Pimitespib in patients with advanced gastrointestinal stromal tumor (CHAPTER-GIST-301): a randomized, double-blind, placebo-controlled phase III trial.

BACKGROUND: Prognosis of advanced gastrointestinal stromal tumors (GIST) refractory to tyrosine kinase inhibitors (TKIs) is poor. This randomized, placebo-controlled, phase III trial evaluated the efficacy and safety of pimitespib, a novel heat shock protein 90 inhibitor, in advanced GIST refractory to standard TKIs. PATIENTS AND METHODS: Patients with histologically confirmed GIST refractory to imatinib, sunitinib, and regorafenib were randomized 2 : 1 to oral pimitespib 160 mg/day or placebo for 5 consecutive days per week in 21-day cycles. Following disease progression by blinded central radiological review (BCRR), cross-over to open-label pimitespib was permitted. The primary endpoint was progression-free survival (PFS) by BCRR in the full analysis set. Secondary endpoints included overall survival (OS) adjusted using the rank-preserving structural failure time (RPSFT) method to reduce the expected confounding impact of cross-over. RESULTS: From 31 October 2018 to 30 April 2020, 86 patients were randomized to pimitespib (n = 58) or placebo (n = 28). Median PFS was 2.8 months [95% confidence interval (CI) 1.6-2.9 months] with pimitespib versus 1.4 months (0.9-1.8 months) with placebo [hazard ratio (HR) 0.51 (95% CI 0.30-0.87); one-sided P = 0.006]. Pimitespib showed an improvement in cross-over-adjusted OS compared with placebo [HR 0.42 (0.21-0.85), one-sided P = 0.007]. Seventeen (60.7%) patients receiving placebo crossed-over to pimitespib; median PFS after cross-over was 2.7 months (95% CI 0.7-4.1 months). The most common (≥30%) treatment-related adverse events (AEs) with pimitespib were diarrhea (74.1%) and decreased appetite (31.0%); the most common (≥10%) grade ≥3 treatment-related AE was diarrhea (13.8%). Treatment-related AEs leading to pimitespib discontinuation occurred in three (5.2%) patients. CONCLUSIONS: Pimitespib significantly improved PFS and cross-over-adjusted OS compared with placebo and had an acceptable safety profile in patients with advanced GIST refractory to standard TKIs.

Morphological lymphocytic reaction, patient prognosis and PD-1 expression after surgical resection for oesophageal cancer.

BACKGROUND: Immune checkpoint inhibitors, such as antibody against programmed cell death protein (PD-1), have demonstrated antitumour effects in patients with malignancies, including oesophageal cancer. A lymphocytic reaction observed by pathological examination is a manifestation of the host immune response to tumour cells. It was hypothesized that a stronger lymphocytic reaction to tumours might be associated with favourable prognosis in oesophageal cancer. METHODS: Using a database of resected oesophageal cancers, four morphological components of lymphocytic reactions (peritumoral, intranest, lymphoid and stromal) to tumours were evaluated in relation to clinical outcome, PD-1 expression by immunohistochemistry and total lymphocyte count in blood. RESULTS: Resected oesophageal cancer specimens from 436 patients were included in the study. Among the four morphological components, only peritumoral reaction was associated with patient prognosis (multivariable P for trend <0·001); patients with a higher peritumoral reaction had significantly longer overall survival than those with a lower reaction (multivariable hazard ratio 0·48, 95 per cent c.i. 0·34 to 0·67). The prognostic effect of peritumoral reaction was not significantly modified by other clinical variables (all P for interaction >0·050). Peritumoral reaction was associated with total lymphocyte count in the blood (P < 0·001), supporting the relationship between local immune response and systemic immune competence. In addition, higher morphological peritumoral reaction was associated with high PD-1 expression on lymphocytes in tumours (P = 0·034). CONCLUSION: These findings should help to improve risk-adapted therapeutic strategies and help stratify patients in the future clinical setting of immunotherapy for oesophageal cancer.

