Identification of characteristic subepithelial surface granulomatosis in immune-related adverse event-associated enterocolitis.

Immune checkpoint inhibitors (ICIs) have provided an additional treatment option for various types of human cancers. However, ICIs often induce various immune-related adverse events (irAEs). Enterocolitis is a major irAE with poorly understood histopathological characteristics. In this study, we retrospectively investigated the histopathology of colon tissue samples from 17 patients treated with ICIs. There were two major histological patterns of colitis: an ulcerative colitis-like pattern and a graft vs host disease-like pattern. Although these two patterns of colitis were mutually exclusive, both patterns often showed a characteristic that we call "subepithelial surface granulomatosis" (SSG), which has not been reported in other types of colitis. SSG was found even in colon tissue without symptoms or endoscopic findings of colitis. Given the increasing reports of sarcoid reaction or exacerbation of tuberculosis after treatment with ICIs, granuloma formation could be a histological hallmark of systemic immune activation by ICIs. Although statistical significance was not obtained, probably because of the small sample size, SSG may be a surrogate biomarker of systemic anticancer immune activation. We propose that a prospective study with larger sample size be performed.

Occult carcinoma confirmed to be a diffuse sclerosing variant of papillary thyroid carcinoma with unusual immunohistochemical features: A pitfall of clinicopathological diagnosis.

Immunocytochemistry in a 78-year-old man diagnosed as having systemic metastatic cancer of unknown primary origin revealed atypical cells positive for napsin A and TTF-1, suggesting adenocarcinoma of the lung. However, there was no evidence of a primary lesion in the lung on positron emission tomography/computed tomography or at autopsy. Meanwhile, both the left and right thyroid lobes were firm and grayish white with marked fibrosis. Histology identified a diffuse sclerosing variant of papillary thyroid carcinoma that was positive for TTF-1 and napsin A but negative for PAX8. This disease entity is often misdiagnosed clinically as chronic thyroiditis. This is the first report of napsin A-positive and PAX8-negative thyroid carcinoma and highlights the pitfalls of clinicopathological diagnosis.

Essential Role of GATA2 in the Negative Regulation of Type 2 Deiodinase Gene by Liganded Thyroid Hormone Receptor ß2 in Thyrotroph.

The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the ß subunit of TSH (TSHß). We previously reported that T3-dependent repression of the TSHß gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived TαT1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with TαT1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TRß2. The inhibition by T3-bound TRß2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TRα1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSHß gene.

[Polyorchidism: a case report and review of the literature].

A 10-year-old boy presented with a painless left scrotal mass that had been present for 2 years. Ultrasound examination revealed 2 left testes measuring 2.4 ml and 1.0 ml with the same echogenicity. Atsurgery, there were 2 similarly sized testes connecting with other spermatic vascular, but the supernumerary testis had no connection with the vas deferens. Histological findings showed immature testicular tissue without malignancy. We report the 26th case of polyorchidism in Japan and discuss this condition.

Validity and reproducibility of Ki-67 assessment in gastrointestinal stromal tumors and leiomyosarcomas.

With the aim of standardizing Ki-67 immunohistochemistry, we assessed interobserver and interlaboratory variability of the Ki-67 labeling index and Ki-67 score among eight general pathologists for 24 gastrointestinal stromal tumors (GISTs) and 12 leiomyosarcomas, which were predominantly of the gastrointestinal (GI) tract, mesentery and retroperitoneum, based on a review of a tissue microarrays subjected to immunohistochemistry with antibodies for Ki-67. For Ki-67 immunostaining of mesenchymal tumors of the GI tract, including GISTs, differences were seen in the scores given by regional hospitals. Conversely, for two categories of the Ki-67 labeling index, namely <10% and ≥10%, concordance of the Ki-67 score between microscopic observation and image analysis, and between the observers, was good, but it was not good for the other four categories of the index for <5%, 5-9%, 10-29%, and ≥30%. The concordance of the Ki-67 scores between the observers in two categories was higher using the Ki-67 pre-stained tissue microarrays (TMAs) within each participating institute than that using the Ki-67 stained TMAs between the participating institutes. The reproducibility of a 10% cut-off value for the Ki-67 labeling index to predict the prognosis of GISTs was relatively high, but there is an urgent need to standardize the staining technique.

