Murine Cytotoxic T Lymphocytes Research Articles

C-Met+ Cytotoxic T Lymphocytes Exhibit Enhanced Cytotoxicity in Mice and Humans In Vitro Tumor Models.

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.

Cytotoxic T Lymphocyte Activation Signals Modulate Cytoskeletal Dynamics and Mechanical Force Generation.

Cytotoxic T lymphocytes (CTLs) play an integral role in the adaptive immune response by killing infected cells. Antigen presenting cells (APCs), such as dendritic cells, present pathogenic peptides to the T cell receptor on the CTL surface and co-stimulatory signals required for complete activation. Activated CTLs secrete lytic granules containing enzymes that trigger target cell death at the CTL-target contact, also known as the immune synapse (IS). The actin and microtubule cytoskeletons are instrumental in the killing of CTL targets. Lytic granules are transported along microtubules to the IS, where granule secretion is facilitated by actin depletion and recovery. Furthermore, actomyosin contractility promotes target cell death by mediating mechanical force exertion at the IS. Recent studies have shown that inflammatory cytokines produced by APCs, such as interleukin-12 (IL-12), act as a third signal for CTL activation and enhance CTL proliferation and effector function. However, the biophysical mechanisms mediating such enhanced effector function remain unclear. We hypothesized that the third signal for CTL activation, IL-12, modulates cytoskeletal dynamics and force exertion at the IS, thus potentiating CTL effector function. Here, we used live cell total internal reflection fluorescence (TIRF) microscopy to study actomyosin and microtubule dynamics at the IS of murine primary CTLs activated in the presence of peptide-MHC and co-stimulation alone (two signals), or additionally with IL-12 (three signals). We found that three signal-activated CTLs have altered actin flows, myosin dynamics and microtubule growth rates as compared to two signal-activated CTLs. We further showed that lytic granules in three-signal activated CTLs are less clustered and have lower velocities than in two-signal activated CTLs. Finally, we used traction force microscopy to show that three signal-activated CTLs exert greater traction forces than two signal-activated CTLs. Our results demonstrate that activation of CTLs in the presence of IL-12 leads to differential modulation of the cytoskeleton, thereby augmenting the mechanical response of CTLs to their targets. This indicates a potential physical mechanism via which the third signal can enhance the CTL response.

Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21.

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory activity of NK and CD8+ T-cells. Here we describe four formats of novel IL21-based antibody–cytokine fusion proteins, targeting the extra domain A (EDA) of fibronectin and explore their potential for cancer treatment. The fusion proteins were designed, expressed, and characterized. F8 in single-chain diabody (scDb) format fused to IL21 at its C-terminus exhibited a promising profile in size exclusion chromatography (SEC) and SDS-PAGE. The lead candidate was further characterized in vitro. A cell-based activity assay on murine cytotoxic T-cells showed that human IL21, compared to murine IL21 partially cross-reacted with the murine receptor. The prototype was able to recognize EDA as demonstrated by immunofluorescence analysis on tumor sections. In an in vivo quantitative biodistribution experiment, F8(scDb)-murine IL21 did not preferentially accumulate at the site of disease after intravenous injection, suggesting that additional protein engineering would be required to improve the tumor-homing properties of IL21-based product.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

The synergistic antitumor effect of arsenic trioxide combined with cytotoxic T cells in pulmonary metastasis model of colon cancer.

Adoptive T cell therapy, including cytotoxic T lymphocytes (CTLs), represents a promising non-toxic anticancer strategy. The effects of this therapy can be impaired by tumor-infiltrated regulatory T cells (Tregs). Autologous murine CTLs acquired using cryopreservation exhibited a cytotoxic effect equivalent to that of conventional CTLs. The killing activity of CTLs was enhanced significantly using arsenic trioxide (ATO), accompanied by reduction in Tregs in vitro. Results using a pulmonary metastasis model of colon cancer indicated that compared with the control group, ATO group, and CTLs group, metastatic node number decreased significantly (p<0.001, p<0.001, p<0.001, respectively) and survival time was prolonged (p<0.001, p=0.669, p=0.158, respectively) in the ATO plus CTLs group. The number of infiltrated Foxp3+ Tregs decreased in the tumor center, but increased in the peri-tumor tissue. Our results indicate that this approach represents a practical protocol for acquiring autologous CTLs and a feasible strategy that uses a synergistic combination of ATO plus CTLs to treat pulmonary metastases of colon cancer.

