In vitro antitumor activity of tumor-infiltrating lymphocytes from human gastric carcinoma

Tumor-infiltrating lymphocytes (TIL) isolated from 11 gastric carcinoma were studied. TIL could grow for a long-term in medium containing recombinant interleukin-2(rIL-2). The mean expansion fold achieved in 6 long-term cultures of 11 specimens was 15.1 RIL-2 expanded gastric TIL exhibited significant cytotoxicity against K562, BGC823, MCF-7 and more effective antitumor cytotoxicity against fresh autologous tumor targets and human gastric cancer cell line. Peak cytotoxicity was shown in the third or fourth week after cultures. Cryopreservation of gastric TIL didn’t influence their expansion capacity and antitumor activity. Phenotypic analysis was demonstrated in this study. The results of present study indicate that TIL from human gastric carcinoma could be expanded and reach high levels of antitumor effector function in long-term cultures with rIL-2. Their function may be of clinical importance.     

Proliferative and cytolytic potentials of purified human tumor-infiltrating T lymphocytes. Impaired response to mitogen-driven stimulation despite T-cell receptor expression

Using limiting dilution analysis (LDA) we have previously shown that in most instances, the frequency (F) of proliferative T lymphocyte precursors (PTL-P) was strikingly reduced in tumor-infiltrating lymphocytes (TIL). In this study involving 19 cases, we show that the impaired clonogenic potential of CD2+ TILs is primarily caused by an intrinsic defect rather than to suppressor T cells or to a direct effect of the tumor cells usually present in the culture system. This was demonstrated by experiments in which the F of PTL-Ps was quantitated both in highly purified CD2+ TILs (using a cell-sorter) and in non-purified TIL suspensions (still containing tumor cells), which originated from the same biopsy specimen. The F of PTL-Ps was virtually identical in either sorted or nonsorted suspensions and the data from LDA were always consistent with the single-hit Poisson model, indicating that no suppressor cells interfered with growth of CD2+ TIL. Stimulation of sorted CD2+ TIL in low-density cultures by either phytohemagglutinin or anti-CD3-monoclonal antibody (MAb) indicated that the antigen-dependent activation pathway was impaired, although structurally intact T-cell receptor (TCR) complexes were apparently expressed, as assessed by immunofluorescence. The depressed proliferative response of CD2+ TIL could not be reversed in vitro when phorbol-esters were used in combination with ionomycin, which bypass the TCR. Nevertheless, 180 clones obtained from 8 cases were analyzed for their cytolytic activity. The majority mediated specific lytic activity (against unknown antigens), as assessed by lectin-dependent cell cytotoxicity, whereas only 6% of them manifested lymphokine-activated killing on appropriate targets.     

Phenotypic analysis of tumor-infiltrating lymphocytes from human breast cancer.

Suspensions of fresh tumor-infiltrating lymphocytes (TIL) were prepared from 30 human breast ductal adenocarcinomas. To evaluate the phenotypic pattern of the isolated TIL, lymphocyte surface markers including CD19, CD3, CD4, CD8, CD16 and HLA-DR were examined by flow cytometry. Lymphocyte recovery ranged from 1.1% to 44%, independent of tumor size. TIL most often scored high for CD3+ with a varying number of CD4+ and CD8+ cells. Three samples out of 30 expressed up to 44% of CD19+ B cells, while CD3-CD16+ NK cells were rare. CD4 and CD8 expression was significantly different between the lymph node metastases group and the lymph node negative group (p < 0.01). 67% of the TIL with a CD4/CD8 ratio greater than 1 showed lymph node metastases. Furthermore, the CD4 expression of TIL and CD4/CD8 ratio correlated with tumor size (p < 0.01), but not with tumor differentiation and hormone receptor expression. Although there was considerable diversity of TIL among breast tumors, our data suggest that a high expression of CD4+ T cells may imply progression of the tumor, and an increased CD4/CD8 ratio of the TIL isolated from human breast adenocarcinoma may indicate development of metastases.     

