Cytotoxicity Of Cytotoxic T Lymphocytes Research Articles

Abstract 34: Wee1 inhibition sensitizes tumor cells with variable antigenicity to T cell mediated lysis

Abstract Antigen-specific elimination of tumor cells by cytotoxic T-lymphocytes (CTL) inherently relies upon the sensitivity of target cells to effector products such as granzyme B, TNFa, TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand (FasL). Cell cycle progression is critically required for granzyme B and TNF superfamily-induced apoptosis. Thus, we hypothesized that inhibition of Wee1 with AZD1775 would release the G2/M cell cycle checkpoint and enhance sensitivity of oral cavity cancer cells to CTL killing. To explore this hypothesis, we stably transduced murine oral cancer 1 (MOC1) cells with a construct encoding the fluorescent protein mKate2 and SIINFEKL, the H2-Kb-restricted epitope of ovalbumin (MOC1mK2SIIN). Using in-vitro generated SIINFEKL-specific OT-1 CTLs as effectors, we demonstrated in a real-time, impedance-based assay of cytotoxicity that physiologic concentrations of AZD1775 (250 nM) significantly sensitized MOC1mK2SIIN cells to CTL killing at low E:T ratios. The same concentration of AZD1775 had negligible effects on the MOC1mK2SIIN cells alone, and OT-1 CTL cytotoxicity was antigen-specific as OT-1 effectors did not kill parental MOC1 cells with or without AZD1775 treatment. This effect was partially reversed with the perforin inhibitor concanamycin A, implicating involvement of granzyme B. Further, by mixing MOC1mK2SIIN and parental MOC1 cells, we could alter the ratio of antigen clonality present within the target cell population. Inhibition of Wee1 with AZD1775 significantly enhanced OT-1 CTL control of target cell populations where 5, 10, 15, 20, 25, and 50% lack SIINFEKL antigen. Treatment of MOC1mK2SIIN and parental MOC1 cells with recombinant TNFa, TRAIL and FasL with and without AZD1775 confirmed enhanced TNF superfamily-induced tumor cell control following Wee1 inhibition. These data suggest that Wee1 inhibition broadly sensitizes antigen positive and negative cells to CTL effector products and that AZD1775 may induce “bystander killing” of antigen negative cells in target cell populations with mixed antigenicity. Experiments combining AZD1775 with immune-activating therapies in vivo are underway. Citation Format: Ellen Moore, Lillian Sun, Jay Friedman, Carter Van Waes, Clint Allen. Wee1 inhibition sensitizes tumor cells with variable antigenicity to T cell mediated lysis [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 34.

Zebularine Treatment Induces MAGE-A11 Expression and Improves CTL Cytotoxicity Using a Novel Identified HLA-A2-restricted MAGE-A11 Peptide.

Melanoma-associated antigen-A11 (MAGE-A11) is frequently expressed in breast cancer and is associated with poor prognosis. Therefore, MAGE-A11 is a potential immunotherapy target in breast cancer. MAGE-A11 expression, however, is downregulated in many patients, thus constraining the application of immunotherapy. The induction of MAGE-A11 expression is crucial for the recognition and killing of breast cancer cells by cytotoxic T lymphocytes (CTL). In this study, a series of HLA-A2-restricted candidate MAGE-A11 peptides were predicted, synthesized, and tested. Of the selected peptides, p350 (FLFGEPKRL) elicited peptide-specific CTLs from healthy HLA-A*0201-positive donors. The induced CTLs can lyse MAGE-A11-expressing breast cancer cells but not MAGE-A11-negative tumor cells. To improve antitumor immune response, zebularine, a DNA methyltransferase inhibitor, was used to induce MAGE-A11 expression and upregulate the cytotoxicity of antigen-specific T cells in breast cancer cell lines and primary breast cancer cells. The present findings suggested that peptide p350 induces peptide-specific cytolytic activity and is thus a potential candidate for tumor vaccination or T-cell therapy. Epigenetic modulation by zebularine can induce MAGE-A11 expression in breast cancer cells and facilitate cytotoxicity via MAGE-A11-specific CTL.

