Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death.

BACKGROUND: The Akt/mammalian target of rapamycin (mTOR) signalling pathway serves as a critical regulator of cellular growth, proliferation and survival. Akt aberrant activation has been implicated in carcinogenesis and anticancer therapy resistance. Piperlongumine (PL), a natural alkaloid present in the fruit of the Long pepper, is known to exhibit notable anticancer effects. Here we investigate the impact of PL on Akt/mTOR signalling. METHODS: We examined Akt/mTOR signalling in cancer cells of various origins including prostate, kidney and breast after PL treatment. Furthermore, cell viability after concomitant treatment with PL and the autophagy inhibitor, Chloroquine (CQ) was assessed. We then examined the efficacy of in vivo combination treatment using a mouse xenograft tumour model. RESULTS: We demonstrate for the first time that PL effectively inhibits phosphorylation of Akt target proteins in all tested cells. Furthermore, the downregulation of Akt downstream signalling resulted in decrease of mTORC1 activity and autophagy stimulation. Using the autophagy inhibitor, CQ, the level of PL-induced cellular death was significantly increased. Moreover, concomitant treatment with PL and CQ demonstrated notable antitumour effect in a xenograft mouse model. CONCLUSIONS: Our data provide novel therapeutic opportunities to mediate cancer cellular death using PL. As such, PL may afford a novel paradigm for both prevention and treatment of malignancy.

Zinc chelation induces rapid depletion of the X-linked inhibitor of apoptosis and sensitizes prostate cancer cells to TRAIL-mediated apoptosis.

The X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family of endogenous caspase inhibitors, blocks the initiation and execution phases of the apoptotic cascade. As such, XIAP represents an attractive target for treating apoptosis-resistant forms of cancer. Here, we demonstrate that treatment with the membrane-permeable zinc chelator, N,N,N',N',-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces a rapid depletion of XIAP at the post-translational level in human PC-3 prostate cancer cells and several non-prostate cell lines. The depletion of XIAP is selective, as TPEN has no effect on the expression of other zinc-binding members of the IAP family, including cIAP1, cIAP2 and survivin. The downregulation of XIAP in TPEN-treated cells occurs via proteasome- and caspase-independent mechanisms and is completely prevented by the serine protease inhibitor, Pefabloc. Finally, our studies demonstrate that TPEN promotes activation of caspases-3 and -9 and sensitizes PC-3 prostate cancer cells to TRAIL-mediated apoptosis. Taken together, our findings indicate that zinc-chelating agents may be used to sensitize malignant cells to established cytotoxic agents via downregulation of XIAP.

Renal cell carcinoma-derived gangliosides suppress nuclear factor-kappaB activation in T cells.

Activation of the transcription factor nuclear factor-kappaB (NFkappaB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, the suppression of NFkappaB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IkappaBalpha. Tumor-derived soluble products from cultured RCC explants inhibit NFkappaB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IkappaBalpha degradation in these cells. The inhibitory agent has several features characteristic of a ganglioside, including sensitivity to neuraminidase but not protease treatment; hydrophobicity; and molecular weight less than 3 kDa. Indeed, we detected gangliosides in supernatants from RCC explants and not from adjacent normal kidney tissue. Gangliosides prepared from RCC supernatants, as well as the purified bovine gangliosides G(m1) and G(d1a), suppressed NFkappaB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-gamma. Taken together, our findings suggest that tumor-derived gangliosides may blunt antitumor immune responses in patients with RCCs.

Dead or dying: necrosis versus apoptosis in caspase-deficient human renal cell carcinoma.

