Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

Epstein-Barr virus genome studies in Burkitt's and non-Burkitt's lymphomas in Uganda.

Burkitt's lymphoma (BL) has been widely investigated and has attracted attention because of the possible etiologic role of the Epstein-Barr virus (EBV). To further determine the role of EBV in the causation of this tumor, we measured EBV-specific nuclear antigen (EBNA) and EBV DNA using immunofluorescence and nucleic acid hybridization techniques, respectively. Of 34 BL biopsies, 27 tissues (79%) were EBNA-positive, whereas none of the 25 non-BL biopsy tissues were EBNA-positive. Of 15 BL tumors tested, 14 (93%) were EBV DNA-positive with a mean of 39 (range, 8-86) EBV genome equivalents per cell. Each of the 15 non-BL biopsy specimens subjected to nucleic acid hybridization had less than two virus genome equivalents per cell, although all had serologic evidence of past EBV infection. The findings further supported the possible etiologic role of EBV in African BL and negated the passenger hypothesis. The EBV genome could, therefore, be used as a separating marker between African BL and non-BL lymphomas.

A new murine model of autoimmune orchitis induced by immunization with viable syngeneic testicular germ cells alone. II. Immunohistochemical findings of fully-developed inflammatory lesion.

Previous studies demonstrated that experimental autoimmune orchitis (EAO) was produced in C3H/He mice with very high incidence by two or three subcutaneous injections of viable syngeneic testicular germ cells, without the use of any adjuvants or immunopotentiators and that the disease induced was characterized by a complete lack of epididymitis despite a definite orchitis with hypospermatogenesis. In this report, immunohistochemical characterization of immune cells in the fully-developed orchitic lesion was carried out using monoclonal antibodies and immunoperoxidase staining. Thy-1.2+ cells, Mac-1+ cells, B220+ cells and cytoplasmic Ig-bearing cells in the lesion were estimated to be approximately 30, 15, 20 and 30% of all inflammatory cells, respectively. Major phenotype of T cells in the lesion was CD4+ (approximately 85%) with the remainder (approximately 15%) being CD8+. The percentages of cytoplasmic IgG-, IgA- and IgM-bearing cells were estimated as approximately 35, 60 and 5% of all cytoplasmic Ig-bearing cells, respectively. Deposits of immunoglobulins and third component of complement were identified on the basement membrane of the seminiferous tubules, interstitium between the tubules, vessel endothelium and degenerated germ cells in the lesion. Circulating antibodies directed against the acrosomal portion of germ cells were detected in IgG and IgM classes but not in IgA class. Inflammatory cells (including macrophages, B cells and, probably, activated T cells) in the lesion were Ia+, but Leydig cells, Sertoli cells and germ cells did not stain for Ia at all.

Mesh-and-glue technique to prevent leakage of cerebrospinal fluid after implantation of expanded polytetrafluoroethylene dura substitute--technical note.

Expanded polytetrafluoroethylene (ePTFE) can be used as a dura substitute but is associated with leakage of cerebrospinal fluid (CSF) through the suture line. Fibrin glue alone may not prevent this problem. This new method for sealing the suture line in ePTFE membrane uses an absorbable polyglycoic acid mesh soaked with fibrinogen fluid placed on the suture line. Thrombin fluid is then slowly applied to the wet mesh, forming a large fibrin membrane reinforced by the mesh over the suture line. Only one of 33 patients in whom this technique was used had CSF leakage, whereas 12 of 59 patients in whom a dural defect was closed with ePTFE alone showed postoperative subcutaneous CSF collection (p < 0.05). Our clinical experiences clearly show the efficacy of the mesh-and-glue technique to prevent CSF leakage after artificial dural substitution. Mesh and glue can provide an adequate repair for small dural defect. The mesh-and-glue technique may also be used for arachnoid sealing in spinal surgery.

The pleiotrophin-ALK axis is required for tumorigenicity of glioblastoma stem cells.

Increasing evidence suggests that brain tumors arise from the transformation of neural stem/precursor/progenitor cells. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma. Here we show that anaplastic lymphoma kinase (ALK) and its ligand pleiotrophin are required for the self-renewal and tumorigenicity of glioblastoma stem cells (GSCs). Furthermore, we demonstrate that pleiotrophin is transactivated directly by SOX2, a transcription factor essential for the maintenance of both neural stem cells and GSCs. We speculate that the pleiotrophin-ALK axis may be a promising target for the therapy of glioblastoma.

