Search

CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in anti-tumor immunity. In a previous study, we identified a subset of murine effector CTLs expressing the hepatocyte growth factor (HGF) receptor, c-Met (c-Met+ CTLs), that are endowed with enhanced cytolytic capacity. HGF directly inhibited the cytolytic function of c-Met+ CTLs, both in 2D in vitro assays and in vivo, leading to reduced T cell responses against metastatic melanoma. To further investigate the role of c-Met+ CTLs in a three-dimensional (3D) setting, we studied their function within B16 melanoma spheroids and examined the impact of cell–cell contact on the modulation of inhibitory checkpoint molecules' expression, such as KLRG1, PD-1, and CTLA-4. Additionally, we evaluated the cytolytic capacity of human CTL clones expressing c-Met (c-Met+) and compared it to c-Met− CTL clones. Our results indicated that, similar to their murine counterparts, c-Met+ human CTL clones exhibited increased cytolytic activity compared to c-Met− CTL clones, and this enhanced function was negatively regulated by the presence of HGF. Taken together, our findings highlight the potential of targeting the HGF/c-Met pathway to modulate CTL-mediated anti-tumor immunity. This research holds promise for developing strategies to enhance the effectiveness of CTL-based immunotherapies against cancer. [ABSTRACT FROM AUTHOR]

CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T (Th) cells play a critical role in protective immune responses to tumor cells. Particularly, Th9 cells exert anti-tumor activity by producing IL-9. TNF receptor (TNFR)-associated factor 6 (TRAF6) is an adaptor protein that mediates the signals from both the TNFR superfamily and Toll-like receptors (TLRs). We have previously reported that T cell-specific TRAF6-deficent (TRAF6ΔT) mice spontaneously developed systemic inflammatory diseases. However, the physiological role of TRAF6 in T cells in controlling anti-tumor immune responses remains largely unclear. Here, we found that tumor formation of syngeneic colon cancer cells inoculated in TRAF6ΔT mice was accelerated compared to that in control mice. Although TRAF6-deficient naïve T cells showed enhanced differentiation of Th9 cells in vitro , these T cells produced lower amounts of IL-9 in response to a specific antigen. Moreover, CD4+ tumor-infiltrating lymphocytes (TILs) in tumor-bearing TRAF6ΔT mice expressed lower levels of IL-9 than those in WT mice. Importantly, administration of recombinant IL-9 (rIL-9) strongly suppressed tumor progression in TRAF6ΔT mice. Furthermore, expression levels of the T-box transcription factor Eomesodermin (Eomes) and its target molecules IFN-γ, granzyme B and perforin, as well as cytotoxic activity, were reduced in TRAF6-deficient CD8+ T cells in vitro. TRAF6-deficient T cells were found to express significantly increased levels of immune checkpoint molecules, CTLA-4 and PD-1 on the cell surface. These results demonstrate that the TRAF6 signaling pathway in T cells regulates anti-tumor immunity through the activation of tumor specific Th9 cells and CTLs in a tumor microenvironment. • Tumor growth inoculated in T cell-specific TRAF6-deficent mice is accelerated. • TRAF6 is required for optimal production of IL-9 by Th9 cells responding to antigen. • TRAF6 is required for cytotoxic activity of CTLs responding to antigen. • Eomes and its target molecules IFN-γ, granzyme B and perforin are regulated by TRAF6. • Immune checkpoint molecules CTLA-4, PD-1 are negatively regulated by TRAF6 in T cell. [ABSTRACT FROM AUTHOR]

Purpose: CD8 cytotoxic T cells (CTLs) play a critical role in the clearance of virally infected cells. SARS-CoV-2-specific CD8 T cells and functional CTLs in natural infections and following COVID-19 vaccine in primary antibody deficiency (PAD) have not been reported. In this study, we evaluated T cell response following COVID-19 or COVID-19 mRNA vaccination in patients with PADs by assessing SARS-CoV-2 tetramer-positive CD8 T cells and functional CTLs. Methods: SARS-CoV-2-specific CD8 and functional CTLs were examined in a patient with X-linked agammaglobulinemia (XLA) and a patient with common variable immunodeficiency (CVID) following COVID-19 infection, and in 5 patients with CVID and 5 healthy controls 1 month following 2nd dose of COVID-19 mRNA vaccine (Pfizer-BioNTech). Cells were stained with SARS-CoV-2 spike protein–specific tetramers, and for functional CTLs (CD8+ CD107a+ granzyme B+ perforin+), with monoclonal antibodies and isotype controls and analyzed by flow cytometry. Results: SARS-CoV-2-specific tetramer + CD8 T cells and functional CTLs in the patient with XLA following COVID-19 infection were higher, as compared to healthy control subject following COVID-19 infection. On the other hand, SARS-CoV2-tetramer + CD8 T cells and functional CTLs were lower in CVID patient following COVID19 infection as compared to healthy control following COVID-19 infection. SARS-CoV2-tetramer + CD8 T cells and functional CTLs were significantly lower in SARS-CoV2-naive CVID patients (n = 10) following vaccination when compared to SARS-CoV-2-naive healthy vaccinated controls (n = 10). Conclusions: CVID is associated with reduced SARS-CoV-2-specific CD8 T cells and functional CTLs in both natural SARS-CoV-2 infection and in response to SARS-CoV-2 mRNA vaccine, whereas natural infection in XLA is associated with a robust SARS-CoV-2-specific CD8 and functional CTL responses. [ABSTRACT FROM AUTHOR]