Characterization of T-cell memory phenotype after in vitro expansion of tumor-infiltrating lymphocytes from melanoma patients

Memory T-cell populations in human antitumor tumor-infiltrating lymphocytes (TILs) for adoptive cell transfer have not been fully characterized. Our studies demonstrated that CD62L, CD27 and CD28 positive effector memory T-cells were present in the TIL samples from the tumor tissues of melanoma patients and T-cell expansion led to the significant loss of memory T-cells. CD27- and CD28-positive T-cells had high levels of CD44 expression. T-Cell expansion resulted in significant down-regulation of CD44 expression. Interleukin-2 (IL-2) and anti-CD3 antibody stimulation may be responsible for CD44 down-regulation on CD8(+) T-cells during expansion. Furthermore, CD44 down-regulation using small interfering RNA (siRNA) on TILs dramatically reduced interferon-gamma and IL-2 release upon tumor stimulation. These results suggest that the regulation of CD44 expression in TILs may play an important role in memory T-cell maintenance and antitumor immune response.     

Development of tumor-infiltrating lymphocytes in breast cancer after neoadjuvant paclitaxel chemotherapy.

PURPOSE: Neoadjuvant chemotherapy for breast cancer creates new possibilities for the analysis of biological factors in the tumor and/or host, which may play a role in the response to treatment. In this study we analyzed whether changes in local antitumor immunity take place after neoadjuvant paclitaxel therapy and if they correlate with response to treatment. EXPERIMENTAL DESIGN: Neoadjuvant chemotherapy (paclitaxel, 200 mg/m2 q2w, 4 treatments) was followed by definitive surgical management. Histological sections from the pre- and post-treatment surgical specimens of 25 patients were analyzed for the extent of lymphocytic infiltration and presence of tumor infiltrating lymphocytes (TILs). The cumulative apoptotic response in the tumor after the first dose of paclitaxel was also studied in 10 of 25 patients. RESULTS: Pretreatment lymphocytic infiltrate in the tumor was minimal in the majority of patients and showed no relationship with clinical response. In the patients without TILs before treatment, development of TILs after treatment was noted in 0/3 (0%) patients with stable disease, 3/12 (25%) patients with clinical partial response, and 4/6 (67%) patients with clinical complete response and pathological residual disease. These correlated with the tumor cell apoptotic response to the first dose of paclitaxel. CONCLUSIONS: These results suggest that development of TILs after treatment correlates with clinical response to neoadjuvant paclitaxel therapy. The possible mechanism(s) whereby neoadjuvant chemotherapy may lead to induction of antitumor T cells is discussed. Immunological processes may influence the response of breast cancer patients to neoadjuvant treatment.     

In vitro migration of lymphocytes through collagen matrix: arrested locomotion in tumor-infiltrating lymphocytes

Antitumor immunity requires (a) extravasation of lymphocytes from the blood stream to interstitium, (b) locomotion through extracellular matrix to the site of the tumor, (c) effector cell recognition of the tumor target with cell/cell contact and binding of adhesion receptors, (d) T-cell receptor binding to histocompatibility and tumor antigens, and (e) tumor cell lysis. We hypothesize that the tumor microenvironment inhibits lymphocyte locomotion through extracellular matrix as one mechanism by which tumors may avert host defense. Lymphocyte locomotion was investigated in vitro using a three-dimensional collagen gel model. Fresh tumor-infiltrating lymphocytes (TIL) were obtained by enzymatic digestion of melanomas and renal cell carcinoma, and mononuclear cells were isolated by discontinuous Ficoll-Hypaque gradient. The lymphocytes were analyzed for motility from a point of origin between basal and overlay layers of collagen gel. Results showed that TIL migration was almost completely inhibited, compared with migration of normal and cancer patient peripheral blood leukocytes and lymphocytes from lymph nodes. Short-term (24-h) exposure of lymphocytes to cytokines during the assay in the collagen gel matrix had no effect on locomotor ability. Long-term (19, 30, or 35 days) culture of TIL in 200 units/ml of interleukin 2 reinstated locomotor ability. Short-term exposure of any of the lymphocyte populations to interleukin 1-alpha, interleukin 1-beta, interleukin 2, interleukin 3, interleukin 4, alpha-interferon, or gamma-interferon had no effect on migration. Thus, TIL display a uniquely arrested ability to locomote through collagen gel. Inhibition of the locomotion of infiltrating effector cells is possibly a mechanism by which the tumor evades the host immune system.     