Identification of a novel population of highly cytotoxic c-Met-expressing CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are critical mediators of anti-tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen-presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c-Met (c-Met+ CTLs). Phenotypic and functional analysis of c-Met+ CTLs reveals that they display enhanced cytolytic capacities compared to their c-Met- CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c-Met+ CTLs in cell-mediated cytotoxicity reactions in vitro and in vivo and abrogates T-cell responses against metastatic melanoma in vivo Finally, we establish in three murine tumor settings and in human melanoma tissues that c-Met+ CTLs are a naturally occurring CD8+ T-cell population. Together, our findings suggest that the HGF/c-Met pathway could be exploited to control CD8+ T-cell-mediated anti-tumor immunity.

Expression of JAK3 Sensitive Na+ Coupled Glucose Carrier SGLT1 in Activated Cytotoxic T Lymphocytes

Background: Similar to tumor cells, activated T-lymphocytes generate ATP mainly by glycolytic degradation of glucose. Lymphocyte glucose uptake involves non-concentrative glucose carriers of the GLUT family. In contrast to GLUT isoforms, Na⁺-coupled glucose-carrier SGLT1 accumulates glucose against glucose gradients and is effective at low extracellular glucose concentrations. The present study explored expression and regulation of SGLT1 in activated murine splenic cytotoxic T cells (CTLs) and human Jurkat T cells. Methods: FACS analysis, immunofluorescence, confocal microscopy, chemiluminescence and Western blotting were employed to estimate SGLT1 expression, function and regulation in lymphocytes, as well as dual electrode voltage clamp in SGLT1 ± JAK3 expressing Xenopus oocytes to quantify the effect of janus kinase3 (JAK3) on SGLT1 function. Results: SGLT1 is expressed in murine CTLs and also in human Jurkat T cells. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake was significantly decreased by SGLT1-blocker phloridzin (0.2 mM) and by pharmacological inhibition of JAK3 with WHI-P131 (156 µM), WHI-P154 (11.2 µM) and JAK3 inhibitor VI (0.5 µM). Electrogenic glucose transport (I_glucose) in Xenopus oocytes expressing human SGLT1 was increased by additional expression of human wild type JAK3, active ^A568VJAK3 but not inactive ^K851AJAK3. Coexpression of JAK3 enhanced the maximal transport rate without significantly modifying affinity of the carrier. I_glucose in SGLT1+JAK3 expressing oocytes was significantly decreased by WHI-P154 (11.2 µM). JAK3 increased the SGLT1 protein abundance in the cell membrane. Inhibition of carrier insertion by brefeldin A (5 µM) in SGLT1+JAK3 expressing oocytes resulted in a decline of I_glucose, which was similar in presence and absence of JAK3. Conclusions: SGLT1 is expressed in murine cytotoxic T cells and human Jurkat T cells and significantly contributes to glucose uptake in those cells post activation. JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, an effect enforcing cellular glucose uptake into activated lymphocytes and thus contributing to the immune response.

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.