Use of potassium channel tetramerization domain-containing 12 as a biomarker for diagnosis and prognosis of gastrointestinal stromal tumor.

Previously, we showed that the expression of potassium channel tetramerization domain-containing 12 (KCTD12), which was discovered by a proteomics approach, is associated with high-risk behavior of gastrointestinal stromal tumors (GISTs). Here, we examined the distribution and expression of this protein by immunostaining with a commercially available polyclonal KCTD12 antibody in GISTs (n = 64) and other types of malignancy (n = 168) to clarify its diagnostic and clinical significance. Diffuse KCTD12 immunoreactivity was found in most GISTs (52 cases; 81%). KCTD12 expression was observed primarily in vascular endothelial cells, Purkinje cells of the cerebellum, and some neurons scattered throughout the cerebral cortex. KCTD12 was absent from not only the interstitial cells of Cajal but also interstitial cells of Cajal hyperplasia that was encountered incidentally in colon diverticulitis. KCTD12 immunostaining was also seen in malignant peripheral nerve sheath tumors (2/10 cases; 20%), synovial sarcomas (2/10; 20%), solitary fibrous tumor (1/8; 13%), angiosarcoma (1/7; 14%), and colon adenocarcinoma (1/24; 4%). In survival analyses, the 5-year recurrence-free survival rate of patients without KCTD12 expression was only 16.7% compared with 95.6% in those with KCTD12 expression (P < .0001). Ki-67 and KCTD12 were significant predictors of recurrence-free survival, and KCTD12 expression provided additional information about recurrence-free survival after accounting for Ki-67 status. Overall, KCTD12 expression was specific for GISTs from neoplastic and nonneoplastic adult tissues other than brain and served as a predictor of GIST recurrence. These findings suggest that KCTD12 is a useful and reliable biomarker for both the diagnosis and prognosis of GIST.

Liganded thyroid hormone receptor inhibits phorbol 12-O-tetradecanoate-13-acetate-induced enhancer activity via firefly luciferase cDNA.

Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin ß gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity, which is inhibited by T3/TR.

GATA2 mediates thyrotropin-releasing hormone-induced transcriptional activation of the thyrotropin ß gene.

Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHß and α-glycoprotein (αGSU) subunit genes. TSHß expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHß gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHß gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHß promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHß gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHß promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHß expression.

Syndrome of inappropriate secretion of thyrotropin associated with thymoma-related peripheral nerve hyperexcitability.

Syndrome of inappropriate secretion of thyrotropin (SITSH) is a clinical state of inappropriately elevated secretion of thyrotropin (TSH) in the presence of elevated free thyroid hormones. Peripheral nerve hyperexcitability (PNH) is a rare disorder characterized by muscle twitching at rest. No relation between them is known. A 49-year-old man was referred to our hospital because of elevated serum free thyroxine (2.6 ng/dL; normal range, 0.9-1.7) and normal TSH (2.7 mIU/L; normal range, 0.5-5.0). Genetic analysis revealed no mutations of the thyroid hormone receptor ß gene. Magnetic resonance imaging visualized no pituitary adenoma. He complained of appetite loss, weight loss, myokymia, paraesthesia, hyperhydrosis and insomnia. Chest X ray and computed tomography (CT) scan showed a mediastinal tumor diagnosed as a thymoma by CT-guided biopsy. Electromyography disclosed fasciculations and myokymic discharges. Nerve conduction studies showed prolonged after-discharges following evoked compound muscle action potential. The patient was diagnosed with thymoma-associated PNH based on neurological manifestations and neurophysiological findings, and was treated with pulse therapy with methylprednisolone after thymectomy. Interestingly, the SITSH state became less prominent as his neurological manifestations improved. This is the first case of SITSH possibly caused by thymoma-associated PNH.

A novel correlation between LINE-1 hypomethylation and the malignancy of gastrointestinal stromal tumors.