体外硫芥对人外周血淋巴细胞DNA的损伤

目的与方法：利用碱性单细胞凝胶电泳（ＳＣＧＥ）技术在体外检测硫芥对人外周血淋巴细胞的损伤情况。结果与结论：结果表明：在硫芥的作用下细胞ＤＮＡ的迁移率和迁移长度增加,且呈显著剂量效应关系。     

Occupational and in vitro exposure to styrene assessed by unscheduled DNA synthesis in resting human lymphocytes

Lymphocytes from 38 individuals occupationally exposed to styreneconcentrations in workroom air of 1 p.p.m. to 40 p.p.m. wereexamined for any genotoxic effects using unscheduled DNA synthesis(UDS) as the indicator of DNA damage. The mean level of N-acetoxy-2-acetylaminofluorene(NA-AAF) induced UDS was significantly increased (p < 0.001)for the styrene exposed group when compared to the mean levelfor the unexposed controls. There was no significant effecton u.v.-induced UDS from the in vivo styrene exposure. Lymphocytecultures exposed in in vitro to styrene concentrations up to100 µM have confirmed the UDS data collected on individualsoecupationally exposed to styrene. In addition, the in vitrostudy has also shown that the increased NA-AAF induced UDS resultingfrom styrene exposure was paralleled by a similar increase inNA-AAF binding to DNA. Taken together these results indicatethat styrene exposure does not inhibit DNA repair synthesis,but rather it predisposes lymphocytes to an increased risk forDNA damage induction from subsequent genotoxic exposures.     

Modulation of microenvironment acidity reverses anergy in human and murine tumor-infiltrating T lymphocytes

Stimulating the effector functions of tumor-infiltrating T lymphocytes (TIL) in primary and metastatic tumors could improve active and adoptive T-cell therapies for cancer. Abnormal glycolysis, high lactic acid production, proton accumulation, and a reversed intra-extracellular pH gradient are thought to help render tumor microenvironments hostile to roving immune cells. However, there is little knowledge about how acidic microenvironments affect T-cell immunity. Here, we report that lowering the environmental pH to values that characterize tumor masses (pH 6-6.5) was sufficient to establish an anergic state in human and mouse tumor-specific CD8(+) T lymphocytes. This state was characterized by impairment of cytolytic activity and cytokine secretion, reduced expression of IL-2Rα (CD25) and T-cell receptors (TCR), and diminished activation of STAT5 and extracellular signal-regulated kinase (ERK) after TCR activation. In contrast, buffering pH at physiologic values completely restored all these metrics of T-cell function. Systemic treatment of B16-OVA-bearing mice with proton pump inhibitors (PPI) significantly increased the therapeutic efficacy of both active and adoptive immunotherapy. Our findings show that acidification of the tumor microenvironment acts as mechanism of immune escape. Furthermore, they illustrate the potential of PPIs to safely correct T-cell dysfunction and improve the efficacy of T-cell-based cancer treatments.     

Properties of recombinant interleukin 2-cultured tumor-infiltrating lymphocytes in human lung cancer

It is thought that TIL can be activated in vitro by rIL-2 and acquire specific anti-tumor activity. In this study, we investigated this possibility, using lymphocytes isolated from primary lung cancer tissues. In a first series of experiments, TILs and autologous PBLs from 16 patients were cultured in rIL-2 from 7 to 14 days under identical conditions, and were compared for proliferation (16 cases), cytolytic activity (11 cases), gamma interferon (IFN-gamma) production (8 cases), and phenotypes (10 cases). TILs grew in response to rIL-2 as well as PBLs. However, the induced cytolytic activity of TIL was significantly lower than that of PBL against autologous tumor cells and 2 human tumor cell lines. IL-2-mediated IFN-gamma production by TILs was also significantly lower than that of PBLs. TILs were phenotypically characterized by their high CD4/CD8 ratio and lack of Leu11-positive cells. Further investigations with 7 other cases showed that exogenous addition of IFN-gamma to rIL-2 cultures of TILs enhanced cytolytic activity in 4 cases. Our results indicate that IL-2 alone is sufficient for TILs to proliferate but not to acquire new functions (cytotoxicity and production of IFN-gamma).     

