed IFN-γ production upon secondary antigen stimulation. Addition of anti-IFN-γ mAb to the culture suppressed generation of IFN-γ-producing CD4⁺ T cells, suggesting that IFN-γ is important in the induction of IFN-γ-producing CD4⁺ T cells. Furthermore, our results showed that depletion of NK cells from spleen cells by anti-asialo GM1 antibody plus complement before culture enhanced induction of IFN-γ-producing CD4⁺ T cells. Although NK cells are known to produce IFN-γ, the results indicate that NK cell-derived IFN-γ may not be important in induction of the Listeria-specific IFN-γ-producing CD4⁺ T cells in the culture system. In addition, we demonstrated that IFN-γ expression was high in CD4⁺ T cells from cultures of spleen cells with VLM at the primary culture level. These results suggest that IFN-γ derived from T cells may enhance production of IFN-γ by CD4⁺ T cells, while NK cells rather suppress the induction of IFN-γ-producing CD4⁺ T cells.     

Enhanced Antitumor Immunity Contributes to the Radio-Sensitization of Ehrlich Ascites Tumor by the Glycolytic Inhibitor 2-Deoxy-D-Glucose in Mice

Two-deoxy-D-glucose (2-DG), an inhibitor of glycolysis differentially enhances the radiation and chemotherapeutic drug induced cell death in cancer cells in vitro, while the local tumor control (tumor regression) following systemic administration of 2-DG and focal irradiation of the tumor results in both complete (cure) and partial response in a fraction of the tumor bearing mice. In the present studies, we investigated the effects of systemically administered 2-DG and focal irradiation of the tumor on the immune system in Ehrlich ascites tumor (EAT) bearing Strain “A” mice. Markers of different immune cells were analyzed by immune-flow cytometry and secretary cytokines by ELISA, besides monitoring tumor growth. Increase in the expression of innate (NK and monocytes) and adaptive CD4⁺cells, and a decrease in B cells (CD19) have been observed after the combined treatment, suggestive of activation of anti-tumor immune response. Interestingly, immature dendritic cells were found to be down regulated, while their functional markers CD86 and MHC II were up regulated in the remaining dendritic cells following the combination treatment. Similarly, decrease in the CD4⁺ naïve cells with concomitant increase in activated CD4⁺ cells corroborated the immune activation. Further, a shift from Th2 and Th17 to Th1 besides a decrease in inflammatory cytokines was also observed in the animals showing complete response (cure; tumor free survival). This shift was also complimented by respective antibody class switching followed by the combined treatment. The immune activation or alteration in the homeostasis favoring antitumor immune response may be due to depletion in T regulatory cells (CD4⁺CD25⁺FoxP3⁺). Altogether, these results suggest that early differential immune activation is responsible for the heterogenous response to the combined treatment. Taken together, these studies for the first time provided insight into the additional mechanisms underlying radio-sensitization by 2-DG in vivo by unraveling its potential as an immune-modulator besides direct effects on the tumor.     

A simple but effective cancer vaccine consisting of an antigen and a cationic lipid

Developing a cancer vaccine with a potent adjuvant, which is safe for human use, remains to be an unmet need. In this study, we developed a simple, safe, yet efficient, peptide-based therapeutic cancer vaccine, DOTAP/E7 complex, which comprises only two molecules: a DOTAP cationic lipid and a peptide antigen derived from E7 oncoprotein of human papillomavirus (HPV) type 16. The anti-cancer activity of DOTAP/E7 against existing HPV positive TC-1 tumor was compared to that of our previous LPD/E7 formulation, which contains bacterial DNA CpG motifs. Tumor-bearing mice showed significant tumor inhibition following a single vaccination of either formulation at the optimal lipid dose, suggesting that DOTAP liposome alone can provide a potent adjuvant activity without plasmid DNA. E7 peptide formulated with DOTAP induced migration of activated dendritic cells (DC) to the draining lymph node (DLN) and efficiently generated functional antigen-specific CD8⁺ T lymphocyte responses. Accumulation of CD8⁺ tumor infiltrating T cells and apoptosis at tumor sites were observed after treatment with DOTAP/E7 complexes, which was also associated with a decreased amount of CD25⁺Foxp3⁺ regulatory T cells in treated animals. Reactive oxygen species (ROS) induced by DOTAP cationic lipid in DLN revealed a plausible mechanism of the initial interaction between DC and DOTAP. An adequate amount of ROS generation was apparently required for the initiation of the vaccine mechanism; however, an overdose of DOTAP induced massive ROS production and apoptosis of DC in DLN, which led to diminished anti-cancer immunity. Overall, these results indicate that cationic lipid DOTAP alone serves as an efficient vaccine adjuvant for the induction of a therapeutic, antigen-specific anti-cancer activity.     