TNF-related apoptosis inducing ligand (TRAIL) is expressed on cytotoxic T lymphocytes (CTLs) and TRAIL is linked to progression of diabetes. However, the impact of high glucose on TRAIL expression and its related killing function in CTLs still remains largely elusive. Here, we report that TRAIL is substantially up-regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. Non-mitochondrial reactive oxygen species, NFκB and PI3K/Akt are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Treatment with metformin and vitamin D reduces HG-enhanced expression of TRAIL in CTLs and coherently protects 1.4E7 cells from TRAIL-mediated apoptosis. Our work suggests that HG-induced TRAILhigh CTLs might contribute to the destruction of pancreatic beta cells in a hyperglycemia condition. [ABSTRACT FROM AUTHOR]

Metabolic and nutrient-sensing pathways play an important role in controlling the efficacy of effector T cells. Oxygen is a critical regulator of cellular metabolism. However, during immune responses T cells must function in oxygen-deficient, or hypoxic, environments. Here, we used high resolution mass spectrometry to investigate how the proteome of primary murine CD8+ cytotoxic T lymphocytes (CTLs) is reconfigured in response to hypoxia in vitro. We identified and quantified over 7,600 proteins and discovered that hypoxia increased the abundance of a selected number of proteins in CTLs. This included glucose transporters, metabolic enzymes, transcription factors, cytolytic effector molecules, checkpoint receptors and adhesion molecules. While some of these proteins may augment the effector functions of CTLs, others may limit their cytotoxicity. Moreover, we determined that hypoxia could inhibit IL-2-induced proliferation cues and antigen-induced pro-inflammatory cytokine production in CTLs. These data provide a comprehensive resource for understanding the magnitude of the CTL response to hypoxia and emphasise the importance of oxygen-sensing pathways for controlling CD8+ T cells. Additionally, this study provides new understanding about how hypoxia may promote the effector function of CTLs, while contributing to their dysfunction in some contexts. [ABSTRACT FROM AUTHOR]

Efficacy of cytotoxic T lymphocyte (CTL)-based immunotherapy is still unsatisfactory against solid tumors, which are frequently characterized by condensed extracellular matrix. Here, using a unique 3D killing assay, we identify that the killing efficiency of primary human CTLs is substantially impaired in dense collagen matrices. Although the expression of cytotoxic proteins in CTLs remained intact in dense collagen, CTL motility was largely compromised. Using light-sheet microscopy, we found that persistence and velocity of CTL migration was influenced by the stiffness and porosity of the 3D matrix. Notably, 3D CTL velocity was strongly correlated with their nuclear deformability, which was enhanced by disruption of the microtubule network especially in dense matrices. Concomitantly, CTL migration, search efficiency, and killing efficiency in dense collagen were significantly increased in microtubule-perturbed CTLs. In addition, the chemotherapeutically used microtubule inhibitor vinblastine drastically enhanced CTL killing efficiency in dense collagen. Together, our findings suggest targeting the microtubule network as a promising strategy to enhance efficacy of CTL-based immunotherapy against solid tumors, especially stiff solid tumors. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) mediate the cross‐talk between cancer cells and the cells of the surrounding Tumour Microenvironment (TME). Professional killer cells include Natural Killer (NK) cells and CD8+ Cytotoxic T‐lymphocytes (CTLs), which represent some of the most effective immune defense mechanisms against cancer cells. Recent evidence supports the role of EVs released by NK cells and CTLs in killing cancer cells, paving the road to a possible therapeutic role for such EVs. This review article provides the state‐of‐the‐art knowledge on the role of NK‐ and CTL‐derived EVs as anticancer agents, focusing on the different functions of different sub‐types of EVs. We also reviewed the current knowledge on the effects of cancer‐derived EVs on NK cells and CTLs, identifying areas for future investigation in the emerging new field of EV‐mediated immunotherapy of cancer. [ABSTRACT FROM AUTHOR]

It remains a major challenge to prime the cytotoxic T lymphocytes (CTLs) response for the treatment of Hepatitis B virus (HBV) infection. Inspired by an important natural biological behavior, membrane fusion, we constructed a pH-responsive nanocarrier (HBsAg&CpG@Lip) with a membrane fusion capacity for HBsAg intracellular delivery and subsequent process of CTLs. In the in vitro experiments, HBsAg&CpG@Lip greatly promoted the cellular uptake of HBsAg and the activation of BMDCs. It induced the cytosolic release of HBsAg in BMDCs, which was in conformity with the membrane fusion capacity of HBsAg&CpG@Lip. Such a capacity was improved by 4.4-fold in pH = 5.0 than that in pH = 7.4 at 60 min, which was calculated through fluorescence resonance energy transfer (FRET) efficiency. Furthermore, the cross-presentation activity induced by liposome at 1 μg/mL was equivalent to that by soluble antigen at 2000 μg/mL, which indicated an advantage for the liposome in cytosolic antigen delivery and potent CTLs activation. In respect of the transport of liposomes into draining lymph nodes (DLNs), the recruitment of liposome+migratory DCs at 24 h raised 11-fold than that at 3 h, suggesting that liposomes were mainly carried by migratory DCs. Meanwhile, HBsAg&CpG@Lip also elicited the activation of DCs and the generation of T fh cells in DLNs. After immunization, HBsAg&CpG@Lip induced a higher anti-HBsAg IgG and ratio of IgG2c/IgG1 than that by HBsAg or Alum+HBsAg group. It also augmented a strong CTLs response as expected. Specifically, stimulation with HBsAg&CpG@Lip increased the number of IFN-γ-secreting splenocytes, the proportion of CD107α+CD8+and FasL+CD8+ T cells and the secretion of Granzyme B, which verified the design of membrane fusion in vivo. In summary, this design for HBsAg cytosolic delivery exhibits great benefits for triggering CTLs, opening an avenue for the development of a potent therapeutic HBV vaccine. Unlabelled Image • The HBsAg delivery system was endowed with a pH-responsive liposome and a synergistic strategy with CpG. • The membrane fusion event of liposome was uncovered in a dynamical and quantitative way. • The liposome improved HBsAg cytosolic trafficking and cross-presentation activity. • An enhanced CTLs response was elicited. [ABSTRACT FROM AUTHOR]