Clonal analysis of tumor-infiltrating lymphocytes from human primary and metastatic liver tumors

Phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) obtained from human primary and metastatic liver tumors were studied. Lymphocytes isolated from 18 tumors and autologous (A) peripheral blood (6 cases) were phenotyped by 2-color flow cytometry and cloned in a limiting dilution system, which allows virtually all normal T lymphocytes to proliferate; 70-80% of fresh TIL were T cells (i.e., CD3+), and the ratio of CD4+/CD8+ cells was 1.2 in both primary and metastatic liver tumors. TIL contained significantly more CD56+ (NKHI+) cells, half of which were CD3+CD56+, CD3+CD25+ cells and CD3+HLA-DR+ cells, than A-PBL. The frequencies of proliferating T-cell precursors (PTL-p) and cytolytic T-lymphocyte precursors (CTL-p) reactive with K562, allogeneic tumor cells and autologous tumor cells, were determined. Mean PTL-p frequencies for TIL from hepatocellular carcinomas, cholangiocarcinomas and metastatic liver tumors were 0.52 (0.22-0.83), 0.10 (0.05-0.16) and 0.16 (0.01-0.30), respectively. The frequency of CTL-p with natural-killer-like activity was lower in TIL than in A-PBL. The frequency of CTL-p for autologous tumor cells in fresh TIL isolated from primary liver tumors was 0.02-0.13 and 12/81 clones were reactive against autologous tumor. In contrast, only 1/66 TIL clones obtained from colon carcinomas metastatic to liver showed autotumor reactivity. No clones reactive with autologous tumor were obtained from peripheral blood of patients with liver cancer. These data indicate that substantial differences in anti-tumor functions of TIL between primary and metastatic liver tumors exist, which can be detected at a clonal level.     

Activation of human tumor-infiltrating lymphocytes by monoclonal antibodies directed to the CD3 complex

We have used high concentrations of recombinant-methionyl human interleukin 2 (rIL-2) for the initial growth and expansion of human tumor-infiltrating lymphocytes (TIL). Early in the life of the TIL bulk culture, cytotoxicity was non-major histocompatibility complex restricted. Under these culture conditions antitumor cytotoxicity was observed to decline with increasing age of the bulk culture. In addition, TIL became refractory to rIL-2-induced expansion. We have used solid-phase anti-CD3 antibodies for TIL activation followed by culture in reduced concentrations of rIL-2 to reactivate TIL previously grown in high concentrations of rIL-2. TIL refractory to rIL-2 in terms of growth and antitumor cytotoxicity proved sensitive to anti-CD3 activation. The use of solid-phase anti-CD3 was also more effective than high concentrations of rIL-2 in the expansion of TIL when used at the start of culture. Finally, TIL could be induced to secrete IL-2 following solid-phase activation with anti-CD3. These data suggest that human TIL are susceptible to activation by signals directed at the CD3 complex of the TIL cell surface.     

Mitogenesis in human lymphocytes following brief exposure to hyperthermia

Whole blood obtained from normal human donors was incubated in vitro at temperatures ranging from 37°C to 51°C for 30 min. Immedlately following this incubation, whole blood cultures were established for assaying lymphocyte transformation in response to phytohemagglutinin (PHA) pokeweed mitogen (PWM) and concanavalin A (Con A). These cultures were maintained throughout the remainder of the experiment at 37°. Mitogenesis was minimally affected with pretreatment at 41°, significantly impaired at 43°, and totally abolished following brief exposure to temperatures in excess of 44°. In contrast, lymphocyte viability by trypan blue exclusion remained >99 % after a 30 min exposure to temperatures as high as 47° and was approximately 50% after 30 min at 53.5°. The length of exposure time (within the range of 30–120 min) became a critical variable at threshold temperatures at which impairment of mitogenesis was detectable with this assay system. This threshold temperature appears to pie within a very narrow range, similar to that in which tumor cell damage occurs. The necessity for accurate monitoring of both intratumor and core temperatures during treatment is apparent from these data. The response of heat-treated and control lymphocytes to optimal mitogen concentrations was compared to the response to sub-optimal (50% of optimal) concentrations. No differential effects of time (30, 60 min) and temperature (41°, 43°) were evident with the suboptimal concentrations.     