The antitumor effect of immuno- and chemotherapeutic agents is executed through stimulation of apoptotic programs in susceptible cells. Apoptosis induced in tumor cells requires activation of members of the caspase family of proteases. Deficient expression or activation of caspases may account in part for the failure of many current anticancer therapies. However, recent studies suggest that cell death can proceed in the absence of caspases. We investigated the susceptibility of human renal cell carcinoma (RCC) lines to two distinct modes of cell death, apoptosis and necrosis. RCC lines displayed almost complete resistance to apoptosis in response to the intracellular zinc chelator, N,N,N'N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), which instead induced dramatic accumulation of nonapoptotic necrotic cells. Conversely, TPEN was a potent inducer of apoptosis in caspase-competent normal kidney cells (NK-72) and Jurkat T lymphocytes. Resistance to apoptosis in RCC lines correlated with almost complete loss of caspase-3 expression and variable down-regulation of caspase-7, caspase-8, and caspase-10. These data may explain the resistance of RCC to drugs inducing apoptosis and have important consequences for further attempts to manipulate tumor cell death.

The T cell death knell: immune-mediated tumor death in renal cell carcinoma.

The antitumor effect of T cells is executed either through CD95 or Perforin (PFN)/Granzyme B (GrB) pathways. Induction of apoptosis by either mode requires activation of caspase family members. However, recent studies have suggested that cell death can proceed in the absence of caspase induction and apoptotic events. We investigated the contribution of CD95 and PFN/GrB-mediated cytotoxicity to apoptotic and necrotic mechanisms of cell death in human renal cell carcinoma. Although freshly isolated and cultured tumors expressed CD95 on their surface, they were resistant to CD95-mediated apoptosis. CD95 resistance coincided with decreased levels of FADD protein and diminished caspase-3-like activity. In contrast, we demonstrated that tumor cell death mediated by PFN/GrB can be achieved in the absence of functional caspase activity and is accompanied by a dramatic accumulation of nonapoptotic necrotic cells.

Tumor-induced sensitivity to apoptosis in T cells from patients with renal cell carcinoma: role of nuclear factor-kappaB suppression.

Antitumor immunity fails to adequately develop in many cancer patients, including those with renal cell carcinoma (RCC). A number of different mechanisms have been proposed to explain the immune dysfunction observed in cancer patient T cells. Here we show that T cells from RCC patients display increased sensitivity to apoptosis. Tumor-infiltrating lymphocytes (TILs) display the most profound sensitivity, because 10-15% of those cells are apoptotic when assessed by terminal deoxynucleotidyltransferase-mediated nick end labeling in situ, and the number of apoptotic TILs further increases after 24 h of culture. Peripheral blood T cells from RCC patients are not directly apoptotic, although T lymphocytes derived from 40% of those individuals undergo activation-induced cell death (AICD) upon in vitro stimulation with phorbol myristate acetate and ionomycin. This is in contrast to T cells from normal individuals, which are resistant to AICD. TILs and peripheral blood T cells from RCC patients also exhibit impaired activation of the transcription factor, nuclear factor (NF)-kappaB. Additional findings presented here indicate that the heightened sensitivity of patient T cells to apoptosis may be tumor induced, because supernatants from RCC explants sensitize, and in some instances directly induce, normal T cells to apoptosis. These same supernatants also inhibit NF-kappaB activation. RCC-derived gangliosides may represent one soluble tumor product capable of sensitizing T cells to apoptosis. Pretreatment with neuraminidase, but not proteinase K, abrogated the suppressive effects of tumor supernatants on both NF-kappaB activation and apoptosis. Additionally, gangliosides isolated from tumor supernatants not only inhibited NF-kappaB activation but also sensitized T cells to AICD. These findings demonstrate that tumor-derived soluble products, including gangliosides, may contribute to the immune dysfunction of T cells by altering their sensitivity to apoptosis.

Mechanisms of apoptosis in T cells from patients with renal cell carcinoma.