Depletion of tumor-associated macrophages enhances the anti-tumor immunity induced by a Toll-like receptor agonist-conjugated peptide

It has been reported that lipopeptides can be used to elicit cytotoxic T lymphocyte (CTL) responses against viral diseases and cancer. In our previous study, we determined that mono-palmitoylated peptides can enhance anti-tumor responses in the absence of adjuvant activity. To investigate whether di-palmitoylated peptides with TLR2 agonist activity are able to induce anti-tumor immunity, we synthesized a di-palmitic acid-conjugated long peptide that contains a murine CTL epitope of HPV E749–57 (Pam2IDG). Pam2IDG stimulated the maturation of bone marrow-derived dendritic cells (BMDCs) through TLR2/6. After immunization, Pam2IDG induced higher levels of T cell responses than those obtained with its non-lipidated counterpart (IDG). In the prophylactic model, Pam2IDG immunization completely inhibited tumor growth, whereas IDG immunization was unable to inhibit tumor growth. However, Pam2IDG immunization could not effectively inhibit the growth of established tumors. Therefore, we further investigated whether the depletion of immunosuppressive factors could improve the therapeutic effects of Pam2IDG. Our data indicate that treatment with Pam2IDG combined with clodronate/liposome delays tumor growth and increases the survival rate. We also observed that the therapeutic effects of Pam2IDG are improved by diminishing the function of tumor-associate macrophages (TAMs) and through the use of an IL10 receptor blocking antibody or a Cyclooxygenase 2 (Cox-2) inhibitor. In conclusion, the depletion of TAMs may enhance the anti-tumor immunity of a TLR2 agonist-conjugated peptide.

Investigating the role of inducible T cell kinase in CD8+ T-lymphocyte cytolytic effector function (HUM8P.343)

Abstract CD8+ cytotoxic T lymphocytes (CTLs) are critical for eliminating virally infected cells, and defects in CTL responses are implicated in lymphoproliferative diseases. Patients with mutations in inducible T cell kinase (Itk), a tyrosine kinase that serves as an amplifier of T cell receptor (TCR) signaling, develop lymphoproliferative disease associated with ineffective responses against EBV, a virus that infects B cells. This phenotype is similar to X-linked lymphoproliferative syndrome, where mutations in the adaptor SAP lead to fatal EBV infections associated with specific defects in B cell cytolysis. Unlike SAP-deficient CTLs however, we found CTLs from Itk-deficient mice exhibit impaired killing of multiple targets, suggesting Itk-deficiency leads to global defects in cytolysis. Killing by CTLs begins when TCR signaling triggers adherence to targets, centrosome polarization, and release of secretory granules inducing cytolysis of targets. Although early events such as adhesion, actin ring formation, and polarization of lytic machinery were intact in Itk-deficient murine CTLs, we found defects in granule secretion suggesting Itk may play an unappreciated role in the final stages of killing. Exploration of the role of Itk in CTL function may provide new insight into the contribution of early TCR signaling to late stages of cytolysis, and why mutations in Itk lead to lymphoproliferative diseases.

Pharmacological Inhibition of TPL2/MAP3K8 Blocks Human Cytotoxic T Lymphocyte Effector Functions

CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens. During development, antigen-presenting cells secrete innate cytokines such as IL-12 and IFN-α, which drive CTL differentiation into diverse populations of effector and long-lived memory cells. Using whole transcriptome analyses, the serine/threonine protein kinase Tpl2/MAP3K8 was found to be induced by IL-12 and selectively expressed by effector memory (TEM) CTLs. Tpl2 regulates various inflammatory pathways by activating the ERK mediated MAP kinase pathway in innate immune cells such as macrophages and dendritic cells. In this study, we found that a specific small molecule Tpl2 inhibitor blocked IFN-γ and TNF-α secretion as well as cytolytic activity of human CTLs. This pathway was specific for human effector CTLs, as the Tpl2 inhibitor did not block IFN-γ and TNF-α secretion from murine effector CTLs. Further, IL-12 failed to induce expression of Tpl2 in murine CTLs, and Tpl2 deficient murine CTLs did not exhibit any functional deficiency either in vitro or in vivo in response to L. monocytogenes infection. In summary, we identified a species-specific role for Tpl2 in effector function of human CTLs, which plays a major role in adaptive immune responses to intracellular pathogens and tumors.

Different Munc13 Isoforms Function as Priming Factors in Lytic Granule Release from Murine Cytotoxic T Lymphocytes

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.

