A comparison of blind intubation with the intubating laryngeal mask FASTRACH™ and the intubating laryngeal mask Ambu Aura-i™ a prospective randomised clinical trial.

BACKGROUND: The intubating laryngeal mask Fastrach™ is considered a gold standard for blind intubation as well as for fibreoptic guided intubation via a laryngeal mask. Recently, a single use version of the mask has been introduced. We compared the Fastrach single use with the new, low-priced single use intubating laryngeal mask Ambu Aura-i™. We hypothesised that the LMA Ambu Aura-i and the LMA Fastrach are comparable with respect to success rates for mask placement and blind tracheal intubation through the LMA device. METHODS: A prospective, randomised clinical trial. University Hospital Schleswig-Holstein, Campus Kiel, from April 2011 to April 2012. Eighty patients undergoing general anaesthesia with planned tracheal intubation were randomised and enrolled in the study. Blind intubation was performed with either laryngeal mask using two different tracheal tubes (Rüsch Super Safety Silk™ and LMA ETT™). A crossover-design was performed after an unsuccessful procedure. Primary outcome measure was the overall success rate of blind intubation. Secondary outcome measures were the time to the first adequate ventilation, a subjective handling score, and a fibreoptic control of placement, as well as the success rate of mask placement, time for mask removal after successful intubation, differences in airway leak pressure, and the incidence of postoperative sore throat and hoarseness. RESULTS: The success rate of tracheal intubation with the Fastrach for the first and second attempt was significantly better compared with the Ambu Aura-i. Tracheal intubation was also significantly faster (14.1 s. ±4.4 versus 21.3 s. ±9.0; p < 0.01), and the time interval for mask removal after successful intubation was significantly shorter using the Fastrach device (24.0 s. ±8.2 versus 29.4 s. ±7.5; p < 0.001). There were no significant differences between groups regarding the incidence of postoperative sore throat and hoarseness. CONCLUSION: Both laryngeal mask devices are suitable for ventilation and oxygenation. Blind intubation remains the domain of the LMA Fastrach, the Ambu Aura-i is not suitable for blind intubation. TRIAL REGISTRATION: Clinicaltrials.gov Identification Number NCT03109678 , retrospectively registered on April 12, 2017.

Health related quality of life after extracorporeal cardiopulmonary resuscitation in refractory cardiac arrest.

BACKGROUND: Recent data identifies extracorporeal cardio-pulmonary resuscitation (eCPR) as a potential addendum of conventional cardiopulmonary-resuscitation (cCPR) in highly specified circumstances and selected patients. However, consented criteria indicating eCPR are lacking. Therefore we provide first insights into the health-related quality of life (HRQoL) outcomes of patients treated with eCPR in a real world setting. METHODS: Retrospective single-center experience of 60 consecutive patients treated with eCPR between 01/2014 and 06/2016 providing 1-year survival- and HRQoL data obtained through the Short-Form 36 Survey (SF-36) after refractory out-of-hospital- (OHCA) and in-hospital cardiac arrest (IHCA) of presumed cardiac etiology. RESULTS: Resuscitation efforts until initiation of eCPR averaged 66 ±â€¯35 min and 63.3% of the patients suffered from OHCA. Fifty-five (91.7%) of the overall events were witnessed and bystander-CPR was performed in 73.3% (n = 44) of cases. Cause of arrest was dominated by acute myocardial infarction (AMI, 66.7%) and initial rhythm slightly outbalanced by ventricular fibrillation/tachycardia (VF/VT 53.3%). 12-month survival was 31%. Survivors experienced more often bystander-CPR (p = .001) and a shorter duration of cCPR (p = .002). While mid-term survivors' perceived HRQoL was compromised compared to controls (p ≦ .0001 for PF, RP, RE and BP; p = .007 for GH; p = .016 for SF; p = .030 for MH; p = .108 for VT), scores however resembled HRQoL of subjects on hemodialysis, following cardiogenic shock or pulmonary failure treated with extracorporeal membrane oxygenation (ECMO). CONCLUSIONS: While HRQoL scores of our survivors ranged markedly below controls, compared to patients on chronic hemodialysis, following ECMO for cardiogenic shock or pulmonary failure most of the discrepancies ameliorated. Thus, successfull eCPR in properly selected patients does translate into an encouraging HRQoL approximating chronic renal failure.

Dexamethasone and cyclosporin A do not inhibit interleukin-15 expression in the human lung carcinoma cell line A549.