Cystatins, cysteine peptidase inhibitors, as regulators of immune cell cytotoxicity

Cystatins comprise a superfamily of evolutionarily related proteins, present in all living organisms, from protozoa to mammals. They act as inhibitors of cysteine peptidases although they can also function independently of their inhibitory function. Cysteine cathepsins are implicated in various physiological and pathological processes. In the immune response they are involved in antigen processing and presentation, the cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), migration and adhesion of immune cells, cytokine and growth factor regulation and toll-like receptor signalling. Cystatins are probably involved in the regulation of all these processes; importantly, cystatin F has a crucial role in the regulation of immune cell cytotoxicity. NK cells and CTLs exploit the granzyme/perforin pathway for target cell killing, with perforin and granzymes as crucial effector molecules. Granzymes are synthesized as inactive pro-granzymes and need to be proteolytically activated by cathepsins C and H. Cystatin F is the main regulator of the activity of cathepsins C and H in cytotoxic cells and, consequently, regulates their cytotoxicity. The role of cystatins and cysteine cathepsins in the immune response is presented, with emphasis on their role in the regulation of cytotoxicity of NK cells and CTLs.

Improvement of Antigen-Specific CTL Persistence By Transduction of Novel Artificial Adapter Molecule

Introduction: Endogenous or gene-modified T cells with T cell receptors (TCRs) are major designs of cytotoxic T lymphocytes (CTL) therapies. However, gene-modified TCR-CTL therapies targeting on malignant tumors have been generally unsuccessful in previous clinical trials. The major reasons were poor expansion and short persistence of CTL. To overcome these problems, TCR-affinity have been genetically enhanced, but often resulted in life-threatening toxicities. We aimed to modify TCR-CTL in order to improve the function by enhancing intracellular signaling after TCR ligation without affinity modulation.Method: We generated three artificial T cell activating adapter molecules (ATAM), which are CD3z, CD3z/CD28, and CD3z/4-1BB. CD28 or 4-1BB intracellular domain was inserted in the middle of CD3z intracellular domain, so that they can assemble with internal TCR complex when stimulated with corresponding antigen. ATAM-T2A-tEGFR was packaged into retrovirus vector to transduce the T cells. In order to evaluate the functional alteration, we established ATAM-CMV CTL model. We stimulated T cells with CMV pp65 peptide to induce endogenous CMV CTL. On day 4 after stimulation, ATAMs were retrovirally transduced into CMV CTL. After a selection with anti-EGFR mAb, we achieved more than 90% purity of ATAM positive CMV CTL and used in 51Cr release assay, cytokine release assay, and the growth assay. In the growth assay, we stimulated the ATAM CMV-CTL weekly and assayed the viable cell number, tetramer positivity, phenotype, and apoptosis. Regarding these assay, we used HLA transduced K562 (HLA-K562) as APC.Results: First, we confirmed the expression of each ATAM on CD8+ T cells, which showed similar expression among each ATAM. To determine the cytotoxicity of CMV CTL according to each ATAM or control tEGFR transduction, we performed 51Cr release assay against HLA-K562 pulsed with or without CMV peptide. In the presence of CMV peptide, each ATAM-CMV CTL showed similar cytolytic activities to control CMV CTL. [specific lysis (%): tEGFR 32.0+-6.0, CD3z 32.4+-2.6, CD3z/CD28 31.1+-8.8, and CD3z/4-1BB 37.4+-1.5: at an E:T ratio = 3:1, respectively}. In the intracellular cytokine assay against HLA-K562 pulsed with CMV peptide, similar proportions of responder were positive for IFN-g and IL-2 in control and ATAM-CMV CTL. [IFN-g (%): tEGFR, 67.3+-9.4, CD3z, 69.0+-7.4, CD3z/CD28, 66.5+-8.0, and CD3z/4-1BB, 69.6+-7.9; IL-2 (%): tEGFR, 17.5+-5.0, CD3z, 17.7+-4.3, CD3z/CD28, 15.9+-4.3, and CD3z/4-1BB, 18.9+-4.8, respectively]. The cytotoxicity and cytokine secretion upon irrelevant stimulation were completely negative among each ATAM-CMV CTL. Although the surface phenotype was not changed in control CMV-CTL, that of ATAMs-CMV CTL was significantly expanded after antigen stimulation [central memory (CD45RA-CD62L+) (%): tEGFR, 6.0+-2.9, CD3z, 26.8+-11.1, CD3z/CD28, 19.4+-9.5, and CD3z/4-1BB, 35.8+-26.8: at 7 days after stimulation]. Regarding the cell growth, CD3z/CD28 and CD3z/4-1BB showed greater CTL expansion upon weekly antigen stimulation. The fold expansion of tEGFR, CD3z, CD3z/CD28, and CD3z/4-1BB-CMV CTL were 4.9+-0.7, 8.2+-2.4, 12.3+-1.1, and 13.0+-1.6 at day7, 1.6+-0.4, 8.0+-2.5, 18.6+-3.9, and 21.3+-7.7 at day14, and 0.4+-0.2, 1.4+-0.7, 5.1+-3.0, and 30.4+-22.9 at day21, respectively (Fig). Comparing with control, CD3z/4-1BB-CMV CTL showed continuous greater expansion after several stimulations (p<0.01: during day0 to day7, p<0.05: during day7 to day14). To examine the reason for the growth advantage, we investigated the apoptosis after the stimulation with DAPI/Annexin V staining. It revealed that the live cells after specific stimulation elevated in ATAMs-CMV CTL compared with control CMV CTL.Conclusion: We generated three ATAMs and successfully transduced into CMV CTL. Particularly, the whole cell growth and continuance of growth after weekly stimulation were significantly improved in CD3z/4-1BB CTL. This greater cell expansion may improve in vivo persistence and further effectiveness of TCR-CTL therapy. ATAM-CMV CTL did not recognize any APC without corresponding peptide, indicating that ATAM transduction could improve the CTL effectiveness without altering CTL specificity. Thus we believe that we could potentially apply ATAM transduction to other TCR-CTLs targeting malignant tumors safely. [Display omitted] DisclosuresKiyoi:Alexion Pharmaceuticals: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding; Celgene Corporation: Consultancy.