Eradication of hepatoma and colon cancer in mice with Flt3L gene therapy in combination with 5-FU

We developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter to investigate the biological efficacy of Flt3L in combination with chemotherapeutical drug, 5-FU, in eliciting an effective anti-cancer immunity in mouse hepatoma and colon cancer model systems. The constructed Ad-mFlt3L efficiently infected hepatoma and colon cancer cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCs, NK cells and lymphocytes in local tumor tissues. Administration of Ad-mFlt3L can protect bone marrow injury caused by 5-Fu and stimulates proliferation and maturation of lymphocytes, APCs and NKs. Intratumoral injection of Ad-mFlt3L followed by an intraperitoneal administration of 5-Fu significantly inhibited tumor growth and cured established tumors. Adenovirus mediated Flt3L gene therapy synergies with chemotherapeutic drug, 5-Fu, in elicitation of long-lasting antitumor immunity. The tumor specific immunity can be adoptively transferred into naïve animals successfully by transfusion of CD3⁺CD8⁺ T cells from the treated mice. The data suggests that adenovirus mediated Flt3L gene therapy in combination with 5-Fu chemotherapy may open a new avenue for development of anti-cancer chemogenetherapy.     

GMP-Compliant,Large-Scale Expanded Allogeneic Natural Killer Cells Have Potent Cytolytic Activity against Cancer Cells In Vitro and In Vivo

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3⁺ T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3⁻CD16⁺CD56⁺ NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.     

Structurally characterized arabinogalactan from Anoectochilus formosanus as an immuno-modulator against CT26 colon cancer in BALB/c mice

In this study, the innate immuno-modulatory effects and anti-cancer action of arabinogalactan (AG), a derivative of a well-known orchid, Anoectochilus formosanus, were investigated. The innate immuno-modulatory effects of AG were determined in vitro using RAW 264.7 cells for microarray analysis, and in vivo using BALB/c mice administrated with AG at 5 and 15 mg/kg intra-peritoneally for 3 weeks. The anti-cancer activity of AG was evaluated by CT26 colon cancer-bearing BALB/c mice. The microarray analysis was performed to evaluate the innate immunity and demonstrated that AG significantly induced the expression of cytokines, chemokines, and co-stimulatory receptors, such as IL-1α, CXCL2, and CD69. An intraperitoneal injection of AG in mice increased the spleen weight, but not the body weight. The treatment of mitogen, LPS significantly stimulated splenocyte proliferation in AG treated groups. The AG treatment also promoted splenocyte cytotoxicity against YAC-1 cells and increased the percentage of CD3⁺CD8⁺ cytotoxic T cells in innate immunity test. Our experiments revealed that AG significantly decreased both tumour size and tumour weight. Besides, AG increased the percentage of DC, CD3⁺CD8⁺ T cells, CD49b⁺CD3⁻ NK cells among splenocytes, and cytotoxicity activity in tumour-bearing mice. In addition, the immunohistochemistry of the tumour demonstrated that the AG treatments increased the tumour-filtrating NK and cytotoxic T-cell. These results demonstrated that AG, a polysaccharide derived from a plant source, has potent innate immuno-modulatory and anti-cancer activity. AG may therefore be used for cancer immunotherapy.     