Tumors may escape immune recognition and destruction through the induction of apoptosis in activated T lymphocytes. Results from several laboratories suggest that FasL (L/CD95L) expression in tumors may be responsible for this process. In this study of patients with renal cell carcinoma (RCC), we provide evidence for two mechanisms of T-cell apoptosis. One mechanism involves the induction of apoptosis via FasL expression in tumor cells. This is supported by several observations, including the fact that tumor cells in situ as well as cultured cell lines expressed FasL mRNA and protein by a variety of techniques. The FasL in RCC is functional because in coculture experiments, FasL+ tumors induced apoptosis in Fas-sensitive Jurkat T cells and in activated peripheral blood T cells but not in resting peripheral blood T cells. Most importantly, antibody to FasL partially blocked apoptosis of the activated T cells. Moreover, Fas was expressed by T cells derived from the peripheral blood (53% median) and tumor (44.3% median) of RCC patients. Finally, in situ staining for DNA breaks demonstrated apoptosis in a subset of T cells infiltrating renal tumors. These studies also identified a second mechanism of apoptosis in RCC patient peripheral T cells. Whereas these cells did not display DNA breaks when freshly isolated or after culture for 24 h in medium, peripheral blood T cells from RCC patients underwent activation-induced cell death after stimulation with either phorbol 12-myristate 13-acetate/ionomycin or anti-CD3/CD28 antibodies. Apoptosis mediated by exposure to FasL in tumor cells or through T-cell activation may contribute to the failure of RCC patients to develop an effective T-cell-mediated antitumor response.

23 assessment of T-cell immune dysfunction in patients with renal cell carcinoma.

Functional T cells are the central component of an effective antitumor immune response. However, in patients with renal cell carcinoma (RCC), the growth of antigenic tumors proceeds in the absence of significant T-cell responses, posing a distinct obstacle to the development of effective immunotherapy strategies and cancer vaccines. The minimum required elements of a functional antitumor immune T-cell response have been identified, including T cells that can preferentially recognize tumor-associated antigens (1). However, despite increasing evidence that T-cells recognize discrete tumor antigen, transformed cells continue to evade immune destruction, and tumors thereby progress. There is now little doubt that the immune response to tumor antigens is altered in patients with cancer (2). This rarely manifests clinically as generalized immune suppression, which may reflect the antigen specificity of the immune dysfunction in the initial stages of the disease.

[The regulating action of oxygen on the nutritional requirements of Neisseria meningitidis]. / Upravliaiushchee deistvie kisloroda na pitatel'nye potrebnosti Neisseria meningitidis.

The study has revealed regularities in changing nutritional requirements of Neisseria meningitidis with changes in the degree of the oxygen saturation of the culture medium in a fermenter under the conditions of the controlled cultivation of N. meningitidis in a synthetic culture medium in the process of batch, semicontinuous and continuous flow cultivation. As shown in this study, when oxygen supply is limited, the consumption of carbohydrates prevails, while in the presence of surplus oxygen the prevalence of the consumption of amino nitrogen is observed.

[The antibody response of animals to a corpuscular meningococcal group-B preparation with different methods of immunization]. / Antitel'nyi otvet zhivotnykh na korpuskuliarnyi meningokokkovyi serogruppy B preparat pri razlichnykh metodakh immunizatsii.

As revealed in animal experiments, the formation of antibodies to group-B N. meningitidis antigens (group-specific polysaccharide, lipopolysaccharide and outer membrane proteins) in response to administration of meningococcal corpuscular preparations depends on the method of administration, the dose, and the number of administrations. In the sera of rabbits, immunized orally, antibodies to all three antigens in sufficiently high titers have been detected.

Caspase-dependent and -independent death pathways in cancer therapy.

The majority of current anticancer therapies induce tumor cell death through the induction of apoptosis. Alterations in the apoptotic pathways may determine tumor resistance to these therapies. Activation of the proteolytic cascade involving caspase family members is a critical component of the execution of cell death in apoptotic cells. However, recent studies suggest that cell death can proceed in the absence of caspases. In this review we describe the role of caspase-dependent and -independent pathways as targets for anticancer treatment. A better understanding of diverse modes of tumor cell death will help to avoid ineffective treatment and provide a molecular basis for the new strategies targeting caspase-independent death pathways in apoptosis-resistant forms of cancer.

Inhibition of NFkappaB induces caspase-independent cell death in human T lymphocytes.