PURPOSE: Gastrointestinal stromal tumors (GIST) are the most important mesenchymal tumors of the gastrointestinal tract. The vast majority of GISTs exhibit activating mutations of KIT or PDGFRA, but epigenetic alteration of GISTs is largely unknown. In this study, we aimed to clarify the involvement of DNA methylation in GIST malignancy. EXPERIMENTAL DESIGN: A total of 106 GIST specimens were studied. Levels of LINE-1 methylation were analyzed using bisulfite pyrosequencing. In addition, methylation of three other repetitive sequences (Alu Yb8, Satellite-α, and NBL2) was similarly analyzed, and CpG island hypermethylation was analyzed using MethyLight. Array-based comparative genomic hybridization (array CGH) was carried out in 25 GIST specimens. RESULTS: LINE-1 hypomethylation was significantly correlated with risk, and high-risk GISTs exhibited significantly lower levels of LINE-1 methylation than low-risk (61.3% versus 53.2%; P = 0.001) or intermediate-risk GISTs (60.8% versus 53.2%; P = 0.002). Hypomethylation of Satellite-α and NBL2 was also observed in high-risk GISTs. By contrast, promoter hypermethylation was relatively infrequent (CDH1, 11.2%; MLH1, 9.8%; SFRP1, 1.2%; SFRP2, 11.0%; CHFR, 9.8%; APC, 6.1%; CDKN2A, 0%; RASSF1A, 0%; RASSF2, 0%) and did not correlate with LINE-1 methylation or risk. Array CGH analysis revealed a significant correlation between LINE-1 hypomethylation and chromosomal aberrations. CONCLUSIONS: Our data suggest that LINE-1 hypomethylation correlates significantly with the aggressiveness of GISTs and that LINE-1 methylation could be a useful marker for risk assessment. Hypomethylation may increase the malignant potential of GISTs by inducing accumulation of chromosomal aberrations.

[A case of advanced gastric cancer responding to S-1/paclitaxel/lentinan as neoadjuvant chemoimmunotherapy].

A 71-year-old man suffering from epigastric discomfort and dizziness was admitted to our hospital and diagnosed with advanced gastric cancer with bulky lymph node metastases and liver metastasis. We thought a complete resection would be difficult, so he was treated with neo-adjuvant immunochemotherapy in combination with S-1 80 mg/m2 (2 weeks administration and 2-week rest), paclitaxel (PTX) 50 mg/m2 (day 1, 8, 15) and Lentinan (LNT) 2 mg/body (day 1, 8, 15). After 5 courses of this treatment, swollen lymph nodes decreased in size and the metastatic liver tumor disappeared. Total gastrectomy with lymph node dissection was performed. The histological diagnosis was pT2 pN0, Stage I B. Histological effects of primary tumor and lymphnodes were judged to be grade 2 and grade 3, respectively. We considered that the combination of S-1, PTX and LNT can be effective and safe for advanced gastric cancer.

High incidence of thyroid cancer in focal thyroid incidentaloma detected by 18F-fluorodeoxyglucose [corrected] positron emission tomography in relatively young healthy subjects: results of 3-year follow-up.

As 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is becoming a common imaging modality, the number of thyroid incidentalomas identified by FDG-PET (PET incidentaloma) is increasing. The purpose of this study was to elucidate the risk of cancer in focal thyroid PET incidentaloma in healthy subjects of relatively younger age as well as the usefulness of repeated FDG-PET. The study was conducted with an observation period of three years. A total of 1,501 healthy volunteers (mean age, 43.5+/-9.7 years) underwent the first FDG-PET from August 2003 to July 2004. When focal thyroid PET incidentaloma was found, further diagnostic examination was conducted. When thyroid cancer was suspected, surgical resection was performed with the patient' s agreement. Patients with PET incidentaloma without surgery were offered annual US and FDG-PET and finally FNAB was performed in the fourth year. Focal thyroid PET incidentaloma was observed in 20 subjects. The final diagnoses in 20 subjects were malignant in 11 (ten papillary thyroid carcinoma (PTC) and one thyroid carcinoma showing thymus-like differentiation), indeterminate in one, and benign in eight subjects. Seven patients not treated surgically at the first examination had annual FDG-PET. One patient with PTC showed increasing SUVmax, but another with a benign nodule exhibited a similar increase. Others (one with PTC, one with an indeterminate nodule, and three with benign nodules) exhibited negligible SUVmax changes. When closely examined, focal thyroid PET incidentaloma in relatively young healthy adults has a high probability of malignancy. Repeated FDG-PET to follow up patients with thyroid nodules is ineffective.

Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin beta promoter.

Thyrotropin (TSH) is a heterodimer consisting of alpha and beta chains, and the beta chain (TSHbeta) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHbeta expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHbeta promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHbeta promoter without PIT1. The deleted region (nt -82/-52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHbeta promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

Inhibition of GATA2-dependent transactivation of the TSHbeta gene by ligand-bound estrogen receptor alpha.

Transcriptional repression of the TSH-specific beta subunit (TSHbeta) gene has been regarded to be specific to thyroid hormone (tri-iodothyronine, T(3)) and its receptors (TRs) in physiological conditions. However, TSHbeta mRNA levels in the pituitary were reported to decrease in the administration of pharmacologic doses of estrogen (17-beta-estradiol, E(2)) and increase in E(2) receptor (ER)-alpha null mice. Here, we investigated the molecular mechanism of inhibition of the TSHbeta gene expression by E(2)-bound E(2)-estrogen receptor 1 (E(2)-ERalpha). In kidney-derived CV1 cells, transcriptional activity of the TSHbeta promoter was stimulated by GATA2 and suppressed by THRBs and ERalpha in a ligand-dependent fashion. Overexpression of PIT1 diminished the E(2)-ERalpha-induced inhibition, suggesting that PIT1 may protect GATA2 from E(2)-ERalpha targeting by forming a stable complex with GATA2. Interacting surfaces between ERalpha and GATA2 were mapped to the DNA-binding domain (DBD) of ERalpha and the Zn finger domain of GATA2. E(2)-dependent inhibition requires the ERalpha amino-terminal domain but not the tertiary structure of the second Zn finger motif in E(2)-ERalpha-DBD. In the thyrotroph cell line, TalphaT1, E(2) treatment reduced TSHbeta mRNA levels measured by the reverse transcription PCR. In the human study, despite similar free thyroxine levels, the serum TSH level was small but significantly higher in post- than premenopausal women who possessed no anti-thyroid antibodies (1.90 microU/ml+/-0.13 S.E.M. vs 1.47 microU/ml+/-0.12 S.E.M., P<0.05). Our findings indicate redundancy between T(3)-TR and E(2)-ERalpha signaling exists in negative regulation of the TSHbeta gene.

Influence of body mass index and total testosterone level on biochemical recurrence following radical prostatectomy.

OBJECTIVE: A high body mass index (BMI) and a low testosterone level were recently reported to be prognostic factors for prostate-specific antigen (PSA) recurrence following radical prostatectomy (RP). The goal of this study was to clarify their relationship and influences on biochemical recurrence after RP. METHODS: We analysed 126 patients whose data, including the pre-operative BMI and pre-operative serum total testosterone level, were available. All patients underwent RP at our institution between March 1998 and April 2006 without any adjuvant therapy or pelvic lymph node metastasis. The Cox proportional hazards model was used for the multivariate analysis regarding PSA recurrence for the variables of age, operation period, BMI, clinical stage, PSA, Gleason's sum, pre-operative serum total testosterone level and margin status. RESULTS: There were no internal correlations among the parameters we used, even between BMI and the total testosterone level. The total testosterone level was not different between two BMI groups (BMI <26.4 and >/=26.4 kg/m(2): the cut-off is the mean + 1 SD). BMI, PSA and Gleason's sum were found to be independent predictors for PSA recurrence through the multivariate analysis. PSA recurrence-free survival rates at 2 years were 77% for BMI <26.4 kg/m(2), and 31% for BMI >/=26.4 kg/m(2) (P = 0.002, log-rank test, 95% CI: 1.489-7.726). CONCLUSIONS: The current study suggests that high BMI independently contributes to PSA recurrence but that the total testosterone level does not. Although the mechanism by which obesity promotes PSA recurrence in RP patients has not been established, careful observation is needed for patients with high BMI.