Human anti-pig NK cell and CD8+ T-cell responses in the presence of regulatory dendritic cells.

Dendritic cells (DC) play a major role in natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activation leading to cell-mediated xenogeneic responses. In contrast, the use of in vitro differentiated regulatory DC may represent an attractive approach to protect porcine endothelial cells (pEC) from human cell-mediated immune responses. In this study, we evaluated the potential of human regulatory DC to reduce xenogeneic NK cell and CTL responses to pEC. Human monocytes were differentiated into DC with GM-CSF and IL-4 in the absence or presence of rapamycin or IL-10. The effect of regulatory DC on xenogeneic NK cell and CTL responses was evaluated by analyzing phenotype, IFNγ production, degranulation, and cytotoxicity by flow cytometry and cytotoxicity assays. Upon maturation with LPS, Rapa-DC and IL-10-DC displayed different phenotypes and cytokine production profiles. In contrast to untreated DC, both Rapa-DC and IL-10-DC induced significantly less IFNγ production and NK cell degranulation in response to pEC, but did not affect NK cell-mediated pEC lysis. Low production of IL-18 by Rapa-DC, and of IL-12 by IL-10-DC were linked to the deficient IFNγ production by NK cells as shown by partial reversion of IFNγ production upon cytokine reconstitution. In contrast to untreated DC efficiently generating xenoantigen-specific CTL, priming of CTL in the presence of IL-10-DC was impaired as shown by lower IFNγ production and cytotoxicity of CTL in response to pEC. Both Rapa-DC and IL-10-DC controlled human anti-porcine NK cell responses, in particular IFNγ production, whereas IL-10-DC presented stronger regulatory properties of anti-porcine CTL responses. These in vitro findings indicate that regulatory DC could be a useful tool to promote xenograft tolerance in vivo.

Immunotherapeutic effects of cytokine-induced killer cells combined with CCL21/IL15 armed oncolytic adenovirus in TERT-positive tumor cells

The effective antitumor immune responses are dependent on coordinate interaction of various effector cells. Thus, the combination of adoptive immunotherapy and target gene therapy is capable of efficiently generating a productive antitumor immune response. We investigated whether combination of cytokine-induced killer (CIK) cells adoptive immunotherapy and CCL21/IL15 armed oncolytic adenovirus could induce the enhanced antitumor activity. The CCL21/IL15 co-expression oncolytic adenoviruses were constructed by using the AdEasy system, which uses homologous recombination with shuttle plasmids and full length Ad backbones. This conditionally replicating adenoviruses CRAd-CCL21-IL15 could induce apoptosis in TERTp-positive tumor cells for viral propagation, but do not replicate efficiently in normal cells, because the E1A promoter was replaced by telomerase reverse transcriptase promoter (TERTp). Our results showed that the combination of CIK cells and CRAd-CCL21-IL15 could induce higher antitumor activity than either CIK cells or CRAd-CCL21-IL15 alone. This combined treatment could induce the tumor specific cytotoxicity of CTLs (cytotoxic T lymphocytes) in vitro. Moreover, the treatment of established tumors with the combined therapy of CIK cells and CRAd-CCL21-IL15 resulted in tumor regression. This study suggests that the combined treatment by adoptive immunotherapy and gene therapy is a promising strategy for the therapy of tumor.

Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway.

CD8+ cytotoxic T lymphocyte (CTL) exhaustion is a chief issue for ineffective virus elimination in chronic infectious diseases. We generated novel ovalbumin (OVA)-specific OVA-Texo and HIV-specific Gag-Texo vaccines inducing therapeutic immunity. To assess their therapeutic effect in chronic infection, we developed a new chronic infection model by i.v. infecting C57BL/6 mice with the OVA-expressing adenovirus AdVova. During chronic AdVova infection, mouse CTLs were found to express the inhibitory molecules programmed cell-death protein-1 (PD-1) and lymphocyte-activation gene-3 (LAG-3) and to be functionally exhausted, showing a significant deficiency in T-cell proliferation, IFN-γ production and cytolytic effects. Naive CD8+ T cells upregulated inhibitory PD-ligand 1 (PD-L1), B- and T-lymphocyte attenuator and T-cell anergy-associated molecules (Grail and Itch) while down-regulating the proliferative response upon stimulation in mice with chronic infection. Remarkably, the OVA-Texo vaccine counteracted T-cell anergy and converted CTL exhaustion. The latter was associated with (i) the upregulation of a marker for CTL functionality, diacetylated histone-H3 (diAcH3), (ii) a fourfold increase in CTLs, occurring independent of host DCs or CD4+ T cells, and (iii) the restoration of CTL IFN-γ production and cytotoxicity. In vivo OVA-Texo-stimulated CTLs upregulated the activities of the mTORC1 pathway-related molecules Akt, S6, eIF4E and T-bet, and treatment of the CTLs with an mTORC1 inhibitor, rapamycin, significantly reduced the OVA-Texo-induced increase in CTLs. Interestingly, OVA-Texo-mediated CD40L signaling played a critical role in the observed immunological effects. Importantly, the Gag-Texo vaccine induced Gag-specific therapeutic immunity in chronic infection. Therefore, this study should have a serious impact on the development of new therapeutic vaccines for human immunodeficiency virus (HIV-1) infection.

Syntaxin 8 is required for efficient lytic granule trafficking in cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL.

Artificial antigen-presenting cells expressing AFP(158-166) peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes.

Professional antigen-presenting cells (APCs) are potent generators of tumor antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy; however, generation of APCs is cumbersome, expensive, and subject to the tumor microenvironment. Artificial APCs (aAPCs) have been developed as a cost-effective alternative to APCs. We developed a cellular aAPC that efficiently generated alpha-fetoprotein (AFP)-specific CTLs. We genetically modified the human B cell lymphoma cell line BJAB with a lentiviral vector to establish an aAPC called BA15. The expression of AFP158-166-HLA-A*02:01 complex, CD80, CD86, and interleukin (IL)-15 in BA15 cells was assessed. The efficiency of BA15 at generating AFP-specific CTLs and the specific cytotoxicity of CTLs against AFP+ cells were also determined. BA15 cells expressed high levels of AFP158-166 peptide, HLA-A2, CD80, CD86, and IL-15. BA15 cells also exhibited higher efficiency in generating AFP-specific CTLs than did dendritic cells. These CTLs had greater cytotoxicity against AFP+ hepatocellular carcinoma cells than did CTLs obtained from dendritic cells in vitro and in vivo. Our novel aAPC system could provide a robust platform for the generation of functional AFP-specific CTLs for adoptive immunotherapy of hepatocellular carcinoma.

Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.

Specific antitumour immunity of HIFU-activated cytotoxic T lymphocytes after adoptive transfusion in tumour-bearing mice