Regeneration of O6-alkylguanine-DNA alkyltransferase in human lymphocytes after nitrosourea exposure

Mitogen-stimulated human lymphocytes have an increased capacity to repair many forms of DNA damage caused by UV, ionizing radiation, and chemical carcinogens. Human lymphocytes rely on a particular DNA repair protein, O6-alkylguanine-DNA alkyltransferase (alkyltransferase) to repair efficiently O6-alkylguanine, an important mutagenic adduct formed by nitrosoureas and other N-nitroso compounds. The alkyltransferase is a "suicide" protein which becomes inactivated during the repair process. Thus, basal activity and the ability to synthesize new protein activity are important compounds of O6-alkylguanine repair. We compared basal and regenerated alkyltransferase activity in resting and mitogen-stimulated human lymphocytes. During stimulation with L-phytohemagglutinin, alkyltransferase activity increased by a mean of 70% over resting cells. Following exposure to N-nitroso-N-methylurea (MNU), alkyltransferase activity was consumed in a dose-dependent manner in both resting and L-phytohemagglutinin-stimulated cells by the repair of MNU-induced O6-methylguanine-DNA adducts. Recovery of alkyltransferase activity began within 1 day of MNU exposure in the L-phytohemagglutinin-stimulated lymphocytes but did not occur in resting cells. Enzyme induction was not observed. When the alkyltransferase was only partially inactivated by low dose MNU, resting lymphocytes still failed to recover alkyltransferase activity. The rate of recovery of alkyltransferase activity in proliferating cells was dependent on the basal level of activity, which varied about 3-fold among donors. These data indicate that mitogen-stimulated lymphocytes develop an increased capacity to repair nitrosourea-induced DNA damage and are able to regenerate activity following nitrosourea exposure. In contrast, resting lymphocytes do not rapidly synthesize new alkyltransferase molecules after nitrosourea exposure and appear susceptible to DNA damage caused by persistent O6-alkylguanine adducts. Thus, both basal alkyltransferase activity and the proliferative state of normal lymphocytes influence the response to nitrosourea exposure.     

Cisplatin modulates tumor cell susceptibility to tumor-infiltrating lymphocytes in vivo

We investigated the in vivo augmentation of susceptibility of tumor cells to tumor-infiltrating lymphocytes (TILs) with cisplatin (CDDP). TILs showed cytotoxicity against autologous and established tumor cells. Pretreatment of tumor cells with CDDP 2 mu g/ml for 12 h enhanced the susceptibility of tumor cells to TILs in vitro. TILs and autologous tumor cells were obtained from malignant ascites of patients, before and after the intraperitoneal administration of CDDP. TILs had higher cytotoxicity against autologous tumor cells of CDDP treated as compared to untreated control tumor cells, providing direct evidence of in vivo immunomodulatory effect of CDDP in cancer patients.     

Effects of HC antibody in autologous tumor-specific cytotoxicity by human melanoma tumor-infiltrating lymphocytes