Nuclear factor kappaB (NFkappaB) regulates the expression of various genes essential for cell survival. Here we demonstrate that suppression of NFkappaB nuclear import with SN50 peptide carrying the nuclear localization sequence (NLS) of the NFkappaB p50 subunit induces apoptosis in human peripheral blood T lymphocytes (T-PBL), which can be blocked with the pan-caspase inhibitor Z-VAD.fmk. However, even when caspase function is blocked, the addition of SN50 induces irreversible cell loss due to the reduction in the mitochondrial transmembrane potential (DeltaPsim) followed by disruption of the cell membrane, hallmarks of necrosis. These observations demonstrate that although inhibition of NFkappaB nuclear translocation by SN50 peptide can induce caspase-dependent apoptosis in T-PBL, cell death may still proceed in the absence of functional caspase activity. The availability of downstream caspases appears to determine the mode of cell death in NFkappaB defective cells.

Inhibition of NF-kappa B activity in human T lymphocytes induces caspase-dependent apoptosis without detectable activation of caspase-1 and -3.

NF-kappa B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-kappa B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-kappa B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kappa B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk, suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kappa B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45. These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cells resulting in NF-kappa B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-kappa B in survival of naive PBL.

Mechanism of apoptosis induced by zinc deficiency in peripheral blood T lymphocytes.

Alterations in intracellular Zn(2+) concentrations are believed to play a crucial role in modulating apoptosis. The observation that Zn(2+) deficiency can induce cell death both in vivo and in vitro has been attributed to the fact that exchange of Zn(2+) for Ca(2+) and Mg(2+) within the nuclei may directly activate endogenous endonucleases therefore inducing DNA fragmentation independent of cytoplasmic factors. Here we show that the membrane-permeable zinc chelator, N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces translocation of cytochrome c from the mitochondrial intramembranous space into the cytosol in human peripheral blood T lymphocytes (PBL) with subsequent activation of caspases-3, -8, and -9. Pretreatment of T lymphocytes with caspase inhibitors Z-VAD.fmk or DEVD.fmk prevented DNA fragmentation in response to TPEN indicating that apoptosis triggered by zinc deficiency is entirely dependent on activation of caspase family members. The release of cytochrome c and activation of downstream caspases precedes changes in the mitochondrial transmembrane potential (Delta Psim). Therefore, cytoplasmic and mitochondrial events are critical to this process.

T cells infiltrating non-Hodgkin's B cell lymphomas show altered tyrosine phosphorylation pattern even though T cell receptor/CD3-associated kinases are present.

Although tumor infiltrating lymphocytes (T-TIL) from B cell non-Hodgkins lymphoma patients contain tumor-reactive T cells, they display poor proliferation and IFN-gamma production when stimulated through the TCR-CD3. To determine if there was altered signaling linked to TCR-CD3 ligation, tyrosine phosphorylation was examined in T-TIL because it represents an early and critical event in T cell activation. After stimulation with anti-CD3 Ab, Western blotting with anti-phosphotyrosine showed reduced phosphorylation in T-TIL when compared with peripheral blood-derived T cells from normal individuals. The altered phosphorylation was not due to the reduced expression of signaling elements linked to the TCR-CD3 complex. T-TIL expressed normal levels of CD3 epsilon, TCR zeta chain, and the three tyrosine kinases, p56lck (Lck), p59fyn, and ZAP-70. However, in T-TIL, anti-Lck Ab reacted with a 60-kDa protein, which appears to be the phosphorylated form of Lck. Binding of anti-Lck Ab to the 60-kDa protein was blocked by Lck peptide. In addition, anti-Lck Ab immunoprecipitated a phosphorylated 60-kDa protein from gamma-32P-labeled T-TIL that was not seen in normal resting T cells. In vitro kinase assay studies also demonstrated that TCR-CD3 engagement increased the kinase activity of Lck in normal T cells but not in T-TIL. These results suggest that although T-TIL from B cell non-Hodgkins lymphoma patients contain the signal transduction molecules associated with TCR-CD3 activation pathway, they are impaired in tyrosine phosphorylation and Lck activity, which may contribute to the functional defects of these cells.