Background: Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in cytotoxic T lymphocytes (CTLs) in the tumor microenvironment is largely unknown. Methods: Tumor tissues and CTLs of human colorectal cancer patients were analyzed for interferon (alpha and beta) receptor 1 (IFNAR1) expression. IFNAR1 knock out (IFNAR-KO), mixed wild type (WT) and IFNAR1-KO bone marrow chimera mice, and mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO) were used to determine IFN-I function in T cells in tumor suppression. IFN-I target genes in tumor-infiltrating and antigen-specific CTLs were identified and functionally analyzed. Results: IFNAR1 expression level is significantly lower in human colorectal carcinoma tissue than in normal colon tissue. IFNAR1 protein is also significantly lower on CTLs from colorectal cancer patients than those from healthy donors. Although IFNAR1-KO mice exhibited increased susceptibility to methylcholanthrene-induced sarcoma, IFNAR1-sufficient tumors also grow significantly faster in IFNAR1-KO mice and in mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO), suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Strikingly, tumor-infiltrating CTL levels are similar between tumor-bearing WT and IFNAR1-KO mice. Competitive reconstitution of mixed WT and IFNAR1-KO bone marrow chimera mice further determined that IFNAR1-deficient naïve CTLs exhibit no deficiency in response to vaccination to generate antigen-specific CTLs as compared to WT CTLs. Gene expression profiling determined that Gzmb expression is down-regulated in tumor-infiltrating CTLs of IFNAR1-KO mice as compared to WT mice, and in antigen-specific IFNAR1-KO CTLs as compared to WT CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that binds to the Gzmb promoter to activate Gzmb transcription in CTLs. Conclusion: IFN-I induces STAT3 activation to activate Gzmb expression to enhance CTL effector function to suppress tumor development. Human colorectal carcinoma may use down-regulation of IFNAR1 on CTLs to suppress CTL effector function to evade host cancer immunosurveillance. [ABSTRACT FROM AUTHOR]

Abstract Background Recent fundamental and clinical studies have confirmed the effectiveness of utilizing the potential of the immune system to remove tumor cells disseminated in a patient’s body. Cytotoxic T lymphocytes (CTLs) are considered the main effectors in cell-mediated antitumor immunity. Approaches based on antigen presentation to CTLs by dendritic cells (DCs) are currently being intensively studied, because DCs are more efficient in tumor antigen presentation to T cells through their initiation of strong specific antitumor immune responses than other types of antigen-presenting cells. Today, it has become possible to isolate CTLs specific for certain antigenic determinants from heterogeneous populations of mononuclear cells. This enables direct and specific cell-mediated immune responses against cells carrying certain antigens. The aim of the present study was to develop an optimized protocol for generating CTL populations specific for epitopes of tumor-associated antigen HER2/neu, and to assess their cytotoxic effects against the HER2/neu-expressing MCF-7 tumor cell line. Methods The developed protocol included sequential stages of obtaining mature DCs from PBMCs from HLA A*02-positive healthy donors, magnet-assisted transfection of mature DCs with the pMax plasmid encoding immunogenic peptides HER2 p369–377 (E75 peptide) and HER2 p689–697 (E88 peptide), coculture of antigen-activated DCs with autologous lymphocytes, magnetic-activated sorting of CTLs specific to HER2 epitopes, and stimulation of isolated CTLs with cytokines (IL-2, IL-7, and IL-15). Results The resulting CTL populations were characterized by high contents of CD8+ cells (71.5% in cultures of E88-specific T cells and 90.2% in cultures of E75-specific T cells) and displayed strong cytotoxic effects against the MCF-7 cell line (percentages of damaged tumor cells in samples under investigation were 60.2 and 65.7% for E88- and E75-specific T cells, respectively; level of spontaneous death of target cells was 17.9%). Conclusions The developed protocol improves the efficiency of obtaining HER2/neu-specific CTLs and can be further used to obtain cell-based vaccines for eradicating targeted tumor cells to prevent tumor recurrence after the major tumor burden has been eliminated and preventing metastasis in patients with HER2-overexpressing tumors.