Generation and characterization of a continuous line of CD8+ suppressively regulatory T lymphocytes which down-regulates experimental autoimmune orchitis (EAO) in mice.

We have previously shown that two injections with viable syngeneic testicular germ cells (TC) alone developed experimental autoimmune orchitis (EAO) in C3H/He mice, and that the induction of antigen-specific tolerance in this EAO model is associated with the generation of antigen-specific suppressively regulatory T (Ts) cells. For the elucidation of the nature of these Ts cells, a murine Ts cell line (designated Ts-A) was established. This line was generated from the spleen cells of C3H/He mice which had received three i.v. injections of a soluble (deaggregated) form of murine testicular antigen (mTA), followed by the repeated selection of these spleen lymphocytes in vitro by stimulation with mTA. Adoptive transfer of Ts-A cells into naive syngeneic mice immediately before the first TC injection was found to downgrade EAO in actively immunized recipients. The transferred Ts-A cells significantly inhibited the cellular immune response to TC in the recipients in an antigen-specific manner, but these cells had no inhibitory effect on the humoral immune response to TC. This line could also inhibit in vitro syngeneic TC-driven proliferation of orchitogenic lymphocytes. Surface phenotype of this line was CD8+, CD4-, Thy-1.2+, CD3+, and TCR alpha beta+. These findings may suggest an in vivo role for suppressively regulatory lymphocytes, capable of inhibiting helper T cells, in the regulation of EAO.

The induction of renal autoantigen-specific T cells by a local Listeria monocytogenes infection.

In order to examine the possibility that a local chronic infection can induce organ-specific autoimmune disease, we inoculated unilateral kidneys with viable Listeria monocytogenes (intrarenal infection). The delayed footpad reaction against syngeneic kidney homogenate (KH) became positive from 1 week after initiating the intrarenal infection. A proliferative response of the spleen T cells from the infected mice was also observed against KH from 1 week after initiating the intrarenal infection, but no such response was seen against liver homogenate (LH). In contrast, an intravenous Listeria infection did not induce a delayed footpad reaction or proliferative response against KH, suggesting that these autoimmune responses were not caused by molecular mimicry between renal antigens and Listeria antigens. Furthermore, the ability to transfer the autoimmune response of spleen T cells from intrarenally infected mice was examined. The transferred mice showed a positive delayed footpad reaction against KH and an interstitial infiltration of mononuclear cells in their kidneys. These results demonstrate that the intrarenal Listeria infection induced renal autoantigen-specific T cells, which subsequently induced an autoimmune interstitial nephritis (AIN). The autoreactive T cells were all induced without immunization by autoantigens mixed with complete Freund's adjuvant. Based on these findings, we propose that a local bacterial infection may induce an autoimmune response against autoantigens in the infected organ and subsequently trigger organ-specific autoimmune disease.

Inflammation alone evokes the response of a TCR-invariant mouse gamma delta T cell subset.

Whether gamma delta T lymphocytes respond to microbial Ags or to inducible host Ags remains a matter of controversy. Using several different disease models and mouse strains, we and others have seen that V gamma 6/V delta 1 gamma delta T cells preferentially increase among the gamma delta T cells infiltrating inflamed tissues. However, it was not clear whether bacteria are necessary to bring about this response. Therefore, we have reexamined this question using a disease model in which inflammation is induced by a purely autoimmune process involving no bacteria, bacterial products, or other foreign material: testicular cell-induced autoimmune orchitis. Using this model we found that gamma delta T cells were still plentiful among the infiltrating T lymphocytes, being 9- to 10-fold more prevalent than in spleen, and that V gamma 6/V delta 1+ cells again represented the predominant gamma delta T cell type. This finding shows that the response of the V gamma 6/V delta 1+ subset does not, in fact, depend upon the presence of bacteria or bacterial products. The stimulus triggering the response of the V gamma 6/V delta 1 gamma delta T cells appears to be neither foreign nor organ-specific in origin, but instead consists of a self-derived host Ag or signal induced during the inflammatory process.

Antigen non-specific tissue damage in T cell-mediated experimental autoimmune orchitis: preliminary characterization of a testis-specific T-cell line by using dermal tissue and cells.