Purpose: The aim of this study was to investigate the specific anti-tumour immunity of cytotoxic T lymphocytes (CTL) activated by high-intensity focused ultrasound (HIFU) after adoptive transfer in a murine tumour model. Materials and methods: H22 tumour-bearing mice were treated by either HIFU or sham-HIFU, while naïve syngeneic mice were used as controls. They were sacrificed and the spleens were harvested 14 days after HIFU. T lymphocytes were obtained from the spleens, and then adoptively transferred into 40 mice each bearing a 3-day implanted H22 tumour. On day 14 after adoptive transfer, 10 mice were sacrificed in each group for assessment of the number of tumour-infiltrating T lymphocytes and interferon-gamma (IFN-γ) secreting cells. The remaining 30 mice were continuously observed for 60 days, and tumour growth, progression and survival were recorded. Results: HIFU significantly increased peripheral blood CD3+, CD4+ levels and CD4+/CD8+ ratio (P < 0.05), CTL cytotoxicity (P < 0.01) and IFN-γ and TNF-α secretion (P < 0.01) in H22 tumour-bearing mice. Adoptive transfer of HIFU-activated T lymphocytes into the autologous tumour-bearing mice induced a significant increase of tumour-infiltrating T lymphocytes and IFN-γ-secreting cells (P < 0.001). Compared to the control and sham-HIFU groups, HIFU-activated lymphocytes elicited significant inhibition of in vivo tumour growth (P < 0.01) and progression (P < 0.0001), and longer survival time in the tumour-bearing mice (P < 0.001). Conclusions: HIFU could enhance CTL’s specific antitumour immunity. Adoptive transfer of HIFU-activated T lymphocytes could increase local antitumour immunity, and elicit stronger inhibition on tumour growth and progression, with more survival benefit in the autologous tumour-bearing mice.

Calreticulin acts as an adjuvant to promote dendritic cell maturation and enhances antigen-specific cytotoxic T lymphocyte responses against non-small cell lung cancer cells

Dendritic cell (DC)-based immunotherapy has promising for treatment of non-small cell lung cancer (NSCLC). Melanoma-associated antigen 3 (MAGE-A3) is a tumor-specific antigen and expressed in approximately 35–40% of NSCLC tissues. Calreticulin (CALR) is a protein chaperone and can enhance DC maturation and antigen presentation. In this study, we evaluated the adjuvant activity of CALR in human DC maturation and their capacity to induce MAGE-A3-specific CD8+ cytotoxic T lymphocyte (CTL) responses to NSCLC in vitro. Infection with recombinant Ad-CALR and/or Ad-MAGE-A3, but not with control Ads, induced CALR and/or MAGE-A3 expression in DCs. Infection with Ad-CALR significantly increased the percentages of CD80+, CD83+, CD86+ and HLA-DR+ DCs and IL-12 secretion, but reduced IL-10 production in DCs. Co-culture of autologous lymphocytes with DC-Ad-CALR or DC-Ad-CM significantly increased the numbers of induced CD8+ CTLs. The percentages of IFNγ-secreting CTLs responding to SK-LU-1 and NCI-H522 NSCLC, but not to non-tumor NL-20 cells in Ad-C-CTL, Ad-M-CTL and Ad-CM-CTL were significantly higher than that of DC-CTL and Ad-null-CTL. Ad-C-CTL, Ad-M-CTL and Ad-CM-CTL, but not control DC-CTL and Ad-null-CTL, induced higher frequency of MAGE-A3+HLA-A2+ NCI-H-522 cell apoptosis, but did not affect the survival of MAGE-A3+HLA-A2- SK-LU-1 and non-tumor NL20 cells in vitro. Treatment with anti-HLA-I antibody, but not with anti-HLA-II, dramatically diminished the cytotoxicity of Ad-CM-CTLs against NCI-H522 cells. Our data indicated that CALR acted as an adjuvant to promote DC maturation, which induced CTL development and enhanced MAGE-A3-specific CTL cytotoxicity against NSCLC.

Abstract PR04: Intravital imaging of cytolytic activity reveals how poor tumor vascularization and hypoxia limit tumor rejection