ANTECEDENTES: Los inhibidores de los puntos de control inmunitario (checkpoints) (p.ej. los anticuerpos anti-PD-1) han demostrado efectos antitumorales en pacientes con tumores malignos, incluido el cáncer de esófago. La reacción linfocítica detectada en estudios anatomopatológicos es una manifestación de la respuesta inmune del huésped a las células tumorales. Se estableció la hipótesis de que una mayor reacción linfocítica a los tumores podría asociarse con un mejor pronóstico en el cáncer de esófago. MÉTODOS: Usando una base de datos de 436 cánceres de esófago resecados, se evaluaron cuatro componentes morfológicos (peritumoral, intra-epitelial, linfoide y estromal) de las reacciones linfocíticas a tumores en relación con los resultados clínicos, la expresión inmunohistoquímica de PD-1 y el recuento total de linfocitos en sangre. RESULTADOS: De los cuatro componentes, solamente la reacción peritumoral se asoció con el pronóstico del paciente (P multivariable para tendencia < 0,001): los pacientes con mayor reacción peritumoral presentaron una supervivencia global significativamente más prolongada que aquellos pacientes con menor reacción peritumoral (cociente de riesgos instantáneos multivariable, hazard ratio, HR: 0,48; i.c. del 95%: 0,34 -0,67; P <0,001). El efecto pronóstico de la reacción peritumoral no se modificó significativamente por otras variables clínicas (todas las P para la interacción > 0,05). La reacción peritumoral se asoció con el recuento total de linfocitos en la sangre (P < 0,001), lo que respalda la relación entre la respuesta inmune local y la competencia inmune sistémica. Además, una elevada reacción morfológica peritumoral se asoció con una alta expresión de PD-1 en linfocitos tumorales (P = 0,034). CONCLUSIÓN: Estos hallazgos deberían ayudar a mejorar las estrategias terapéuticas adaptadas al riesgo y contribuir a estratificar a los pacientes en el entorno clínico futuro de la inmunoterapia para los pacientes con cáncer de esófago.

Prognostic and clinical impact of sarcopenia in esophageal squamous cell carcinoma.

Recently, depletion of skeletal muscle mass (sarcopenia) has been linked to poor prognosis in several types of cancers, but has not been investigated in esophageal squamous cell carcinoma (ESCC). This retrospective study investigates the relationship between sarcopenia and clinical outcome in ESCC patients treated by surgical resection or definitive chemoradiation therapy (dCRT). The study was retrospectively conducted in a single academic hospital in Kumamoto, Japan, and involved 325 ESCC patients (256 surgical cases and 69 dCRT cases) treated between April 2005 and April 2011. Skeletal muscle mass was quantified by radiologic measures using standard computed tomography scans. The skeletal muscle tissue in the 325 ESCC patients was distributed as follows: mean: 47.10; median: 46.88; standard deviation (SD): 7.39; range: 31.48-71.11; interquartile range, 46.29-47.90. Skeletal muscle tissue was greater in male patients than in female patients (P < 0.0001), but was independent of other clinical and tumor features. Sarcopenia was not significantly associated with overall survival (log rank P = 0.54). Lymph node involvement significantly altered the relationship between sarcopenia and survival rate (P for interaction = 0.026). Sarcopenia significantly reduced the overall survival of patients without lymph node involvement (log rank P = 0.035), but was uncorrelated with overall survival in patients with lymph involvement (log rank, P = 0.31). The anastomosis leakage rate was significantly higher in the sarcopenia group than in the non-sarcopenia group (P = 0.032), but other surgical complications did not significantly differ between the two groups. Sarcopenia in ESCC patients without lymph node involvement is associated with poor prognosis, indicating sarcopenia as a potential biomarker for identifying patients likely to experience an inferior outcome. Moreover, sarcopenia was associated with anastomosis leakage but no other short-term surgical outcome.

Influence of preoperative docetaxel, cisplatin, and 5-fluorouracil on the incidence of complications after esophagectomy for resectable advanced esophageal cancer.