A549 cells constitutively expressed IL-15 mRNA which could be upregulated by stimulation with TNF-alpha- or IL-1beta. Constitutive and induced levels of IL-15 mRNA were not decreased in the presence of 10- 6 M dexamethasone. Control experiments revealed that 10- 6 M dexamethasone inhibited the TNF-alpha- or IL-1beta-mediated increase of IL-8 mRNA in A549 cells, which showed that the glucocorticoid was functional. A549 cells did not secrete relevant amounts of IL-15 protein. The constitutive expression and the TNF-alpha- or IL-1beta-mediated upregulation of intracellular IL-15 protein was not inhibited by dexamethasone, in contrast, the release of IL-8 protein was inhibited. Also, cyclosporin A at 250 ng/ml did not inhibit the TNF-alpha-induced upregulation of IL-15 mRNA and intracellular IL-15 protein. The data suggest that the synthesis of IL-15 mRNA and protein is not influenced by immunosuppressive glucocorticoids or by cyclosporin A.

Physician participation in research surveys. A randomized study of inducements to return mailed research questionnaires.

The authors randomly selected 400 physicians from a population of 1,545 practicing physicians providing follow-up care to patients who received bone marrow or blood stem cell transplants at the Fred Hutchinson Cancer Research Center to determine interest in receiving Internet-based transplant information. In a two-factor completely randomized factorial design, the 400 physicians were assigned to receive mailed surveys with either no compensation or a $5 check and either no follow-up call or a follow-up call 3 weeks after mailing. Overall, 51.5% of the physicians returned the mailed surveys. Comparison of logit models showed that inclusion of a $5 check in the mailer significantly (p = .016) increased the probability of returning the surveys (57.5% vs. 45.5%). In contrast, the telephone follow-up had no overall effect. The authors concluded a modest financial reward can significantly improve physician response rates to research surveys but a telephone follow-up may be inefficient and even ineffective.

Induction of IL-15 mRNA and protein in A549 cells by pro-inflammatory cytokines.

Interleukin-15 is a recently discovered cytokine which is functionally similar to IL-2. In order to learn more about possible targets for modulation of the expression of IL-15 we investigated the expression of IL-15 mRNA and protein in the A549 (human lung carcinoma) cell line. Constitutive expression of IL-15 mRNA was detected in A549 cells. Treatment with TNF-alpha or IL-1 beta (10 ng/ml each) induced an about 2-fold increase of IL-15 mRNA; IFN-gamma induced significant effects only at 100 ng/ml. Stimulation with a combination of TNF-alpha and IFN-gamma was not superior to stimulation with TNF-alpha alone. EGF, KGF and the combination thereof were without effects. IL-15 protein was detected in cellular lysates of unstimulated cells and was increased by stimulation with TNF-alpha or IL-1 beta. No significant amounts of IL-15 protein were detected in cellular supernatants.

Matrix protein synthesis by glomerular mesangial cells in culture: effects of transforming growth factor beta (TGF beta) and platelet-derived growth factor (PDGF) on fibronectin and collagen type IV mRNA.

The pathogenesis of glomerular scarring is multifactional; recent evidence suggests that transforming growth factor beta (TGF beta), a pleiotropic cicatricial mediator, may promote mesangial sclerosis by enhancing the production of extracellular matrix proteins. We studied the effect of TGF beta 1 and TFG beta 2 on collagen type IV and fibronectin (FN) synthesis in human glomerular mesangial cells in culture (GMC). Two hours after addition of TGF beta, an up to twofold increase in abundance of collagen type IV mRNA was found, which further increased up to fivefold within 24 h. Addition of cycloheximide did not inhibit the TGF beta effect, but caused by itself an up to twofold increase in the abundance of collagen type IV mRNA after 2 h. Together with collagen mRNA, the mRNA for FN and for platelet-derived growth factor (PDGF) was also enhanced. PDGF was found to enhance abundance of the collagen type IV and fibronectin mRNA in GMC. A neutralizing antibody to PDGF or a PDGF-antisense oligonucleotide partly inhibited the TGF beta-induced increase of collagen type IV mRNA, suggesting that TGF beta can affect the collagen type IV synthesis not only directly but also indirectly via the synthesis of PDGF.

Activation of the mouse IL-2 gene by okadaic acid: synergy with interleukin-1.