Abstract Background: Immunotherapies that harness cytotoxic T lymphocytes (CTLs) to eradicate solid tumors, melanoma in particular, have re-emerged as potent clinical options in recent years. Improving the dispersion and cytotoxicity of intratumoral CTLs may further benefit patients. One of the obstacles to successful treatment is thought to be poor vascularization of tumors, which leads to sub-optimal tumor infiltration. In contrast, the effects of disrupted vascularization and the hypoxia it introduces on CTL migration and function within tumors have not been investigated. Methods: To study whether tumor vascularity can shape anti-tumor CTL responses, we combined immunohistochemistry of human melanoma samples with dynamic two-photon imaging within B16 melanoma tumors expressing ovalbumin (OVA) in live mice. Results: In patients, CD8 T cells were twofold denser around peripheral vessels than around deep vessels, and fourfold denser than in avascular areas. This pattern was clearest in poorly-infiltrated tumors. Correspondingly, in mice, adoptively transferred OT-I CTLs, specific for OVA, preferentially infiltrated B16-OVA tumors through peripheral ICAM1+ vessels. CTLs remained initially at their entry areas, crawling vigorously only within 50µm of flowing blood vessels. These dense CTLs swarms efficiently cleared perivascular areas of tumor cells but left avascular areas intact. In contrast, Foxp3+ regulatory T cells could venture farther away from blood vessels. Target killing was antigen-specific, contact-dependent and perforin-mediated, and required multiple CTLs to engage one target cell to be effective. Several findings converge to suggest that perivascular CTL confinement is oxygen-dependent: occlusion of intratumoral blood vessels resulted in instantaneous and reversible arrest of CTLs. Immunohistology indicated that CTLs avoided hypoxic tumor areas. Hypoxia and mitochondrial uncoupling interfered with CTL migration in vitro and, finally, highly vascular tumors grown in a matrix containing the pro-angiogenic factor bFGF were more easily rejected after CTL injection, displayed delayed growth in untreated mice but were not affected in immunodeficient mice. Although such normalized tumors attracted normal numbers of CTLs, these cells migrated more freely within the tumors. Conclusions: We propose that CTLs depend on oxygen for intratumoral locomotion and cytolysis. Incomplete vascularization promotes patchy infiltration and tumor escape; tumor immunotherapy may thus benefit from an extensive and functional vasculature. Citation Format: Guy Shakhar, Yoav Manaster, Tali Feferman. Intravital imaging of cytolytic activity reveals how poor tumor vascularization and hypoxia limit tumor rejection. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr PR04.

Mesenchymal stromal cells derived from cervical cancer tumors induce TGF-β1 expression and IL-10 expression and secretion in the cervical cancer cells, resulting in protection from cytotoxic T cell activity

Cervical cancer (CeCa) tumors are characterized by increased expression of TGF-β1 and IL-10, which are correlated with downregulated expression of major histocompatibility complex class I antigens (HLA-I) on cancer cells and a reduced immune response mediated by cytotoxic T lymphocytes (CTLs). Mesenchymal stromal cells (MSCs) are important components in the tumor microenvironment that have been suggested to contribute to cancer progression through the induction of TGF-β1 and IL-10. In this study, we provided evidence that MSCs derived from cervical tumors (CeCa–MSCs) cocultured with CeCa cells induced significant expression of TGF-β1 and secretion of IL-10 by CeCa cells compared to MSCs derived from the normal cervix (NCx–MSCs) and normal bone marrow (BM–MSCs; gold standard). This increase in expression was associated with a significant downregulation of HLA-I molecules and protection of the cells against specific CTL lysis. Interestingly, the addition of the neutralizing antibody anti-TGF-β to the CeCa/CeCa–MSCs coculture strongly inhibited the expression and production of IL-10 by CeCa cells. Anti-TGF-β as well as anti-IL-10 also abolished HLA-I downregulation, and reversed the inhibition of CTL cytotoxicity. These results provide evidence that TGF-β1 and IL-10 could play an important role in the downregulation of HLA-I molecules on CeCa cells induced by tumor MSCs. Our findings suggest a novel mechanism through which MSCs may protect tumor cells from immune recognition by specific CTLs.

Retraction Note to: Effect of NS-398, a cyclooxygenase-2 selective inhibitor, on the cytotoxicity of cytotoxic T lymphocytes to ovarian carcinoma cells.

Cyclooxygenase-2 (COX-2) is a key limited enzyme of prostaglandin (PG) synthesis and has been demonstrated to be overexpressed in cancer. N-[2-(cyclohexyloxy)-4-nitropheny]-methane sulfonamide (NS-398) is a special inhibitor of COX-2 and may suppress PGE2 release and promote immune response mechanism of cytotoxic T lymphocytes (CTLs). We aimed to investigate the effect of NS-398 on the PGE2 release, proliferation of ovarian carcinoma cell line CAOV3, and immune cytotoxicity of CTLs. CAOV3 cells were incubated with 0, 50, and 100 μmol/L NS-398 for 24, 48, and 72 h. Cell viability was determined by trypan blue staining. PGE2 was measured using radioimmunoassay. CTLs were generated from peripheral blood mononuclear cells after stimulated by CAOV3 cells or CAOV3 cells treated with NS-398 when IL-2 existed. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to evaluate the cytotoxicity of CTLs. As expected, the amount, cell density, cellular volume, and growth speed of CAOV3 cells incubated with NS-398 were significantly decreased compared with control group. This difference became more obvious with an increase in the NS-398 concentration and incubation time. Similarly, PGE2 released from CAOV3 cells treated with 50 and 100 μmol/L NS-398 was significantly decreased compared with control group (P < 0.05). There was also significance in PGE2 between the two groups treated with 50 and 100 μmol/L NS-398 (P < 0.05). Compared with control group, the killing rates of CTLs to CAOV3 treated with 50 and 100 μmol/L NS-398 were significantly increased (P < 0.05). However, there is no significant difference between them. Together, our results suggest that NS-398 could be useful in prevention and therapy of ovarian carcinoma.