A previous study demonstrated the establishment of a murine testicular antigen (mTA)-specific CD4+ T-cell line (designated BT.1) which was capable of transferring experimental autoimmune epididymo-orchitis to naive recipient mice. The disease transfer was antigen-specific, because no inflammatory lesion was observed in any other organs and tissues of the recipients. In this study, to investigate the local environment of BT.1 cells, the effect of the cells and their culture supernatant on a local tissue integrity was studied. When BT.1 cells were seeded on cultured fibroblastoid cell monolayers, the cells completely disrupted these monolayers in spite of the absence of the specific antigens. Moreover, the culture supernatant of BT.1 cells induced non-specific dermal inflammation when injected into skin tissue of normal syngeneic mice. Therefore, BT.1 cells were shown to devastate a tissue integrity and cause attraction and activation of inflammatory cells of the recipient origin in a local environment. These results suggest that the transferred BT.1 cells will specifically home to the testis and epididymis of recipients but the following devastation of seminiferous tubules and epididymal ducts might be non-specifically produced by the inflammatory cells of both donor and recipient origin in the lesion.

Inhibition of a novel model of murine experimental autoimmune orchitis by intravenous administration with a soluble testicular antigen: participation of CD8+ regulatory T cells.

Recently, we established a novel murine model of experimental autoimmune orchitis (EAO) in C3H/He mice by means of two sc injections of 1 x 10(7) viable syngeneic testicular germ cells (TC) without the use of any adjuvants. Using this model, an effective and reproducible system of immunoregulation in EAO was developed. The induction of this EAO was suppressed by pretreatment with five iv injections of a soluble (deaggregated) form of murine testicular antigen (mTA). The antigen, mTA, was prepared by acid extraction and ammonium sulfate precipitation of defatted testes and epididymides. The development of EAO and relevant delayed-type hypersensitivity was suppressed in an antigen-specific fashion, but anti-TC antibody formation was not affected. A single dose of cyclophosphamide at 2 days after the tolerogenic regime abrogated the unresponsiveness to EAO. Three doses of recombinant interleukin 2 at every other day starting on the next day of the last pretreatment did not overcome the unresponsiveness to EAO. CD8+ T cells isolated from the spleen of deaggregated mTA-pretreated animals could adoptively transfer suppression against EAO into naive recipients, whereas CD4+ T cells failed to transfer the suppression.

Establishment of an experimental model of autoimmune epididymo-orchitis induced by the transfer of a T-cell line in mice.

A murine T-cell line derived from BALB/c mice (designated B.T.1) was established which was capable of adoptively transferring experimental autoimmune orchitis (EAO) in normal recipients. The protocol consisted of preparing lymphocytes obtained from the mice that were immunized with syngenetic testicular germ cells (TGC) and the subsequent repeated selection of the lymphocytes in vitro by stimulation with murine testicular antigens (mTA). Phenotypic analysis revealed that B.T.1 cells were CD4+ T-cells. Intra-peritoneal inoculation of as few as 1 x 10(5) B.T.1 cells, that were stimulated in vitro with mTA before the inoculation, was capable of transferring EAO to naive recipients. In the latter, both delayed type hypersensitivity (DTH) and humoral responses to TGC were augmented. The transferred lesion was characterized by infiltration of inflammatory cells into the epididymis and rete testis and widespread aspermatogenesis in the testis. The transfer of EAO was unsuccessful when the recipients received B.T.1 cells that were maintained in culture medium without stimulation with mTA. In these recipients, anti-TC DTH was not detected, although the specific humoral response was observed. In-vitro characterization of the biological activity of B.T.1 cells revealed that the line had no cytolytic activity against TGC but the culture supernatant had macrophage migration inhibitory activity involved in the DTH response. Therefore, the DTH responsiveness transferred by B.T.1 cells was found to correlate with their orchitis-inducing capacity.

Genetic susceptibility to the induction of murine experimental autoimmune orchitis (EAO) without adjuvant. I. Comparison of pathology, delayed type hypersensitivity, and antibody.