The main effectors in tumor control are the class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). Tumor-specific CTL induction can be regulated by dendritic cells (DCs) expressing both tumor-derived epitopes and co-stimulatory molecules. Immunosuppressive tolerogenic DCs, having down-regulated co-stimulatory molecules, are seen within the tumor mass and can suppress tumor-specific CTL induction. The tolerogenic DCs expressing down-regulated XCR1+CD141+ appear to be induced by tumor-derived soluble factors or dexamethasone, while the immunogenic DCs usually express XCR1+CD141+ molecules with a cross-presentation function in humans. Thus, if tolerogenic DCs can be reactivated into immunogenic DCs with sufficient co-stimulatory molecules, tumor-specific CD8+ CTLs can be primed and activated in vivo. In the present study, we converted human tolerogenic CD141+ DCs with enhanced co-stimulatory molecule expression of CD40, CD80, and CD86 through stimulation with non-toxic mycobacterial lipids such as mycolic acid (MA) and lipoarabinomannan (LAM), which synergistically enhanced both co-stimulatory molecule expression and interleukin (IL)-12 secretion by XCR1+CD141+ DCs. Moreover, MA and LAM-stimulated DCs captured tumor antigens and presented tumor epitope(s) in association with class I MHCs and sufficient upregulated co-stimulatory molecules to prime naïve CD3+ T cells to become CD8+ tumor-specific CTLs. Repeat CD141+ DC stimulation with MA and LAM augmented the secretion of IL-12. These findings provide us a new method for altering the tumor environment by converting tolerogenic DCs to immunogenic DCs with MA and LAM from Mycobacterium tuberculosis. [ABSTRACT FROM AUTHOR]

Intrinsic genetic instability of tumor cells leads to continuous production of mutated proteins referred to as tumor-specific neoantigens. Generally, they are recognized as nonself products by the host immune system. However, an effective adaptive response clearing neoantigen-expressing cells is lost in tumor diseases. Most advanced therapeutic strategies aim at inducing neoantigen-specific immune activation through personalized approaches. They include tumor cell exome sequencing, human leukocyte antigen (HLA) typing, synthesis, and injection of peptides/RNA with adjuvants. Here, we propose an innovative method to induce a CD8+ T cytotoxic lymphocyte (CTL) immune response against tumor neoantigens bypassing the steps needed in current therapeutic strategies of personalized vaccination. We assumed that tumor cells can be the most efficient and precise factory of major histocompatibility complex (MHC) class I-associated, tumor neoantigen-derived peptides. Hence, endowing tumor cells with professional antigen-presenting functions would prime CD8+ T lymphocytes towards a response against nonself tumor antigens. To explore this possibility, both adenocarcinoma and melanoma human cells were engineered to express both CD80 and CD86 costimulatory molecules. HLA-matched lymphocytes were then primed through cocultivation with the engineered tumor cells. The generation of tumor-specific CD8+ T lymphocytes was tested through the combined analysis of cell activation markers, formation of immunologic synapses, generation of tumor antigen-specific CD8+ T lymphocytes, and cytotoxic activity. Our data consistently indicate that tumor cells endowed with professional antigen-presenting functions can generate an effective tumor-specific CTL immune response. This finding may open avenues towards the development of innovative antitumor immunotherapies. Key messages: We established a novel method to induce antitumor CTLs without a need to identify TAAs and/or tumor neoantigens. This strategy relies on transducing tumor cells with a retroviral vector expressing both CD80 and CD86. In this way, tumor cells prime naïve CD8+ T lymphocytes in a way that CTLs killing the same tumor cells are generated. These findings open the way towards preclinical assays in the perspective to introduce this antitumor immunotherapy strategy in clinic. [ABSTRACT FROM AUTHOR]

Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50 µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy. [ABSTRACT FROM AUTHOR]

Cytotoxic T cells (CTLs) bind to peptides presented by MHC I (pMHC) through T cell receptors of various affinities. Low-affinity CTLs are important for the control of intracellular pathogens and cancers; however, the mechanisms by which these lower affinity CTLs are activated and maintained are not well understood. We recently discovered that fully activated CTLs stimulated by strong-affinity peptides in the presence of IL-12 are able to secrete exosomes that, in turn, stimulate bystander CTLs without requiring the presence of antigen. We hypothesized that exosomes secreted by high-affinity CTLs could strengthen the activation of low-affinity CTLs. Naive OT-I CD8+ cells were stimulated with altered N4 peptides of different affinities in the presence or absence of Exo. The presence of Exo preferentially increased cell proliferation and enhanced the production of IFNγ in CTLs stimulated by low-affinity peptides. The expression of granzyme B (GZB) was augmented in all affinities, with higher GZB production in low-affinity stimulated CTLs than in high-affinity stimulated ones. Exosomes promoted the rapid activation of low-affinity CTLs, which remained responsive to exosomes for a prolonged duration. Unexpectedly, exosomes could be induced quickly (24 h) following CTL activation and at a higher quantity per cell than later (72 h). While exosome protein profiles vary significantly between early exosomes and their later-derived counterparts, both appear to have similar downstream functions. These results reveal a potential mechanism for fully activated CTLs in activating lower-affinity CTLs that may have important implications in boosting the function of low-affinity CTLs in immunotherapy for cancers and chronic viral infections. [ABSTRACT FROM AUTHOR]