A limited number of patients with resectable advanced esophageal cancer can be cured by surgery alone. Although a regimen that consists of docetaxel, cisplatin, and 5-fluorouracil (DCF) is a potential preoperative chemotherapy (PCT) option for squamous cell carcinoma of the esophagus, the influence of DCF on subsequent esophagectomies remains unclear. A total of 80 patients who received preoperative DCF chemotherapy, and 174 patients who did not receive any preoperative treatment were retrospectively analyzed. There were no treatment-related deaths. No delays in surgery due to adverse events related to DCF were reported. Although patients who received PCT had significantly more advanced cancers and worse preoperative conditions, the incidence rates of complications did not increase. Although the frequency of severe complications was significantly higher in patients who received PCT, this treatment was not an independent factor for the occurrence of severe complications. PCT with DCF did not negatively affect subsequent esophagectomies with regard to the frequency of complications.

Radiofrequency ablation for pulmonary metastases from esophageal squamous cell carcinoma.

Radiofrequency ablation (RFA) is increasingly being used for the treatment of intrathoracic malignancies. Although RFA has been found to be promising in the treatment of lung metastases from some types of neoplasms, little is known concerning its clinical significance in the treatment of pulmonary metastasis from esophageal squamous cell carcinoma (ESCC). This retrospective study evaluated the feasibility, safety, and effectiveness of computed tomography-guided RFA for pulmonary metastasis from ESCC. A series of 10 ESCC patients with 17 pulmonary tumors were included. Correct placement of the ablation device into the target tumor proved to be feasible in all tumors (100%). The mean visual analog scale score, with values that ranged from 0 (no pain) to 10 (worst pain possible), was 1. This suggested that this procedure was well tolerated. No procedure-related deaths occurred. A pneumothorax needing drainage was a major complication in two patients. Local control of ablated tumor lasting for at least 1 year was achieved in 10 (83%) of 12 assessable tumors. Although locoregional recurrences developed in two tumors, these lesions could be recontrolled by repeat treatment with RFA. Three patients died of recurrent disease. The predicted 1- and 2-year overall survival rates after lung RFA were 77.8% and 62.2%, respectively. Percutaneous computed tomography-guided RFA yielded relatively high levels of local control in patients with pulmonary metastases from ESCC and was associated with an acceptable level of complications. It was concluded that a prospective study will be necessary to evaluate the effectiveness of a combination of systemic therapy and RFA for ESCC lung metastases.

Frequency of HER2 expression of circulating tumour cells in patients with metastatic or recurrent gastrointestinal cancer.

BACKGROUND: The clinical significance of circulating tumour cell (CTC) detection in gastrointestinal (GI) cancer remains controversial and the molecular biological characteristics of CTCs are poorly understood. METHODS: A total of 87 patients with metastatic or recurrent GI cancer were prospectively enrolled. Circulating tumour cells and their HER2 status were assessed using the CellSearch System. RESULTS: Among the 62 CTC-positive cases, we found 22 discordant cases (35.5%). Among the HER2-negative primary tumours, 17 of 54 developed HER2-positive CTCs. Five of eight had HER2-negative CTCs among the HER2-positive primary tumours. CONCLUSION: The findings in the current study suggest that it is critical to evaluate the HER2 status of not only the primary tumour but also the CTCs because the metastasising tumour cells are the primary target of systemic therapy.

Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer.