Interleukin-1 (IL-1) has potent immunoregulatory and inflammatory functions. Its activity is mediated by an 80-kDa receptor on the cell surface and leads to activation of other genes. The underlying molecular events are largely unknown. We investigated the role of phosphatases in activation of the IL-2 gene in EL4 thymoma cells. We found that the protein phosphatase PP1 and PP2A inhibitor okadaic acid (OA) alone was able to significantly stimulate IL-2 production by the IL-1-responsive EL4 subline EL4 5D3 and also by the IL-1-nonresponsive EL4 subline EL4D6/76. In the IL-1-responsive cell line OA strongly synergized with phorbol myristate acetate (PMA) and IL-1. In the IL-1-nonresponsive cell line OA synergized with PMA but not with IL-1. Under suboptimal conditions of PMA/OA synergy an additional synergistic effect of IL-1 was shown. This was true for IL-2 and IL-6 production. Sphingomyelinase or sphingosine had no detectable effect. The kinetics of OA- and PMA-induced expression of IL-2 mRNA and IL-2 protein was different. PMA induced maximal expression between 6 and 12 h and was almost undetectable at 24 h. OA-induced expression was first obvious at 12 h and continued longer than 36 h. In both cases IL-1 caused no shift in kinetics, but potentiated the effects of the different tumor promoters. Utilizing IL-2 promoter-CAT constructs we showed in transfection experiments that the synergistic effect was also evident on the transcriptional level. We conclude from the data that phosphatases play an important role for IL-2 expression and that IL-1 can use additional pathways of activation that are different from events induced by PMA or OA.

In vitro expansion and analysis of T lymphocyte microcultures obtained from the vaccination sites of cancer patients undergoing active specific immunization with autologous Newcastle-disease-virus-modified tumour cells.

In order to understand further the effects of Newcastle-disease-virus(NDV)-modified tumour vaccines we investigated the feasibility of isolating lymphocytes from the site of injection of patients undergoing postoperative active specific immunization (ASI) with autologous NDV-modified tumour cells. Delayed-type-hypersensitivity(DTH)-like reactions from five cancer patients were surgically removed, minced and the tissue particles were digested with collagenase and DNase. Lymphoid cells recovered were expanded in a highly efficient limiting-dilution analysis system optimized for T cell growth [Moretta et al. (1983) J Exp Med 157: 743] and lymphocyte microcultures (clonal probability > 0.8) could be grown for up to 1 year. Analysis of the microcultures for phenotype and function showed that the majority were positive for CD4 (92%) and TCR alpha beta (96%). Concanavalin-A-induced production of interleukin-2 (IL-2), IL-6, interferon gamma and tumour necrosis factor alpha was detected in more than 70% of the microcultures. Lectin-dependent cytotoxicity was only very rarely observed. The general characteristics of the microcultures obtained support the notion of a DTH-like reaction taking place at the site of tumour cell challenge. The possibility of in vitro expansion and cultivation of T lymphocytes from ASI vaccination sites should help to elucidate further the role of these cells in active specific immunization against autologous tumour cells.

Transforming growth factor-beta production and induction of cellular responses in 13762NF rat mammary adenocarcinoma cell clones.

We characterized three clones of different metastatic capacity (MTC, MTLn2 and MTLn3) derived from the 13762NF rat mammary adenocarcinoma for their production and response to TGF-beta. All three clones expressed comparable amounts of TGF-beta 1 mRNA and secreted 100-300 pg/10(6) cells/24 h in a soluble latent form. TGF-beta was found in extracellular matrices produced by all three tumor clones. Addition of exogenous TGF-beta induced different responses. While the low metastatic clone MTC was highly sensitive to the growth inhibitory effect of TGF-beta (ID50 approximately 50 pg/ml), a 6-fold higher dose was necessary for the high metastatic clone MTLn3 (ID50 approximately 300 pg/ml). The clone with intermediate metastatic potential MTLn2 was unresponsive to TGF-beta (1 pg/ml to 3 ng/ml). Our data suggest that tumor cells can modulate their biological properties in an autocrine and/or paracrine fashion by virtue of expression of TGF-beta.

Distinct effects of phorbol esters and exogenous diacylglycerols in the induction of murine thymocyte proliferation.