Non-specific modulation of the immune response with liposomal meningococcal lipopolysaccharide: role of different cells and cytokines.

The immunomodulating action of Neisseria meningitidis lipopolysaccharide (LPS) incorporated into liposomes and the activation of different populations of immunocompetent cells or the secretion of cytokines were studied. LPS stimulated an anti-sheep red blood cell (SRBC) plaque-forming cell response in the spleen of mice after simultaneous injection of LPS and SRBC but if LPS was administered 3 days before the immunization with SRBC the response to SRBC was strongly suppressed. After the incorporation of LPS into liposomes the stimulation index was increased from 6 to 19 and the liposomal LPS did not suppress the immune response to SRBC. The incorporation of LPS into liposomes leads to enhancement of B-mitogenic properties of LPS, as liposomal LPS stimulated the proliferation of splenocytes in mice better than free LPS and has no influence on the thymocytes. The liposomal LPS induced more prolonged and significant accumulation of IgM-secreting cells in the spleen of mice in comparison with the free LPS. Liposomal LPS also induced more active accumulation of IFN-gamma in human peripheral blood mononuclear cells and less active accumulation of monokines, contributing to the realization of the toxic properties of endotoxin (IL-1 alpha, TNF-alpha, IL-6 and GM-CSF). These results demonstrated that the incorporation of N. meningitidis LPS into liposomes dramatically changed its immunomodulating activity. The data obtained are important for the construction of an adjuvant formulation for synthetic immunogens capable of inducing genetically unrestricted immune responses.

Prevalence and patterns of self-initiated nutritional supplementation in men at high risk of prostate cancer.

OBJECTIVE: To define the prevalence and patterns of self-initiated herbal and vitamin supplementation among men at high risk of developing prostate cancer, as there is increasing public awareness of prostate cancer screening, risk-factor assessment and prevention, leading to increasing interest in the use and systematic study of nutritional therapies for prostate cancer prevention. SUBJECTS AND METHODS: Since 1996 our institution has prospectively maintained a prostate cancer-risk registry through its Prostate Cancer Risk Assessment Program (PRAP). Eligibility includes African-American men, any man with at least one first-degree relative or two or more second-degree relatives with prostate cancer, or men who tested positively for the BRCA1 gene mutation. A 420-item self-administered questionnaire was completed and included the use of nutritional supplements and complementary therapies. We divided men into groups who used supplements to lessen their cancer risk and those who did not. The prevalence and patterns of use were evaluated and the two groups then compared for differences in demographic, socio-economic and risk-perception variables. RESULTS: In all, 345 high-risk men were enrolled in the PRAP over a 5-year period. Data on the use of dietary or herbal supplements were available on 333 men (97%), of whom over half (170) reported taking one or more supplements to prevent prostate cancer. Supplement use was divided into eight categories, including vitamins, minerals, extracts from fruits/seeds, organic compounds, flowers/bulbs, leaves/bark, roots, or animal products. Most commonly used for self-initiated chemoprevention were vitamins (95%), minerals (28%), and fruit/seed extracts (18%). More than a quarter of men (27%) took three or more agents. Men taking proactive preventative measures were statistically more likely to be Caucasian and aged > 60 years (P < 0.05). African-Americans were less likely to self-initiate preventative steps. Men taking supplements tended to return more often for follow-up and participate in PRAP longer, while those not taking supplements tended to earn less and report less self-perceived risk. CONCLUSIONS: A significant proportion of men at risk of developing prostate cancer initiate measures they perceive to reduce their risk. Although the chemopreventative efficacy of many of these supplements remains unsubstantiated, they are widely perceived by the public to reduce the risk of developing prostate cancer. These data provide an insight into patient perceptions and misconceptions of chemopreventative strategies, and may help to refine recruitment efforts in multi-institutional prostate cancer prevention trials.