• In MCMV infected Was KO mice, the late differentiated CD8+ T subset (CD27-CD28-) decreased in lungs, compared with those in the spleen and peripheral blood. • IL-2 and granzyme B production of the spleen-derived CD8+ T lymphocytes of Was KO mice declined compared with that in wild- type mice. • WASP may be involved in regulating cytotoxic function and apoptosis in CD8+ T cells following MCMV infection. Wiskott-Aldrich syndrome (WAS) patients are characterized by immunodeficiency and viral infections. T cells derived from WAS patients and WAS protein (WASP)-deficient mice have various defects. However, whether WASP plays a role in immune control of cytomegalovirus (CMV) infection remains unclear. We analyzed the distribution of CD8+ T subsets and the pathological damage to various organs and tissues in MCMV infected Was knockout (KO) mice. A relatively high number of MCMV-specific cytotoxic T cells (CTLs) were observed in the spleen of Was KO mice. In MCMV infected Was KO mice, the late differentiated CD8+ T subset (CD27−CD28−) decreased in lungs, compared with those in the spleen and peripheral blood. Additionally, we found that the most severe pathological lesions occurred in the lungs, the main target organ of MCMV infection. By stimulating the spleen-derived CD8+ T lymphocytes of Was KO mice, we found that IL-2 and granzyme B production declined compared with that in wild- type mice. Moreover, the number of apoptotic CD8+ T cells increased in Was KO mice compared with the number in wild-type mice. Therefore, our results demonstrate that WASP may be involved in regulating cytotoxic function and apoptosis in CD8+ T cells following MCMV infection, which is supported by the distribution and memory compartment of MCMV-specific T cells in MCMV infected WAS mice. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM), a highly lethal brain cancer, is notorious for immunosuppression, but the mechanisms remain unclear. Here, we documented a temporospatial patterning of tumor-associated myeloid cells (TAMs) corresponding to vascular changes during GBM progression. As tumor vessels transitioned from the initial dense regular network to later scant and engorged vasculature, TAMs shifted away from perivascular regions and trafficked to vascular-poor areas. This process was heavily influenced by the immunocompetence state of the host. Utilizing a sensitive fluorescent UnaG reporter to track tumor hypoxia, coupled with single-cell transcriptomics, we revealed that hypoxic niches attracted and sequestered TAMs and cytotoxic T lymphocytes (CTLs), where they were reprogrammed toward an immunosuppressive state. Mechanistically, we identified chemokine CCL8 and cytokine IL-1β as two hypoxic-niche factors critical for TAM trafficking and co-evolution of hypoxic zones into pseudopalisading patterns. Therefore, perturbation of TAM patterning in hypoxic zones may improve tumor control. [Display omitted] • Host immune status influences tumor vasculature and hypoxic-zone formation in GBM • Spatial patterning of TAM and CTL parallels hypoxic-zone maturation to pseudopalisades • Sequestered TAM and CTL in hypoxic zones are reprogrammed toward immunosuppression • TAM/CTL organization involves CCL8 and IL-1β as niche factors in hypoxic zones Glioblastoma is notorious for immunosuppression, but the mechanisms are unclear. Sattiraju et al. report that hypoxic zones in GBM attract and sequester tumor-associated myeloid cells and cytotoxic T cells, where they are reprogrammed into an immunosuppressive state. This process is influenced by the immunocompetence state of the host and involves CCL8 and IL-1B as niche factors in hypoxic zones. [ABSTRACT FROM AUTHOR]

Background: Recent fundamental and clinical studies have confirmed the effectiveness of utilizing the potential of the immune system to remove tumor cells disseminated in a patient's body. Cytotoxic T lymphocytes (CTLs) are considered the main effectors in cell-mediated antitumor immunity. Approaches based on antigen presentation to CTLs by dendritic cells (DCs) are currently being intensively studied, because DCs are more efficient in tumor antigen presentation to T cells through their initiation of strong specific antitumor immune responses than other types of antigen-presenting cells. Today, it has become possible to isolate CTLs specific for certain antigenic determinants from heterogeneous populations of mononuclear cells. This enables direct and specific cell-mediated immune responses against cells carrying certain antigens. The aim of the present study was to develop an optimized protocol for generating CTL populations specific for epitopes of tumor-associated antigen HER2/neu, and to assess their cytotoxic effects against the HER2/neu-expressing MCF-7 tumor cell line. Methods: The developed protocol included sequential stages of obtaining mature DCs from PBMCs from HLA A*02- positive healthy donors, magnet-assisted transfection of mature DCs with the pMax plasmid encoding immunogenic peptides HER2 p369-377 (E75 peptide) and HER2 p689-697 (E88 peptide), coculture of antigen-activated DCs with autologous lymphocytes, magnetic-activated sorting of CTLs specific to HER2 epitopes, and stimulation of isolated CTLs with cytokines (IL-2, IL-7 and IL-15). Results: The resulting CTL populations were characterized by high contents of CD8+ cells (71.5% in cultures of E88-specific T cells and 90.2% in cultures of E75-specific T cells) and displayed strong cytotoxic effects against the MCF-7 cell line (percentages of damaged tumor cells in samples under investigation were 60.2 and 65.7% for E88- and E75-specific T cells, respectively; level of spontaneous death of target cells was 17.9%). Conclusions: The developed protocol improves the efficiency of obtaining HER2/neu-specific CTLs and can be further used to obtain cell-based vaccines for eradicating targeted tumor cells to prevent tumor recurrence after the major tumor burden has been eliminated and preventing metastasis in patients with HER2-overexpressing tumors. [ABSTRACT FROM AUTHOR]