BACKGROUND: LINE-1 methylation level is a surrogate marker of global DNA methylation. LINE-1 methylation in primary colorectal cancers (CRCs) is highly variable and strongly associated with a poor prognosis. However, no study has examined LINE-1 methylation levels of metastatic CRCs in relation to prognosis or assessed the heterogeneity of LINE-1 methylation level within the primary CRCs. METHODS: Pyrosequencing was used to quantify LINE-1 methylation level in 42 liver metastases, 26 matched primary tumours, and 6 matched lymph node (LN) metastases. KRAS, BRAF, and PIK3CA mutation status and microsatellite instability (MSI) status were also examined. RESULTS: The distribution of LINE-1 methylation level in liver metastases was as follows: mean, 67.3; range, 37.1-90.1. Primary tumours showed LINE-1 methylation levels similar to those of matched liver and LN metastases. The difference in LINE-1 methylation level between superficial areas and invasive front areas was within 7.0 in all six cases evaluated. Prognostic impact of LINE-1 hypomethylation in liver metastases on overall survival was not observed. The concordance rate was 94% for KRAS, 100% for BRAF, 88% for PIK3CA, and 97% for MSI. CONCLUSION: Alteration of LINE-1 methylation level may occur in early CRC tumorigenesis, and the LINE-1 methylation level is relatively stable during CRC progression.

[Complete response (CR) in a case of advanced hepatocellular carcinoma by percutaneous multi-ablation therapy].

A 56-year-old male patient was diagnosed with hepatocellular carcinoma by abdominal ultrasonography. The tumor was located in segment 7-1 and was 7 cm in diameter. Two transcatheter arterial chemoembolizations (TACE) were not effective. The patient had experienced more than ten fractures because of fibrous dysplasia of the bone. Laparotomy was very risky, so we decided to perform multi-ablation therapy. This therapy consists of percutaneous radiofrequency ablation (RFA) and percutaneous ethanol injection therapy (PEIT). PEIT was applied to the lesion where extrahepatic the Glisson's capsule was near the tumor. After two sessions with these therapies, the tumor with the surrounding liver parenchyma turned necrotic. A complete response was obtained and the patient has been disease-free for 6 months. We conclude that our multi-ablation therapy is effective for patients with advanced hepatocellular carcinoma who have therapeutic limitations because of some preoperative complications.

Restricted usage of T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation.

We analysed T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo-BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6-7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1-4 (26%, 11 of 41 recipients), VA3-1 (32%) and VB24-1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity-determining region 3 of BV24S1. We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (> 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P < 0.05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T-cell regeneration via a thymic-dependent pathway in children.

Characterization of the protocatechuic acid catabolic gene cluster from Streptomyces sp. strain 2065.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) catalyzes the ring cleavage step in the catabolism of aromatic compounds through the protocatechuate branch of the beta-ketoadipate pathway. A protocatechuate 3,4-dioxygenase was purified from Streptomyces sp. strain 2065 grown in p-hydroxybenzoate, and the N-terminal sequences of the beta- and alpha-subunits were obtained. PCR amplification was used for the cloning of the corresponding genes, and DNA sequencing of the flanking regions showed that the pcaGH genes belonged to a 6. 5-kb protocatechuate catabolic gene cluster; at least seven genes in the order pcaIJFHGBL appear to be transcribed unidirectionally. Analysis of the cluster revealed the presence of a pcaL homologue which encodes a fused gamma-carboxymuconolactone decarboxylase/beta-ketoadipate enol-lactone hydrolase previously identified in the pca gene cluster from Rhodococcus opacus 1CP. The pcaIJ genes encoded proteins with a striking similarity to succinyl-coenzyme A (CoA):3-oxoacid CoA transferases of eukaryotes and contained an indel which is strikingly similar between high-G+C gram-positive bacteria and eukaryotes.

Polyclonal expansion of TCRBV2- and TCRBV6-bearing T cells in patients with Kawasaki disease.