Elucidating immunological synapse organisation: a CAZ-protein study (P1036)

Abstract The highly adaptive and sophisticated network of our immune system ensures a potent defense against a variety of possible threats. As a part of this machinery, cytotoxic T lymphocytes (CTLs), upon antigen recognition, are inducing programmed cell death by forming an immunological synapse (IS) between CTL and target cell, followed by release of granzyme B and perforin from lytic granules into the synaptic cleft. Whereas some mechanisms underlying the fusion of lytic granules with the plasma membrane have already been determined, temporal and spatial organization of these vesicles right before their fusion at the IS still remains elusive. From chemical synapses in the hippocampus it is known, that vesicle fusion occurs only at the active zone which is being formed by an exclusive number of CAZ (cytomatrix of the active zone) proteins like the 420 kDa protein bassoon. Our qRT-PCR data from primary human and murine CTLs show that CAZ proteins are not only expressed, but are specifically upregulated in activated CTLs, suggesting a possible role during the killing process for example as scaffolding proteins. As specific target killing is a process of paramount importance for survival, it seems likely that vesicle fusion at the IS is precisely organized as well. We present data from primary CTLs derived from CAZ-deficient mice that investigate a possible contribution of CAZ proteins in the organisation and function of the IS.

Different Munc13 isoforms function as priming factors in lytic granule release from murine cytotoxic T lymphocytes (P1044)

Abstract In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues like brain and neuroendocrine glands this process is mediated by members of the Munc13 protein family. In human CTLs mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3 (FHL3) suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs. We demonstrate on both mRNA and protein level that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Furthermore mobility studies of LGs performed with TIRF micrscopy indicate that Munc13-4 as well as Munc13-1 are responsible for the priming process of LGs. Even more the domains of the Munc13 protein which are responsible for functional fusion could be identified. We conclude from these data that both isoforms of the Munc13 family are functionally redundant and identify Munc13-1 as an alternative target for immunotherapy in patients suffering from FHL3.

Zinc signals promote IL‐2‐dependent proliferation of T cells

Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T-cell activation by IL-2 was investigated in the murine cytotoxic T-cell line CTLL-2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin-3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL-2-receptor. Activation of the ERK-pathway was blocked by chelation of free zinc with N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK-specific gene expression of c-fos and IL-2-induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK-dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL-2-induced ERK signaling and proliferation of T cells.

Paclitaxel inhibits killing by murine cytotoxic T lymphocytes in vivo but not in vitro

To kill antigen-specific target cells (TCs), cytotoxic T lymphocytes (CTLs) reorganise their microtubule cytoskeleton to deliver lytic granules to the TCs. We used two drugs that stabilise microtubules, paclitaxel and peloruside, to determine how the stabilising microtubule network affects CTL function in vitro and in vivo. In vitro, neither paclitaxel nor peloruside inhibited antigen-specific killing, lytic granule delivery to the cell surface, nor interferon-gamma release by murine CTLs. In contrast, in an in vivo murine model of antigen-induced killing, a single dose of paclitaxel had a significant inhibitory effect on killing by CTLs. Furthermore, the inhibitory effect of paclitaxel was not caused by specific deletion of the effector CTL population in drug-treated mice. The findings reveal that microtubule-stabilising drug treatment can lead to immediate impairment of CTL function without affecting lytic granule release. The results also suggest that patients undergoing taxane anti-cancer therapy may be impaired in their ability to fight infection before the anti-mitotic effects of paclitaxel are apparent.