Despite more than three decades of intense research, a prophylactic HIV-1 vaccine remains elusive. Four vaccine modalities have been evaluated in clinical efficacy studies, but only one demonstrated at least modest efficacy, which correlated with polyfunctional antibody responses to the HIV surface protein Env. To be most effective, a HIV-1 vaccine probably has to induce both, functional antibody and CD8 + T cell responses. We therefore analyzed DNA/DNA and DNA/virus-like particle (VLP) regimens for their ability to induce humoral and cellular immune responses. Here, DNA vaccination of mice induced strong CD8 + responses against Env and Gag. However, the humoral response to Env was dominated by IgG1, a subclass known for its low functionality. In contrast, priming only with the Gag-encoding plasmid followed by a boost with VLPs consisting of Gag and Env improved the quality of the anti-Env antibody response via intrastructural help (ISH) provided by Gag-specific T cells to Env-specific B cells. Furthermore, the Gag-specific CD8 + T cells induced by the DNA prime immunization could still protect from a lethal infection with recombinant vaccinia virus encoding HIV Gag. Therefore, this immunization regimen represents a promising approach to combine functional antibody responses toward HIV Env with strong CD8 + responses controlling early viral replication. [ABSTRACT FROM AUTHOR]

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2 A levels. Loss-of-function mutations in MID1 lead to the human X-linked Opitz G/BBB (OS) syndrome characterized by defective midline development during embryogenesis. We have recently shown that MID1 is strongly up-regulated in murine cytotoxic T lymphocytes (CTLs), and that it has a significant impact on exocytosis of lytic granules and the killing capacity of CTLs. The aims of the present study were to determine the localization of MID1 in migrating CTLs, and to investigate whether MID1 affects CTL polarization and migration. We found that MID1 mainly localizes to the uropod of migrating CTLs and that it has a substantial impact on CTL polarization and migration in vitro. Furthermore, analysis of contact hypersensitivity responses supported that MID1 controls effector functions of CTLs in hapten-challenged skin in vivo. These results provide significant new knowledge on the role of MID1 in CTL biology. [ABSTRACT FROM AUTHOR]

Memory programming of cytotoxic T cells (CTLs) by inflammatory cytokines can be regulated by mammalian target of rapamycin (mTOR). We have shown that inhibition of mTOR during CTL activation leads to the enhancement of memory, but the molecular mechanisms remain largely unknown. Using high-throughput RNA-Seq, we identified genes and functions in mouse CTLs affected by mTOR inhibition through rapamycin. Of the 43,221 identified transcripts, 184 transcripts were differentially expressed after rapamycin treatment, corresponding to 128 annotated genes. Of these genes, 114 were downregulated and only 14 were upregulated. Most importantly, 50 of them are directly related to cell death and survival. In addition, several genes such as CD62L are related to migration. Furthermore, we predicted downregulation of transcriptional regulators based on the total differentially expressed genes, as well as the subset of apoptosis-related genes. Quantitative PCR confirmed the differential expressions detected in RNA-Seq. We conclude that the regulatory function of rapamycin may work through inhibition of multiple genes related to apoptosis and migration, which enhance CTL survival into memory. [ABSTRACT FROM AUTHOR]

Background: Dendritic cell (DC) therapy is a promising therapy for cancer-targeting treatments. Recently, DCs have been used for treatment of some cancers. We aimed to develop an in vitro assay to evaluate DC therapy in cancer treatment using a breast cancer model. Methods: DCs were induced from murine bone marrow mononuclear cells in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with GM-CSF (20 ng/mL) and IL-4 (20 ng/mL). Immature DCs were primed with breast cancer stem cell (BCSC)-derived antigens. BCSCs were sorted from 4T1 cell lines based on aldehyde dehydrogenase expression. A mixture of DCs and cytotoxic T lymphocytes (CTLs) were used to evaluate the inhibitory effect of antigen-primed DCs on BCSCs. BCSC proliferation and doubling time were recorded based on impedance-based cell analysis using the xCELLigence system. The specification of inhibitory effects of DCs and CTLs was also evaluated using the same system. Results: The results showed that impedance-based analysis of BCSCs reflected cytotoxicity and inhibitory effects of DCs and CTLs at 72 hours. Differences in ratios of DC:CTL changed the cytotoxicity of DCs and CTLs. Conclusion: This study successfully used impedance-based cell analysis as a new in vitro assay to evaluate DC efficacy in cancer immunotherapy. We hope this technique will contribute to the development and improvement of immunotherapies in the near future. [ABSTRACT FROM AUTHOR]

Highlights: [•] NNK directly affects CTL activation. [•] NNK enhances the expression of adhesion molecule CD62L in CTLs. [•] NNK reduces CTL memory programming. [ABSTRACT FROM AUTHOR]

Abstract: The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18–24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells. [Copyright &y& Elsevier]