Tumor-induced dysfunction in interleukin-2 production and interleukin-2 receptor signaling: a mechanism of immune escape.

PURPOSE: The development of an effective antitumor immune response is compromised in patients with renal cell carcinoma. Despite significant infiltration by T lymphocytes into renal tumors, no detectable induction of gene expression is associated with the generation of an antitumor immune response. Tumor-induced down-regulation of interleukin (IL)-2 expression may contribute to the impaired development of the T cell-mediated antitumor immune response. Within renal tumors, there is no detectable expression of IL-2 or the IL-2 receptor alpha chain, and only low levels of interferon gamma (IFN-gamma) mRNA are detected. Products in the tumor environment may suppress the expression of these genes, thus inhibiting production of type 1 helper T cell cytokines. METHODS: Peripheral blood lymphocytes obtained from healthy volunteers were exposed to supernatants from renal cell carcinoma explants, and the immunologic consequences of this were assessed using a variety of molecular assays. RESULTS: Soluble products from renal tumor explants can inhibit the production of IL-2 and IFN-gamma by peripheral blood lymphocytes and can suppress T-cell proliferation. Soluble products from renal cell carcinoma explants appear to block the nuclear translocation of nuclear factor kappa B (NFkappaB) proteins p50 and RelA without affecting cytoplasmic levels of these proteins. In some experiments, a reduction in the nuclear translocation of other transcription factors involved in IL-2 gene expression, including nuclear factor of activated T cells and accessory protein-1, was observed. Gangliosides isolated from tumor supernatants blocked the production of IL-2 and IFN-gamma in response to ionomycin plus phorbol myristate acetate stimulation. These gangliosides also inhibited stimulus-dependent activation and nuclear accumulation of NFkappaB. Coculture experiments demonstrated that renal cell carcinoma lines known to express gangliosides could inhibit the activation of NFkappaB in normal T cells and the Jurkat T-cell line. Supernatants from renal cell carcinoma explants and renal cell carcinoma cell lines can also suppress the proliferation of normal T cells, thus reproducing another defect observed in tumor-infiltrating lymphocytes. Supernatants from renal cell carcinoma tumors also appear to inhibit signaling through the IL-2 receptor. Although tumor supernatants had little effect on IL-2 receptor (alpha, beta or gamma) expression, they did block expression of JAK3, a key kinase involved in signaling through the IL-2 receptor pathway. Moreover, downstream events in IL-2 receptor signaling linked to JAK3 were impaired in T cells treated with tumor supernatants. CONCLUSION: These findings suggest that soluble products from renal tumors may suppress T-cell responses by blocking both IL-2 production and normal IL-2 receptor signaling.

Responses to T cell receptor/CD3 and interleukin-2 receptor stimulation are altered in T cells from B cell non-Hodgkin's lymphomas.

T cells infiltrating (T-TIL) B cell non-Hodgkin's lymphomas (NHL) are thought to represent a local host response to the tumor. However, tumor progression in the presence of this T cell infiltrate suggests that the T-TIL may be functionally impaired. To address this issue we determined whether response to stimulation of T-TIL from 25 patients with NHL through the T cell receptor (TCR/CD3) and the interleukin-2 (IL-2) receptor (IL-2R) was intact, since activation of these receptors is important for proliferation and cytokine production. Our results demonstrate defects in response to stimulation via TCR/CD3 and the IL-2R in T-TIL cells from patients with NHL that were not observed with T cells from the peripheral blood. T-TIL showed minimal proliferation to anti-CD3 and only modest proliferation to IL-2 alone or when combined with anti-CD3. Moreover, cytokine production in T-TIL was impaired since stimulation through the TCR/CD3 complex did not induce mRNA for interferon gamma (IFN gamma), IL-2, IL-4 or IL-10. The functional unresponsiveness of these cells may be linked to altered signalling through the TCR/CD3 since an abnormal tyrosine phosphorylation pattern was detected in T-TIL after stimulation with anti-CD3.