Immunosuppressive effect of angiotensin receptor blocker on stimulation of mice CTLs by angiotensin II

While angiotensin II, which is produced by the renin–angiotensin–aldosterone system, is considered to be the major regulator molecule that controls both the blood pressure and fluid system, there is an increasing body of evidence that this bioactive peptide and its receptor might also contribute to the immune system. However, there are few details known about the direct effect that angiotensin type I receptors (AT1R) have on the cytotoxic T cell (CTL). To clarify the relationship between angiotensin II and its CTL receptor, we used murine splenic and antigen-specific CTLs. Murine CTLs constantly expressed AT1R, with the activation of the AT1R expression strengthened by both anti-CD3 Ab and the use of an antigen-specific methodology. Moreover, the production of IFN-γ and TNF-α through CTL stimulation can be inhibited by the selective AT1R inhibitor, Losartan. In particular, the TNF-α production from activated CTL that had been magnified by angiotensin II, was nullified by the AT1R inhibitor. However, a cytotoxic assay indicated it did not have any effect on the cognate interaction of the CTLs. In addition, the antigen-specific CTL induction by immunization with the CTL antigenic peptide was reduced by angiotensin II type 1 receptor blocker (ARB) in vivo. These findings suggest that ARBs might have the ability to suppress excessive antigen-specific activation and induction of CTLs promoted by angiotensin II.

Duodenase, a New Serine Protease of Unusual Specificity from Bovine Duodenal Mucosa

In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation, carboxymethyl-cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29 ± 0.5 kDa by SDS/PAGE and G-2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 ± 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars. The enzyme displays trypsin-like and chymotrypsin-like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9–8.2. Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an activ-site serine in this protease. α-N-Tosyl-l-lysine chloromethane and α-N-tosyl-l-phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin-like and chymotrypsin-like activities of duodenase. The enzyme activity was strongly supressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity. The N-terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T-lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases. The biological role of duodenase is discussed.

Control of mesothelin-expressing ovarian cancer using adoptive transfer of mesothelin peptide-specific CD8+ T cells

Cancer immunotherapy targeting mesothelin represents a potentially plausible approach for the control of ovarian cancer as most ovarian cancers express high levels of mesothelin. In the current study, we created a mesothelin-positive luciferase-expressing ovarian cancer model, MOSEC/luc. This luciferase-expressing tumor model allowed us to quantitate tumor distribution and tumor load in tumor-challenged mice using a non-invasive bioluminescence imaging system. In addition, we identified an H-2D(b)-restricted mesothelin peptide-specific cytotoxic T-lymphocyte (CTL) epitope (amino acid (aa) 406-414) that was endogenously processed and presented by MOSEC/luc tumor cells. We showed that adoptive transfer of mesothelin peptide (aa406-414)-specific CD8(+) T cells led to the control of MOSEC/luc tumor cells. The MOSEC/luc tumor model and the newly identified H-2D(b)-restricted murine mesothelin-specific CTL epitope (aa406-414) will be very useful for the development of immunotherapy for ovarian cancer as well as for the development of quantitative CD8(+) T cell-mediated immunological assays.

Free fatty acid release from human breast cancer tissue inhibits cytotoxic T-lymphocyte-mediated killing

Immune-mediated antitumor activities confront a variety of tumor-mediated defense mechanisms. Here, we describe a new mechanism involving FFA that may allow breast cancer to evade immune clearance. We determined the IC50 at which unbound free fatty acids (FFA(u)) inhibit murine cytotoxic T-lymphocyte (CTL)-mediated killing to assess the physiologic relevance of this phenomenon. We found that the IC50 for unbound oleate is 125 +/- 30 nM, approximately 200-fold greater than normal plasma levels. FFA inhibition, however, may play an important role in breast cancer because we found that large quantities of FFAs are released constitutively into the media surrounding samples of human breast cancer but not normal or benign tissue. FFA(u) concentration ([FFA(u)]) increased to at least 25 nM in 20 of 22 cancer tissue samples and exceeded 100 nM in 11 patients. Media from these samples inhibited CTL-mediated killing. Extrapolation from our in vitro conditions suggests that for tumor interstitial fluid, in vivo [FFA(u)] may be 300-fold greater than we observed in vitro. Although breast cancer release of FFA may suppress effector cell antitumor activity, strategies that reduce interstitial [FFA(u)] may significantly improve antitumor immune therapies.