Murine thymocytes were stimulated with the protein kinase C activating agents Phorbol-12-myristate-13-acetate (PMA) or a more physiological membrane permeant diacylglycerol (dioctanoyl-sn-glycerol, DiC8) in the presence or absence of exogenous lymphokines (rIL-1 beta, rIL-2). Whereas PMA directly induced reactivity to rIL-2, DiC8 did not but had to synergize with the calcium ionophore Ionomycin. Expression of the p55 chain of the IL-2 receptor behaved similarly. In the absence of exogenous rIL-2, thymocytes proliferated in response to a combination of Ionomycin and PMA; however, replacing PMA by a single addition of DiC8 did not result in proliferation. Stimulation with Ionomycin plus repeated addition of DiC8 induced a low level of thymocyte proliferation and further addition of rIL-1 beta resulted in a significant increase. Purified immature (L3T4-Lyt2-) thymocytes behaved similarly, but showed an increased sensitivity to rIL-1 beta. Taken together, the data support the idea that PMA and the more physiological diacylglycerols do not possess totally equivalent activities in lymphocyte stimulation.

Cytotoxic potential despite impaired activation pathways in T lymphocytes infiltrating nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial tumor consistently associated with EBV. The histological picture is characterized by a strikingly abundant lymphocytic infiltrate. Furthermore, the epithelial tumor cells present several immunological characteristics which suggest an important role for tumor-infiltrating lymphocytes (TIL) in the biology of this tumor. The present study reports the phenotypic and functional characterization of TIL from NPC obtained after enzymatic digestion of 15 NPC biopsies. Flow cytometric analysis of TIL suspensions indicated that most TIL were mature CD3+ T lymphocytes (mean = 60%) with a variable CD4/CD8 ratio. Most TIL were TCR alpha/beta-positive (mean = 55%) and only a few TCR gamma-delta-positive cells could be identified. A small percentage (mean = 9%) displayed an activated phenotype (CD25+, HLA class II+). Using limiting dilution analysis, we found that the average frequency of proliferative T-lymphocyte precursors (PTL-P) is lower among TIL (1/40) than in autologous (1/7) or normal PBL (1/1.4). Moreover, sorting experiments have shown that this defect is significantly more pronounced in the CD8+ than in the CD4+ TIL subset. Accordingly, the TCR and the CD2-mediated antigen-independent pathways of activation were impaired. Different types of cytotoxic precursor could be detected. These included lectin-dependent cell cytotoxicity (LDCC) and NK-like or lymphokine-activated killer (LAK) activity. Interestingly, some TIL from NPC were able to lyse an NPC tumor (C15) maintained in nude mice. Thus, despite impaired activation pathways, the cytolytic potential of proliferating TIL in NPC is preserved.

Effect of transforming growth factor beta on the EL4 thymoma variant EL4/6.1: dissociation of inhibition of proliferation from expression of IL-1 and IL-2 receptors.

In order to further characterize the action of transforming growth factor beta (TGF-beta) on lymphoid cells, we investigated the effects of porcine TGF-beta 1 and -2 on the IL-1 sensitive EL4/6.1 thymoma cell line. The proliferation of EL4/6.1 thymoma cells was inhibited by TGF-beta 1 and TGF-beta 2 (1 ng/ml) to a similar degree, the population doubling time was increased by 50-60%, total inhibition was not achieved. This decrease of proliferation was associated with an increase of the number of cells in the G0/G1 compartment of the cell cycle. TGF-beta-mediated inhibition could not be overcome by adding exogenous rIL-1 nor was the binding capacity for IL-1 reduced. In addition, TGF-beta did not interfere with the induction of IL-2 receptors by a combination of Ionomycin+PMA+IL-1. The data suggest that TGF-beta mediated inhibition of thymocyte/lymphocyte proliferation is not associated with an inhibition of the expression or the induction of expression of IL-2 or IL-1 receptors.

Stimulation of FACS-analysed CD4+ and CD8+ human tumour-infiltrating lymphocytes with ionomycin + phorbol-12,13-dibutyrate does not overcome their proliferative deficit.

Human tumour-infiltrating lymphocytes (TIL) were prepared by enzyme digestion from a series of different tumours and were purified on a fluorescence-activated cell sorter (FACS II) according to their CD4+ and CD8+ phenotype. CD4+ and CD8+ TIL were stimulated separately in a low density microculture system with phytohaemagglutinin (PHA) or with ionomycin plus phorbol-12, 13-dibutyrate (PDBu). The PHA-induced proliferation of TIL was highly decreased when compared with control peripheral blood lymphocytes. A decreased proliferation of TIL was also observed when cells were stimulated with ionomycin plus PDBu, a combination which is thought to circumvent early events associated with lymphocyte activation. Some TIL were also plated in limiting dilution where they showed decreased frequencies of proliferating T cell precursors. The data suggest that one component of the inhibition of TIL must be acting 'downstream' of the early events of lymphocyte activation.