We examined T-cell receptor (TCR) usage, cytokine production and antibody responses to superantigens in patients with Kawasaki disease (KD) to facilitate a better understanding of the immunopathogenesis of KD. The mean percentage of VB2- or VB6. 5-bearing T cells in peripheral blood mononuclear cells (PBMC) of patients with acute-phase KD was significantly higher than that of patients in the convalescent phase of KD or in healthy donors. Expansion of VB2- or VB6.5-bearing T cells was polyclonal because DNA sequences in the complementarity determining region 3 of VB2- and VB6.5-positive cDNA clones were all different from each other. The plasma levels of interleukin (IL)-1beta, IL-2, IL-6, IL-8, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and granulocyte colony-stimulating factor (G-CSF) were elevated in the acute phase of KD. We previously reported that streptococcal pyrogenic exotoxin C (SPEC) was a potent stimulator of VB2- and VB6.5-positive T cells and, furthermore, serum levels of anti-SPEC antibodies were significantly higher in patients with acute and convalescent KD than in age-matched controls. The results of the present study, together with those of our previous report, suggest that SPEC induces activation and polyclonal expansion of VB2- and VB6.5-positive T cells, and that SPEC-induced activation of T cells may lead to the pathogenesis of KD.

Development of a syngenic brain-tumor model resistant to chloroethyl-nitrosourea using a methylguanine DNA methyltransferase cDNA.

Chloroethyl-nitrosourea (CENU) is one of the most potent chemotherapeutic agents for brain tumors. However, acquired resistance to this drug has become a serious problem in the treatment of brain tumor patients. The main mechanism of the resistance is a recruitment of the O6-methylguanine-DNA- methyltransferase (MGMT) in tumor cells. Many approaches, including treatment with enzyme-depletions, antibodies, antisenses, and a ribozyme, have been reported to successfully overcome the resistance. In order to evaluate these approaches properly, we designed a syngenic rat brain-tumor model resistant to CENU. The 9L rat gliosarcoma cells were retrovirally transduced with MGMT cDNA and stereotactically implanted into the brain parenchyma. In this model, rats inoculated with resistant cells died significantly earlier than did rats with control cells after treatment with CENU. Because of the limited intracranial space, the animals presented a restricted survival. Since the survival was sensitive and reproducible, this system may have a role in the evaluation of approaches to drug-resistant brain-tumors.

[Inhibitory effects of natural medicines on the enzymes related to the skin].

In this paper, we investigated the inhibitory effects of water extracts from sixty-six natural medicines on the enzymes related to the skin, which were tyrosinase, hyaluronidase and collagenase. To clarify the inhibitory components in water extracts, tannin quantity and the inhibitory activity of the water extracts after removal of phenolic compounds using polyclar AT, were measured. Twelve kinds of natural medicines were found to have tyrosinase inhibitory activity. Six of them showed that tannin, which contains sufficient amounts in extracts, might be major inhibitory compounds due to a significant decrease of inhibition by these samples after removal of phenolic compounds. The inhibitory compound of Aurantii fructus immaturus was thought phenolic compounds except tannin. The inhibitory compounds may include Armeniacae semen, Perillae folium and Persicae semen besides a phenolic compound. Twenty-seven species among the natural medicines studied showed inhibitory activity on hyaluronidase. Phenolic compounds in these extracts except Artemisiae argyi folium, could not be candidates for hyaluronidase inhibitors. Seven kinds of the natural medicines have inhibitory activity on collagenase. It was estimated that these inhibitory compounds were phenolic compounds. These results are to be expected for finding novel compounds for skin disease or skin-care cosmetics.

[On the "system repeatability" specified in the operating conditions for HPLC assay in JP monographs].

The system repeatability tests in the operating conditions for the HPLC assay by absolute calibration method are specified in 9 monographs in JP13 (Part 1). The reasonableness of the specified system repeatability, expressed in relative standard deviation(%), is discussed in consideration with the specified content and also the probability of Type 1 error(alpha). For simplification, it was assumed that the variance due to the analytical system(sigma s2) is equal to that due to other error sources(sigma e2). Based on the above considerations, it was concluded that the present specification of system repeatability(sigma s) in JP monographs is not always reasonable and some of them should be reexamined following to the described considerations in the text.

Analysis of TCRAV and TCRBV repertoires in healthy individuals by microplate hybridization assay.