In the present study, it was demonstrated that there were marked strain differences in susceptibility to the induction of our new murine model of experimental autoimmune orchitis (EAO; definite orchitis with hypospermatogenesis) induced by two or three sc injections with viable syngeneic testicular germ cells (TC) without any adjuvants. Among 12 inbred strains of mice examined, the A/J (H-2a), C3H/He (H-2k), and C3H/HeN (H-2k) strains were highly susceptible, whereas the C57BL/6N (H-2b), C57BL/10Sn (H-2b), BALB/cAnN (H-2d), AKR/N (H-2k), CBA/JN (H-2k), C3H/HeJ (H-2k), and MRL/lpr (H-2k) strains were low susceptible, and the DBA/2N (H-2d) as well as C3H/BiKi (H-2k) strains were resistant. In particular, mice of the H-2k haplotype demonstrated varying degrees of susceptibility, from highly to totally resistant, to the induction of EAO. Disease susceptibility to this type of EAO does not seem to be associated with a particular H-2 haplotype. All mice of the highly susceptible strains that received two injections of TC (TC x 2) developed a significant increase in both levels of delayed footpad reaction (DFR) to TC and anti-TC antibodies measured by ELISA. In the low susceptible and the resistant strains receiving TC x 2 or TC x 3, there was no correlation between the immune responses and the susceptibility to disease in these strains, with the exception of the BALB/cAnN mice receiving TC x 3. The low susceptible and the resistant mice that received TC x 2 were classified into four groups based on the DFR and antibody response: the C57BL/6N, BALB/cAnN, CBA/JN, and C3H/HeJ strains were both positive, and the C57BL/10Sn and AKR/N strains were both negative or very low; the DBA/2N and MRL/lpr strains showed negative DFR and positive antibody response, and the C3H/BiKi strain showed quite the opposite. Almost all mice of the 12 inbred strains that received TC x 3 showed positive antibody response, although its level varied. There seems to be no linkage between the cell-mediated and humoral immune responses and the H-2 locus in our new EAO model.

Evidence that the same gamma delta T cells respond during infection-induced and autoimmune inflammation.

Inflammatory responses are induced in both testes of a mouse following injection of Listeria monocytogenes into one testis. Although the uninjected testis contains no detectable bacteria, it undergoes an autoimmune attack. Normally, the testis lacks lymphocytes, but in the infected and autoimmune state, both gamma delta and alpha beta T cells are found as infiltrates. Here, we have examined the repertoire of the infiltrating gamma delta T cells, using two different methods, and found a high frequency of V gamma 6/V delta 1 gamma delta T cells in both infected and autoimmune testes. All of these expressed the invariant V gamma 6/V delta 1 TCR previously reported. However, secondary gamma and delta transcripts present within V gamma 6/V delta 1 hybridomas indicated nonclonality. Interestingly, some of these secondary transcripts were derived from gamma gene rearrangements not previously found in this gamma delta T cell subset, implying a difference in its origin. The increase in V gamma 6/V delta 1 cells observed here in both infected and autoimmune testes, together with our previous finding of a preferential response by the same subset in Listeria-infected liver, indicates that their response is triggered by the inflammation rather than by the infectious agent or because they are already resident in the tissue. We and others have previously reported that the presence of gamma delta T cells during certain inflammatory conditions correlates with less host tissue damage. This result, together with the evidence presented here, further implies that a response by the V gamma 6/V delta 1 subset in some way exerts a controlling influence on the host inflammatory response.

New experimental model for adoptive transfer of murine autoimmune orchitis.

Previous studies demonstrated that experimental autoimmune orchitis (EAO) was produced in C3H/He mice with very high incidence by subcutaneous (s.c.) injections of viable syngeneic testicular germ cells (TC), without resorting to any adjuvants or immunopotentiators. Using this EAO model, a new and simple protocol was developed for adoptive transfer of EAO. Cell donors were C3H/He mice that received s.c. injections twice with TC alone. Spleen cells from the donors were stimulated in vitro with TC, propagated in interleukin-2 containing medium, then injected i.p. to naive recipient mice. This procedure induced severe orchitis and hypospermatogenesis with or without inflammation in epididymis and vas deferens in the recipients at high incidence. Elimination of all T cells or CD4+ T cells before the transfer produced no histopathological signs in the recipients whereas that of the CD8+ T cells or B cells had no inhibitory effect on the disease transfer, indicating that the effector cells are CD4+ T cells.