Background Data exploring the potential use of effector molecules produced by cytotoxic T lymphocytes (CTLs) in the immunodiagnostics of tuberculosis (TB) are scarce. The present study focused a) to gain an insight into the discriminatory power of CTLs in patients with acute pulmonary or extra-pulmonary TB, or latent tuberculosis infection (LTBI); and b) to evaluate the influence of various anti-TB therapeutic schemes on the immunological profiles of residual CTLs. Methods Immunological signatures of antigen-specific CTLs were explored in patients with active pulmonary and extra-pulmonary TB, LTBI and in those treated for TB decades ago by using ELISPOT, intracellular flow cytometry and extracellular CD107a detection. Results No difference was seen between active TB, LTBI or any of those treated for TB in the ELISPOT analysis of antigen-specific Granzyme B (GrB), Perforin (Prf) and interferongamma (IFN-γ) producing lymphocytes, the FACS analysis of the intracellular expression of IFN-γ, or the surface expression of CD107a degranulation factor of both CD8+ and CD4+ antigen-specific T cell subsets. The effector memory (TEM) phenotype proved predominant in the surface marker profiling both in active TB and LTBI. The proportion of the CD107a degranulation factor proved higher in the central memory (TCM) than in the other cell subsets in all the study groups. Interestingly, functionally and phenotypically similar CTLs profiles were observed in active TB, LTBI and in all the three groups treated for TB. Conclusion The phenotypic and functional profiling of CTLs has a limited potential in the immunodiagnostics of active TB. Antigen-specific CTLs persist in patients treated for TB decades ago regardless of the efficacy of implemented and completed anti-TB therapy. [ABSTRACT FROM AUTHOR]

Abstract: Although TGF-β and IL-6 would turn CD8+ T cells to differentiate into non-cytotoxic state, these treated cells were converted to cytolytic phenotypes after re-exposure to their antigenic epitope in vitro. Here, using spleen cells from TCR transgenic mice expressing TCRαβ genes of clone RT1 recognizing an epitope peptide (P18-I10: RGPGRAFVTI) of HIV-1 gp160, we generated CD8+ cytotoxic T lymphocytes (CTLs) activated by re-exposure to P18-I10 after primarily cultured with TGF-β and IL-6 in vitro to examine their effector function. The CTLs, having strong cytotoxic activity in vitro, were not only resistant to Fas–FasL mediated apoptosis, but also insensitive to the suppression of their cytotoxicity by re-exposure to TGF-β in vitro. Moreover, adoptive transfer experiments indicated that the CTLs are capable of eliminating recombinant vaccinia virus expressing HIV-1 gp160 in vivo. Taken together, our data suggest that TGF-β and IL-6 may play pivotal roles in inducing apoptosis-resistant and TGF-β-insensitive CTLs in vitro. [Copyright &y& Elsevier]

To clarify the mechanism of Seoul orthohantavirus (SEOV) persistence, we compared the humoral and cell-mediated immune responses to SEOV in experimentally and naturally infected brown rats. Rats that were experimentally infected by the intraperitoneal route showed transient immunoglobulin M (IgM) production, followed by an increased anti-SEOV immunoglobulin G (IgG) antibody response and maturation of IgG avidity. The level of SEOV-specific cytotoxic T lymphocytes (CTLs) peaked at 6 days after inoculation and the viral genome disappeared from serum. In contrast, naturally infected brown rats simultaneously had a high rate of SEOV-specific IgM and IgG antibodies (28/43). Most of the IgM-positive rats (24/27) had the SEOV genome in their lungs, suggesting that chronic SEOV infection was established in those rats. In female rats with IgG avidity maturation, the viral load in the lungs was decreased. On the other hand, there was no relationship between IgG avidity and viral load in the lungs in male rats. A CTL response was not detected in naturally infected rats. The difference between immune responses in the experimentally and naturally infected rats is associated with the establishment of chronic infection in natural hosts. [ABSTRACT FROM AUTHOR]

• DCs in Hepa1-6-1 tumours remained tolerogenic even after blockade of PD-1. • Combination therapy with anti-PD-1 mAb and α-GalCer showed an excellent effect. • The amount of anti-PD-1 mAb could be reduced by 90% when used with α-GalCer. Immunotherapy for cancer cells induced by interfering with PD-1/PD-L1 engagement via check-point blockades was initiated by tumour-specific PD-1+ CD8+ cytotoxic T lymphocytes (CTLs) within a tumour mass and eliminate the tumour. Here, we used C57BL/6 (B6) mice implanted with the syngeneic hepatoma cell line Hepa1-6-1, and confirmed that the dendritic cells (DCs) within Hepa1-6-1 tumour mass were tolerogenic with downmodulated co-stimulatory molecules by tumour-derived factors. Although Hepa1-6-1 cells did not prime tumour-specific CTLs within the tumour, specific CTLs primed in the regional lymph nodes seemed to be invaded into the tumour mass. The specific CTLs gained PD-1+ expression when associated with PD-L1+ Hepa1-6-1 cells within the tumour mass. Their cytotoxic activity in vivo was revitalised after intraperitoneal (i.p.) administration of the anti-PD-1 monoclonal antibody (mAb), indicating that PD-1/PD-L1 engagement within the tumour was abrogated by check-point blockade. Nonetheless, the tolerogenic DCs within the Hepa1-6-1 tumour mass remained tolerogenic even after three shots of PD-1-blockade administration, and the suppressed Hepa1-6-1 growth was revisited. In this study, we show here an excellent therapeutic effect consisting of three injections of anti-PD1 mAb and the sequential administration of the CD1d molecule-restricted ligand α-galactosylceramide (α-GalCer), an immuno-potent lipid/glycolipid, which converts tolerogenic DCs into immunogenic DCs with upregulated expression of co-stimulatory molecules. The α-GalCer-activated DCs secreted a large amount of IL-12, which can activate tumour-specific CTLs in vivo. The check-point blockade was not sufficiently effective, but the dose needed for tumour eradication was reduced by 90% when tumour-bearing mice were also administered i.p. α-GalCer. [ABSTRACT FROM AUTHOR]