Effects of extracellular pH and hypoxia on the function and development of antigen-specific cytotoxic T lymphocytes

The major effector cells for cellular adaptive immunity are CD8+ cytotoxic T lymphocytes (CTLs), which can recognize and kill virus-infected cells and tumor cells. Although CTLs exhibit strong cytolytic activity against target cells in vitro, a number of studies have demonstrated that their function is often impaired within tumors. Nevertheless, CTLs can regain their cytotoxic ability after escaping from the tumor environment, suggesting that the milieu created by tumors may affect the function of CTLs.As for the tumor environment, the patho-physiological situation present in vivo has been shown to differ from in vitro experimental conditions. In particular, low pH and hypoxia are the most important microenvironmental factors within growing tumors. In the present study, to determine the effect of these factors on CTL function in vivo, we examined the cytolytic activity of CTLs against their targets using murine CTL lines and the induction of these cells from memory cells under low pH or hypoxic conditions using antigen-primed spleen cells. The results indicated that both cytotoxic activity and the induction of functional CTLs were markedly inhibited under low pH. In contrast, in hypoxic conditions, although cytotoxic activity was almost unchanged, the induction of CTLs in vitro showed a slight enhancement, which was completely abrogated in low pH conditions.Therefore, antigen-specific CTL functions may be more vulnerable to low pH than to the oxygen concentration in vivo. The findings shown here provide new therapeutic approaches for controlling tumor growth by retaining CTL cytotoxicity through the maintenance of higher pH conditions.

Enhanced therapeutic effects against murine colon carcinoma induced by a Colon 26/Ag85A-CD226 tumor cell vaccine.

Genetically modified tumor cells represent one of the most effective cancer vaccine strategies. In the present study, we describe our approach for inducing an immune response against a colon carcinoma in BALB/c mice, using a Colon 26 tumor cell line expressing Ag85A and CD226. We investigated whether CD226 plays a promotive role for Ag85A against Colon 26 colon carcinoma. The therapeutic efficacy was investigated. The cytotoxic T lymphocyte (CTL) and natural killer (NK) cell cytotoxicity were assessed. Dynamic changes in interferon (IFN)-γ levels in the spleen and the number of IFN-γ-producing CD4+ or CD8+ T cells in the spleen or mesenteric lymph nodes were detected by enzyme-linked immunoabsorbent assay or flow cytometry. Extended survival times, delayed appearances of tumors, and reduced tumor volumes were achieved by preventive vaccination with the Colon 26/Ag85A-CD226 tumor cell vaccine. NK cell or CTL cytotoxicity in the spleens of mice immunized with the Colon 26/Ag85A-CD226 tumor cell vaccine was significantly higher than that in the other treatment groups. The numbers of CD4+ IFN-γ+ and CD8+ IFN-γ+ T cells were both significantly increased in mice immunized with the Colon 26/Ag85A-CD226 tumor cell vaccine in both the spleen and mesenteric lymph nodes. Our results indicated that the tumor vaccine expressing Ag85A and CD226 induced more intensive antitumor immunity than tumor vaccine expressing Ag85A or CD226 only. Moreover, the results suggest that Ag85A and CD226 play a synergistic antitumor effect and CD226 could be used as a genetic adjuvant to enhance the effects of Ag85A vaccine against murine colon carcinoma.