Heteroconjugate (HC) antibody has a potential use in cancer biotherapy because of its ability to mimic antigenic specificity and induce cytotoxicity in the activated lymphocytes against various tumor cells. This study investigated the effects of HC antibody (anti-CD3 MAb x anti-p97 melanoma cell MAb) in autologous tumor-specific cytotoxicity by interleukin-2 (IL-2)-activated melanoma tumor-infiltrating lymphocytes (TILs). HC antibody significantly augmented p97pos uncultured autologous tumor cell lysis mediated by effector TILs or cytotoxic T lymphocyte (CTL) clones derived from TIL. It did not significantly increase p97mix autologous tumor-cell lysis and slightly inhibited the lysis only at higher E:T ratios and higher concentrations (greater than or equal to 100 ng/ml). It inhibited p97neg autologous tumor-cell lysis. HC antibody respectively induced potent lysis of p97pos or modest lysis of p97mix tumor cells by allogeneic effector TILs as well as PBMC. In contrast, parental anti-CD3 MAb primarily suppressed the autologous tumor-specific cytotoxicity, and did not induce lysis of uncultured melanoma cells, regardless of differences in expression of p97 antigens on tumor cells. Although parental anti-p97 MAb did not augment or suppress the autologous tumor-specific cytotoxicity, it completely abrogated HC antibody-mediated augmentation of p97pos autologous tumor cell lysis by effector TILs. Anti-class-I MAb, but not anti-DR MAb, suppressed the autologous tumor-specific cytotoxicity, but failed to block HC antibody-mediated augmentation of p97pos autologous tumor-cell lysis. These results suggest that the levels of p97 antigen expression largely influenced HC antibody-mediated modulation of TIL cytotoxicity against uncultured autologous tumor cells.     

Residual gammaH2AX after irradiation of human lymphocytes and monocytes in vitro and its relation to late effects after prostate brachytherapy.

BACKGROUND AND PURPOSE: Retention of gammaH2AX foci in irradiated cells can signify a deficiency in DNA double-strand break repair that may be useful as an indicator of individual radiosensitivity. MATERIALS AND METHODS: To examine this possibility, the retention of gammaH2AX after irradiation was compared using white blood cells from 20 prostate brachytherapy patients who developed late normal tissue toxicity and 20 patients with minimal toxicity. Peripheral blood lymphocytes and monocytes were coded for analysis, exposed in vitro to 4 doses of 0.7 Gy X-rays at 3 hourly intervals, and retention of gammaH2AX was measured by flow cytometry 18 hours after the final irradiation. RESULTS: Excellent reproducibility in duplicate samples and a range in residual gammaH2AX from 7% above background to 244% above background were observed. Residual gammaH2AX in lymphocytes showed a positive correlation with patient age. However, no relation was observed between the level of residual gammaH2AX in peripheral blood mononuclear cells and late normal tissue damage. CONCLUSIONS: We conclude that the method of detection of residual gammaH2AX after in vitro irradiation of lymphocytes and monocytes was simple, reproducible, and sensitive. However, it failed to predict for late normal tissue toxicity after brachytherapy. Possible reasons are discussed.     

Lysis of fresh human tumor cells by autologous peripheral blood lymphocytes and tumor-infiltrating lymphocytes activated by PSK

The protein-bound polysaccharide PSK was tested for the ability to induce in vitro autologous tumor killing (ATK) activity in human cancer patients. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) demonstrated various levels of cytotoxicity against autologous, freshly isolated tumor cells. When PBL and TIL were cultured overnight with PSK, ATK activity was induced in previously non-reactive cases and augmented in previously reactive samples. The PSK effect was observed with PSK concentrations of 10-100 micrograms/ml that could be obtained in the blood of cancer patients who received standard oral administration of PSK. The manifestation of PSK-induced ATK required active cell metabolism and RNA and protein syntheses, but not DNA synthesis of lymphocytes. PSK-induced enhancement of ATK was not abrogated by monoclonal antibodies (mAb) directed against interferon (IFN) alpha or IFN gamma. In addition, mAb that neutralized interleukin-2 (IL-2) or mAb reactive with alpha-chain or beta-chain of IL-2 receptors (IL-2R) had no effect on PSK-induced ATK activity. Supernatants from PSK-stimulated lymphocyte cultures did not induce ATK. Cell fractionation experiments revealed that CD3-CD16+ large granular lymphocytes (LGL) and/or CD3+CD16- T lymphocytes were responsible for both spontaneous and PSK-induced ATK. PSK-activated LGL, but not T lymphocytes expressed lysis of fresh allogeneic tumor cells. These results indicate that PSK activates PBL and TIL to exhibit ATK independently of IL-2/IL-2R systems.