Background: Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in cytotoxic T lymphocytes (CTLs) in the tumor microenvironment is largely unknown. Methods: Tumor tissues and CTLs of human colorectal cancer patients were analyzed for interferon (alpha and beta) receptor 1 (IFNAR1) expression. IFNAR1 knock out (IFNAR-KO), mixed wild type (WT) and IFNAR1-KO bone marrow chimera mice, and mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO) were used to determine IFN-I function in T cells in tumor suppression. IFN-I target genes in tumor-infiltrating and antigen-specific CTLs were identified and functionally analyzed. Results: IFNAR1 expression level is significantly lower in human colorectal carcinoma tissue than in normal colon tissue. IFNAR1 protein is also significantly lower on CTLs from colorectal cancer patients than those from healthy donors. Although IFNAR1-KO mice exhibited increased susceptibility to methylcholanthrene-induced sarcoma, IFNAR1-sufficient tumors also grow significantly faster in IFNAR1-KO mice and in mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO), suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Strikingly, tumor-infiltrating CTL levels are similar between tumor-bearing WT and IFNAR1-KO mice. Competitive reconstitution of mixed WT and IFNAR1-KO bone marrow chimera mice further determined that IFNAR1-deficient naïve CTLs exhibit no deficiency in response to vaccination to generate antigen-specific CTLs as compared to WT CTLs. Gene expression profiling determined that Gzmb expression is down-regulated in tumor-infiltrating CTLs of IFNAR1-KO mice as compared to WT mice, and in antigen-specific IFNAR1-KO CTLs as compared to WT CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that binds to the Gzmb promoter to activate Gzmb transcription in CTLs. Conclusion: IFN-I induces STAT3 activation to activate Gzmb expression to enhance CTL effector function to suppress tumor development. Human colorectal carcinoma may use down-regulation of IFNAR1 on CTLs to suppress CTL effector function to evade host cancer immunosurveillance. [ABSTRACT FROM AUTHOR]

Antitumor cytotoxic T lymphocytes (CTLs) are essential for immune surveillance, yet the blockade of eliciting such CTLs during oncolytic virotherapy remains incompletely understood. Here, we show that oncolysis of mesothelioma by modified vaccinia Tiantan (MVTT) induces damage-associated molecular patterns exposure. Although MVTT leads to regression of established mesothelioma dose-dependently, antitumor CTLs are rarely induced. Mechanistically, MVTT virotherapy generates C-X-C chemokines that recruit CXCR2-expressing polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into tumor microenvironment, where they suppress dendritic cells (DCs) by producing IL-10 and halt CTL responses. During the virotherapy, however, depletion of PMN-MDSCs but not of monocytic (M)-MDSCs results in the induction of potent antitumor CTLs that not only eradicate established mesothelioma but also prevent the second tumor challenge. Our findings suggest that vaccinia virotherapy may combine strategies that prevent the chemotactic recruitment of PMN-MDSCs, block their suppression on DCs or deplete PMN-MDSCs in order to induce potent CTLs for tumor eradication. [ABSTRACT FROM AUTHOR]

Summary How cytotoxic T lymphocytes (CTLs) sense T cell receptor (TCR) signaling in order to specialize an area of plasma membrane for granule secretion is not understood. Here, we demonstrate that immune synapse formation led to rapid localized changes in the phosphoinositide composition of the plasma membrane, both reducing phosphoinositide-4-phosphate (PI(4)P), PI(4,5)P2, and PI(3,4,5)P3 and increasing diacylglycerol (DAG) and PI(3,4)P2 within the first 2 min of synapse formation. These changes reduced negative charge across the synapse, triggering the release of electrostatically bound PIP5 kinases that are required to replenish PI(4,5)P2. As PI(4,5)P2 decreased, actin was depleted from the membrane, allowing secretion. Forced localization of PIP5Kβ across the synapse prevented actin depletion, blocking both centrosome docking and secretion. Thus, PIP5Ks act as molecular sensors of TCR activation, controlling actin recruitment across the synapse, ensuring exquisite co-ordination between TCR signaling and CTL secretion. Graphical Abstract Highlights • Immune synapse formation triggers rapid changes in the membrane composition and charge • PIP5K is a molecular sensor of TCR activation and is rapidly depleted at the synapse • PIP5K distribution controls actin recruitment across the immune synapse • Membrane specialization controls accessibility for centrosome docking and secretion Gawden-Bone et al. show that during immune synapse formation, rapid depletion of PIP5K at the synapse triggers subsequent changes in membrane composition and actin dynamics to establish a zone of localized granule secretion. [ABSTRACT FROM AUTHOR]