Intratumor IL-17-Positive Mast Cells Are the Major Source of the IL-17 That Is Predictive of Survival in Gastric Cancer Patients

Interleukin-17 (IL-17) is prevalent in tumor tissue and suppresses effective anti-tumor immune responses. However, the source of the increased tumor-infiltrating IL-17 and its contribution to tumor progression in human gastric cancer remain poorly understood. In this study, we enrolled 112 gastric cancer patients, immunofluorescence was used to evaluate the colocalization of CD3, CD4, CD56, CD20, CD68, and mast cell tryptase (MCT) with IL-17. Immunohistochemistry was used to evaluate the distribution of microvessel density (CD34), CD66b⁺, CD68⁺, and FoxP3⁺ cells in different microanatomical areas. Prognostic value was determined by Kaplan-Meier analysis and a Cox regression model. The results showed that mast cells, but not T cells or macrophages, were the predominant cell type producing IL-17 in gastric cancer. Significant positive correlations were detected between densities of mast cell-derived IL-17 and microvessels, neutrophils, and regulatory T cells (Tregs). Futhermore, we found that the majority of vascular endothelial cells expressing Interleukin-17 receptor (IL-17R). Kaplan-Meier analysis revealed that increasing intratumor infiltrated mast cells and IL-17⁺ cells, as well as MCT⁺ IL-17⁺ cells, were significantly associated with worse overall survival. These findings indicated that mast cells were the major source of IL-17 in gastric cancer, and intratumor IL-17 infiltration may have promoted tumor progression by enhancing angiogenesis in the tumor microenvironment through the axis of IL-17/IL-17R. IL-17-positive mast cells showed a prognostic factor in gastric cancer, indicating that immunotherapy targeting mast cells might be an effective strategy to control intratumor IL-17 infiltration, and consequently reverse immunosuppression in the tumor microenvironment, facilitating cancer immunotherapy.     

Immune cell phenotype and functional defects in Netherton syndrome

Background

Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete.

Results

We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17?years, and healthy age-matched controls. The proportion of B cells (CD19⁺) and naïve B cells (CD27^?, IgD⁺) were high while memory B cells (CD27⁺) and switched memory B cells (CD27⁺IgM^?IgD^?), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21^low, CD38l^ow) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4⁺ T cells was reduced significantly and the proportion of CD8⁺ T central memory significantly elevated. An increased proportion of CD57⁺ CD8⁺ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16⁺ and CD27^? NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity.The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L⁺ T cells, naïve CD4⁺ and CD27⁺ CD8⁺ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8⁺ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied.

Conclusions

This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.

     

γc cytokine-aided crosstalk between dendritic cells and natural killer cells together with doxorubicin induces a healer response in experimental lymphoma by downregulating FOXP3 and programmed cell death protein 1

Background aimsThe stimulatory natural killer–dendritic cell axis in the tumor microenvironment could play a critical role in stimulating cytotoxic T cells and driving immune responses against cancer.MethodsWe established a novel treatment protocol by adroitly combining chemotherapy with doxorubicin and immunotherapy with dendritic cells and natural killer cells against a highly aggressive and malignant lymphoma called Dalton's lymphoma.ResultsOur data suggest that binary application of adoptive cell therapy and chemotherapy nearly cures (95%) early-stage experimental lymphoma. In the case of mid-stage cancer, the success rate was significantly lower but still impressive (75%). Our results demonstrated that the application of combination therapy in early-stage cancer significantly reduced the tumor volume and extended the lifespan of the experimental animal in addition to reinvigorating the immune system, including restoring the effector functions of dendritic cells and natural killer cells. The novel protocol limits the metastasis of tumor cells in vascularized organs and rearms the adaptive immune response mediated by dendritic cells and CD4⁺ and CD8⁺ T cells.ConclusionsCombination therapy in the early stage alters the cytokine profile, increases interferon-γ and tumor necrosis factor-α in the serum of treated animals and downregulates programmed cell death protein 1 expression in CD8⁺ T cells. Thus, cooperative and cognitive interactions between dendritic cells and natural killer cells in addition to therapy with doxorubicin promote the immune response and tumoricidal activities against lymphoma.