We have developed an adaptor ligation PCR-based microplate hybridization assay (MHA) for analysis of T cell receptor alpha chain variable region (TCRAV) and T cell receptor beta chain variable region (TCRBV) repertoires. Forty three TCRAV and thirty eight TCRBV-specific probes were immobilized onto microplate wells in water-soluble carbodiimide. After hybridization of 5'-biotinylated PCR products, quantitative ELISA was carried out and followed by automated colorimetric reading. The conditions for immobilization and hybridization were optimized using representative TCRBV-specific probes. The sensitivity of MHA allows us to detect as low as 40 pg of biotinylated PCR products. The frequencies of individual V segments obtained by MHA were consistent with those obtained by FACS analysis and reverse dot blot assays. Analysis of the entire TCRAV and TCRBV repertoires could be done using a single 96-well plate, and completed in less than 6 h. Simplicity and reproducibility of this method make it suitable for routine laboratory use. The expression of TCRAV and TCRBV segments was next studied in peripheral blood mononuclear cells (PBMC) of 14 healthy donors using the newly developed MHA method. TCRAV8S1, TCRAV23S1, TCRBV2S1, TCRBV3S1, TCRBV4S1, and TCRBV6S5 were highly expressed in PBMC. Further, the TCRAV repertoires among individuals were less variable compared to the TCRBV repertoires. Interestingly, considerable variations in the expression levels of BV3S1, BV4S1, and BV17S1 were observed among individuals. One polymorphic site was found at the coding region of BV4S1, and there were two alleles. These results suggest that variable expression among individuals may be associated with unknown allelic polymorphism in coding and/or regulatory regions of these TCRBV segments, or with disparity in HLA genes.

Quantitative analysis of the usage of human T cell receptor alpha and beta chain variable regions by reverse dot blot hybridization.

We previously developed an adaptor ligation-mediated PCR method to amplify the T cell receptor (TCR) cDNA pools. In the present study we applied reverse dot blot hybridization to PCR-amplified specimens for quantitative analysis of the usage of TCR alpha and beta chain variable (V) region. 44 VA sequence-specific oligonucleotide probes (SSOPs) and 38 VB SSOPs were synthesized corresponding to unique sequences of VA and VB subfamilies. Peripheral blood lymphocytes of ten healthy donors and five T cell clones established from bone marrow cells were examined for VA and VB usage using this method. The results were consistent with those obtained by a colony hybridization method and those by immunofluorescence staining using monoclonal antibodies to VA and VB. Thus, reverse dot blot hybridization for TCR V(alpha) and Vbeta is a new, easy and dependable technique useful for analysis of VA and VB usage by human T cells.

The role of a conserved tyrosine residue in high-potential iron sulfur proteins.

Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.

Establishment and characterization of nurse cell-like clones from human skin. Nurse cell-like clones can stimulate autologous mixed lymphocyte reaction.

We have established nurse cell-like clones from long-term cultures of the human skin. These human skin nurse cell (HSNC)-like clones were type I collagen+, type IV collagen-, vimentin+, cytokeratin-, CD44+, CD54+, and weakly positive for VCAM-1, and easily identified by the pseudoemperipolesis that allowed T lymphocytes to migrate beneath the HSNCs. HSNCs and various T cell lines formed a typical complex in the hanging drop culture system. The majority of human and murine T cells, and some of the tumor cell lines other than T cells, including B lymphoma and myeloblastoma cells, migrated beneath the HSNC clones. HSNC clones produced various cytokines, including IL-6, IL-7, IL-8, IL-9, granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (CSF-1), TGF-beta 1, and c-kit ligand, but could not produce IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, TNF-alpha, or TNF-beta. These characteristics were similar to those of nurse cells established from the murine thymus. Furthermore, IFN-gamma-pretreated HSNC clones that expressed MHC class II Ags induced autologous mixed lymphocyte reaction (AMLR) in autologous PBMCs to proliferate and exhibit the cytotoxicity against altered autologous cells and various tumor cells. These results suggest that HSNCs play an important role in the immunoregulation at skin tissues.