Comparison of the immunosuppressive properties of milk growth factor and transforming growth factors beta 1 and beta 2.

The effects of the newly isolated bovine milk growth factor (MGF) which shows N-terminal homology to transforming growth factor beta 2 were compared with the effects of porcine transforming growth factor beta 1 and beta 2 (pTGF-beta 1 and -beta 2) on human T lymphocyte activation. Freshly isolated human PBMC were stimulated with either PHA, anti-CD3 + phorbol-12,13-dibutyrate (PDBu), or with a combination of ionomycin + PDBu. MGF, pTGF-beta 1, and pTGF-beta 2 decreased mitogen-induced [3H]thymidine incorporation by 30 to 75% in a dose-dependent manner. The maximum degree of inhibition was obtained at 1 ng/ml (40 pM) and could not be increased by increasing the concentration of teh transforming growth factor 10-fold. Stimulation of fresh T cells with the recall Ag tetanus toxoid was also inhibited (85%) by MGF at pM concentrations as was the proliferation of a human T cell clone specific for purified protein derivative. The effects of MGF and pTGF-beta 1 on anti-CD3-mediated increase of intracellular Ca2+ (Cai2+) was investigated by using the Fura-2 method. Neither MGF nor pTGF-beta 1 inhibited this increase in Cai2+ induced by a mitogenic concentration of anti-CD3 antibody. In order to determine whether TGF-beta preferentially inhibited the CD4+ or CD8+ subpopulation of human T cells, a limiting dilution analysis system, which allows every T cell to proliferate, was used. pTGF-beta 1 at a concentration of 5 ng/ml decreased the frequency of proliferating T cell precursors of both the CD4+ and CD8+ subsets to a similar extent. Furthermore, MGF, pTGF-beta 1, and pTGF-beta 2 also decreased IL-2 mediated [3H]thymidine incorporation into human PBL Con A blasts and the IL-4-mediated [3H]thymidine incorporation of purified T lymphocytes costimulated with PDBu by 70%. In conclusion, bovine MGF exerts suppressive effects on human T cells stimulated with Ag, mitogens, or interleukins, and the degree of T cell suppression is similar (or identical) to those of pTGF-beta 1 or -beta 2.

Transforming growth factors beta slow down cell-cycle progression in a murine interleukin-2 dependent T-cell line.

Transforming growth factors beta (TGF-beta) inhibit the growth of a variety of cell types, including lymphocytes. The immunosuppressive effects of TGF-beta have been attributed to the interference of these molecules with the interleukin-2 (IL-2)-driven component of lymphocyte proliferation. In order to elucidate in more detail the effects of TGF-beta on IL-2-induced proliferation, we investigated the effects of porcine transforming growth factor beta 1 and 2 (pTGF-beta 1 and 2) on the IL-2-driven proliferation of a murine IL-2-dependent T-lymphocyte line (CTLL). The results showed that pTGF-beta 1 and 2 decreased 3H-thymidine incorporation in CTLL cells in a dose-dependent fashion (maximum decrease of 75-85%). Combined-time kinetic analysis of the effects of pTGF-beta on 3H-thymidine incorporation, cell growth, and cell-cycle distribution (monitored as DNA content distribution) revealed that, in the first 48 h of culture, pTGF-beta 1 increased the doubling time from 11.4 to 19.2 h without significantly affecting the cell-cycle distribution of CTLL cells. After 96 h of culture in the presence of pTGF-beta 1, cells started to accumulate in G0/G1, although at this time point 30% of the pTGF-beta 1-treated cells were still in S-G2/M. Furthermore, during the first 48 h, neither the expression of the 55 kd chain of the IL-2 receptor (IL-2R) nor the expression of the transferrin receptor (TfR) was affected by TGF-beta. After 72 h of culture in the presence of pTGF-beta 1, the expression of the IL-2R and TfR was decreased. The data suggest that in CTLL cells TGF-beta initially slows the progression of cells in all phases of cell cycle. In addition, the initial TGF-beta-mediated decrease of IL-2-induced 3H-thymidine incorporation and cell proliferation in CTLL cells is not due primarily to downregulation of the IL-2R and/or TfR.