CD8+ T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

AbstractImmune thrombocytopenia (ITP) is a common autoimmune bleeding disorder characterized by autoantibodies targeting platelet surface proteins, most commonly GPIIbIIIa (αIIbβ3 integrin), leading to platelet destruction. Recently, CD8+ cytotoxic T-lymphocytes (CTLs) targeting platelets and megakaryocytes have also been implicated in thrombocytopenia. Because steroids are the most commonly administered therapy for ITP worldwide, we established both active (immunized splenocyte engraftment) and passive (antibody injection) murine models of steroid treatment. Surprisingly, we found that, in both models, CD8+ T cells limited the severity of the thrombocytopenia and were required for an efficacious response to steroid therapy. Conversely, CD8+ T-cell depletion led to more severe thrombocytopenia, whereas CD8+ T-cell transfusion ameliorated thrombocytopenia. CD8+ T-regulatory cell (Treg) subsets were detected, and interestingly, dexamethasone (DEX) treatment selectively expanded CD8+ Tregs while decreasing CTLs. In vitro coculture studies revealed CD8+ Tregs suppressed CD4+ and CD19+ proliferation, platelet-associated immunoglobulin G generation, CTL cytotoxicity, platelet apoptosis, and clearance. Furthermore, we found increased production of anti-inflammatory interleukin-10 in coculture studies and in vivo after steroid treatment. Thus, we uncovered subsets of CD8+ Tregs and demonstrated their potent immunosuppressive and protective roles in experimentally induced thrombocytopenia. The data further elucidate mechanisms of steroid treatment and suggest therapeutic potential for CD8+ Tregs in immune thrombocytopenia.

Construction and evaluation of DNA vaccine encoding Hantavirus glycoprotein N-terminal fused with lysosome-associated membrane protein

BackgroundHantaviral diseases can have a high case fatality rate within the absence of broadly effective antiviral treatments or vaccines. We developed a DNA vaccine targeting the Hantavirus glycoprotein N-terminal (Gn) to major histocompatibility complex class II compartment by fusing the antigen with lysosome-associated membrane protein 1 (LAMP1), which altered antigen presenting pathway and activated the CD4+ T cells. MethodsThe segments of Gn and LAMP1 were cloned into vector pVAX1, and recombinant plasmid was constructed by inserting Gn sequence into LAMP1, between luminal and the transmembrane/cytoplasmic domains. Subsequently, the protein expression was identified through immunoprecipitation, western blot and Immunofluorescent assay. Adaptive immune responses were assessed by the presence of specific and neutralizing antibodies, interferon (ELISpot results, and cytotoxic T-lymphocyte (CTL) cytotoxicity. Epitope mapping was performed to study the T-cell epitopes. Protective immunity in vivo was evaluated using a novel HTNV-challenging model, and safety evaluation was based on histological and behavioral observations. ResultsNative or LAMP1 targeting HTNV Gn was successfully identified. Humoral immune responses were enhanced, featuring with satisfying titers of specific and neutralizing antibody production. The boosted activities of IFN-γ and CTL cytotoxicity witnessed enhanced cellular immune responses. Effective protection against HTNV in vivo was conferred in all three vaccine groups by the challenge model. Safety was confirmed and one dominant T-cell epitope screened from immunized mice overlapped the specific T-cell hot spot in HFRS patients. ConclusionLAMP1 targeting strategy successfully enhanced the efficacy of HTNV Gn-based vaccine, which is highly immunogenic and safe, showing promise for immunoprophylaxis against HFRS. Further investigations are warranted in the future.

ADAP and SKAP55 deficiency suppresses PD-1 expression in CD8+ cytotoxic T lymphocytes for enhanced anti-tumor immunotherapy.

PD-1 negatively regulates CD8+ cytotoxic T lymphocytes (CTL) cytotoxicity and anti-tumor immunity. However, it is not fully understood how PD-1 expression on CD8+ CTL is regulated during anti-tumor immunotherapy. In this study, we have identified that the ADAP-SKAP55 signaling module reduced CD8+ CTL cytotoxicity and enhanced PD-1 expression in a Fyn-, Ca2+-, and NFATc1-dependent manner. In DC vaccine-based tumor prevention and therapeutic models, knockout of SKAP55 or ADAP showed a heightened protection from tumor formation or metastases in mice and reduced PD-1 expression in CD8+ effector cells. Interestingly, CTLA-4 levels and the percentages of tumor infiltrating CD4+Foxp3+ Tregs remained unchanged. Furthermore, adoptive transfer of SKAP55-deficient or ADAP-deficient CD8+ CTLs significantly blocked tumor growth and increased anti-tumor immunity. Pretreatment of wild-type CD8+ CTLs with the NFATc1 inhibitor CsA could also downregulate PD-1 expression and enhance anti-tumor therapeutic efficacy. Together, we propose that targeting the unrecognized ADAP-SKAP55-NFATc1-PD-1 pathway might increase efficacy of anti-tumor immunotherapy.