Essential role of GATA2 in the negative regulation of thyrotropin beta gene by thyroid hormone and its receptors.

Previously we reported that the negative regulation of the TSHbeta gene by T(3) and its receptor [thyroid hormone receptor (TR)] is observed in CV1 cells when GATA2 and Pit1 are introduced. Using this system, we further studied the mechanism of TSHbeta inhibition. The negative regulatory element (NRE), which had been reported to mediate T(3)-bound TR (T(3)-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSHbeta-D4-chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T(3)-TR. The Zn finger region of GATA2 interacts with the DNA-binding domain of TR in a T(3)-independent manner. The suppression by T(3)-TR was impaired by overexpression of a dominant-negative type TR-associated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, TalphaT1, revealed that T(3) treatment recruited histone deacetylase 3, reduced the acetylation of histone H4, and caused the dissociation of TRAP220 within 15-30 min. The reduction of histone H4 acetylation was transient, whereas the dissociation of TRAP220 persisted for a longer period. In the negative regulation of the TSHbeta gene by T(3)-TR we report that 1) GATA2 is the major transcriptional activator of the TSHbeta gene, 2) the putative NRE previously reported is not required, 3) TR-DNA-binding domain directly interacts with the Zn finger region of GATA2, and 4) histone deacetylation and TRAP220 dissociation are important.

Bilateral diffuse uveal melanocytic proliferation in a patient with cancer-associated retinopathy.

PURPOSE: To describe a patient with bilateral diffuse uveal melanocytic proliferation (BDUMP) and cancer-associated retinopathy (CAR). DESIGN: Interventional case report. METHODS: A 66-year-old woman developed progressive vision loss 4 months after total hysterectomy. Ophthalmologic examination, Western blot test of sera and aqueous humor, and immunohistochemistry of carcinoma cells were performed. RESULTS: Testing revealed BDUMP and severe retinal dysfunction. Autoantibodies against recoverin and heat shock cognate protein 70 (hsc 70) were detected in serum. Cytoplasmic immunoreactivity for recoverin and hsc 70 was observed in endometrioid carcinoma cells. CONCLUSIONS: Simultaneous cases of BDUMP and CAR are rare. Aberrantly expressed recoverin and hsc 70 triggered serum autoantibody production, which caused photoreceptor degeneration.

Five novel monoclonal antibodies to thymic epithelial cell surface antigens in rats.

OBJECTIVE: To establish monoclonal antibodies (mAbs) against thymic epithelial cells and study the function of epithelial cells during T-cell differentiation in the thymus. METHODS: Hybridomas secreting mAbs against thymic epithelial cells were derived by immunization of Balb/c mice with two thymic epithelial cell lines, TaD3 and FTE. The distribution of antigens recognized by these mAbs was detected by immunochemical staining and cytofluorographic analysis, and the molecular weight of the antigens by immunoblotting. RESULTS: Five specific monoclonal antibodies (mAb) were obtained. On the basis of their distribution in the thymus determined by immunochemical staining, mAb RE-4D8 was regarded as clusters of thymic epithelium staining (CTES) type IIA: mAb RE-12B2, which showed a unique distribution pattern only in the medulla, was CTES type V: mAb RE-5C6 was CTES type IV: mAb RE-6D6 might be CTES type IIB: and mAb RE-1D4 was classified as type V. The molecular weight (MW) of antigen RE-4D8, RE-6D6 and RE-12B2 were 120 kDa, 220 kDa and 35 kDa, respectively. Antigen RE-1D4 is a novel marker of cortical epithelium, several established thymic epithelial cell lines were classified and their original intrathymic locations were determined by these mAbs. Thymic cell lines, TuD3 and FTE were cortical phenotypes whereas TaD3 had a medullar phenotype. CONCLUSIONS: These mAbs clearly demonstrate the heterogeneity of the thymic epithelium; they could detect antigens not only in the cytoplasm but also on the surface of thymic epithelial cells. Our data suggest that these newly established mAbs may help elucidate the interaction between thymocytes and epithelial cells during T cell maturation.