Transforming growth factors beta 1 and beta 2 as well as milk growth factor decrease anti-CD3-induced proliferation of human lymphocytes without inhibiting the anti-CD3-mediated increase of [Ca2+]i and the activation of protein kinase C.

Porcine transforming growth factor 1 and 2 (pTGF-beta 1 and -beta 2) and milk growth factor (MGF) at 1 ng/ml significantly inhibited the proliferation of human lymphocytes induced by anti-CD3 antibodies. In contrast, the anti-CD3-mediated increase of intracellular Ca2+ and the activation and translocation of protein kinase C were not affected by the transforming growth factors.

Comparison of phorbol-12-myristate-13-acetate and dioctanoyl-sn-glycerol in the activation of EL4/6.1 thymoma cells.

The present study compared the role of two protein kinase C (PK-C) activating agents, the phorbol ester phorbol-12-acetate-13-myristate (PMA) and the membrane-permeating diacylglycerol dioctanoyl-sn-glycerol (DiC8) in the activation of EL4/6.1 thymoma cells. These cells have been shown to express interleukin-2 receptors (IL-2R) upon stimulation with optimal amounts of PMA (10 ng/ml); also, suboptimal amounts of PMA (1 ng/ml) synergized with the Ca2+ ionophore ionomycin and recombinant interleukin-1 (rIL-1) (Lowenthal et al., 1986). Comparing PMA and DiC8 led to the following results: PMA at 10 ng/ml induced IL-2R; in contrast, DiC8 (30-3 micrograms/ml) alone was unable to induce IL-2R, although it did synergize with ionomycin (0.5 micrograms/ml) and rIL-1. Bihourly additions of DiC8 did not change this pattern. The addition of DiC8 together with rIL-2 also resulted in no IL-2R expression. Furthermore, DiC8 (10 micrograms/ml) effectively translocated PK-C. Therefore, the differences observed between PMA and DiC8 do not seem to be due to differences in metabolism or to an inability to translocate PK-C. Analysis of messenger (m) RNA produced in stimulated EL4/6.1 cells revealed that DiC8 was also unable to induce mRNA for IL-2R. Our data suggest that PMA, especially at "optimal" concentrations, might have effects that cannot be mimicked by diacylglycerol. Furthermore, it seems that the deficient activity of diacylglycerols can be compensated for by a Ca2+ ionophore and, depending on the cellular system, by further signals such as IL-1.

Preferential clonogenic deficit of CD8-positive T-lymphocytes infiltrating human solid tumors.

The vast majority of tumor infiltrating lymphocytes (TIL) are either CD4+ or CD8+ T-lymphocytes. In order to examine directly the functional capabilities of the individual CD4+ and CD8+ TIL subsets we performed cell sorting of double immunofluorescence-labeled TIL recovered from 15 biopsies by enzyme digestion. These CD4+ and CD8+ TIL subsets were compared with similar subsets of T-lymphocytes from peripheral blood of normal subjects. Both CD4+ and CD8+ TIL showed a reduced clonogenicity as assessed quantitatively by limiting dilution analysis in a microculture system which allows every normal T-lymphocyte to undergo clonal expansion. The reduced clonogenic potential was unequally distributed among the CD4+ and CD8+ subsets with the CD8+ TIL showing a significant reduction of the frequency of proliferating T-lymphocyte precursors compared to the CD4+ TIL (with a median of 1/50 proliferating T-lymphocytes in CD8+ TIL versus a median of 1/11 in CD4+ TIL). The reduced response of CD8+ TIL was not caused by suppressor cells, lack of surface expression of CD2 and CD3 antigens nor of the alpha, beta T-cell receptor, nor by an accumulation of CD8+ cells of large granular lymphocyte morphology. Using low density cultures, the highly purified CD4+ and CD8+ TIL were stimulated either via the T-cell receptor or the CD2-mediated antigen-independent pathway of activation. Whereas CD8+ TIL did not respond to either stimulus the CD4+ TIL showed evidence of responder and nonresponder groups. In addition, we show that the deficient response obtained by triggering CD4+ TIL via the TCR can be restored by activation of the antigen-independent pathway. Finally, a total of 94 clones from four different TIL samples were obtained by limiting dilution and examined for their respective helper and cytolytic capabilities: 57% of the CD4+ TIL clones were able to produce interleukin 2 and 93% of the CD8+ TIL clones demonstrated cytolytic activity mediated by the T-cell receptor complex, indicating that the functional potential of proliferating TIL is intact.

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.