Establishment of a murine thymic epithelial cell line capable of inducing both thymic nurse cell formation and thymocyte apoptosis.

A thymic epithelial cell (TEC) line (B/c. TEC-L1) was established from a normal thymus of a 4-week BALB/c mouse. The B/c. TEC-L1 had an epithelial morphology showing a contact-inhibited cobblestone-like arrangement with occasional desmosome-like structures at the adjacent cellular membranes. B/c.TEC-L1 cells showed positive staining for desmosomal glycoprotein, cytokeratin, thymosin alpha 1 beta 3, and I-Ad, and MHC class I antigens. The doubling time was 24 hours, and the chromosome number ranged from 52 to 78 with the mode of 70. Coculture of B/c.TEC-L1 cells with syngeneic, peanut agglutinin-agglutinated (PNA+) thymocytes in suspension at 37 degree C was followed by the formation of TEC thymocyte rosettes, after which the reconstitution of thymic nurse cells ensued. At 4 degrees C, PNA+ thymocytes bound to the B/c.TEC-L1 cell but did not form thymic nurse cells. PNA- thymocytes, although to a lesser degree than PNA+ cells, bound to the TECs at 37 degrees C, but at 4 degrees C few cells bound to the TECs. Allogeneic thymocytes also bound to the TECs at 37 degrees C. When the PNA+ thymocytes were cultured on the B/c.TEC-L1 monolayer, the small ones chiefly adhered on the surface of the TECs, while underneath the TECs the relatively large thymocytes (including cells in mitosis) predominated. Although the PNA- thymocytes bound to the surface of the monolayer within a few hours after coculture, by 24 hours nearly all cells disappeared. It is presumed that the thymocytes creeping underneath the B/c.TEC-L1 monolayer and those enveloped within the thymic nurse cell reconstituted in the suspension culture; both may be placed in circumstances analogous to the thymic microenvironment, wherein immature thymocytes appear to contact TECs directly and to be exposed to higher concentrations of thymic hormones and other soluble factors. Additionally, cell death in the PNA+ thymocytes was also observed in the coculture with B/c.TEC-L1 cells. The PNA+ cells revealed the morphological changes termed "apoptosis" characterized by chromatin condensation and nuclear fragmentation.

Suppression of efferent limb of testicular autoimmune response by a regulatory CD4+ T cell line in mice.

A murine T cell line (designated as C.Ts) as a mediator of suppression of experimental autoimmune orchitis (EAO) was established. The method of establishment of C.Ts cell line was preparing spleen cells from C3H/He mice hyperimmunized with testicular germ cells (TC) and the repeated selection of the lymphocytes in vitro by stimulation with mouse testicular antigens (mTA). The C.Ts cells were Thy1.2+, surface immunoglobulin-, CD3+, CD4+ and CD8-. The cells could suppress the induction of EAO when transferred into actively EAO-sensitized mice only at the pre-clinical stage of the disease (efferent limb of the autoimmune response). The transferred C.Ts cells significantly inhibited both cellular and humoral immune responses to TC in the recipients in an antigen-specific manner. The disease suppression by C.Ts cells was found to depend upon their cell number, and their suppressive activity was markedly augmented by in vitro stimulation with mTA.

Multilobular cavernous malformation: report of two cases.

The authors report two cases of cavernous malformation characterized by a multilobular appearance on magnetic resonance images. At surgery, the malformations consisted of several nests of angiomatous components that were separated by intervening brain tissue and connected with each other by tiny vessels. This basic configuration seems to explain the unexpected postoperative recurrence of cavernous malformations and/or rebleeding from the residual lesions.

Infection of Mycobacterium bovis bacillus Calmette-Guérin in antibody-mediated gamma delta T-cell-depleted mice.

To evaluate the hypothesis that gamma delta T cells participate in protective immunity against mycobacterial infection, we depleted gamma delta T cells from mice by administration of anti-T-cell receptor (TCR)gamma delta monoclonal antibody (mAb) and analysed protection against Mycobacterium bovis bacillus Calmette-Guérin (BCG). The gamma delta T-cell-depleted mice did not show any exaggerated bacterial multiplication compared with control mice. In contrast, alpha beta T-cell-depleted mice, which were administrated anti-TCR alpha beta mAb before BCG infection, showed a depressed protective immunity. These results suggest that gamma delta T cells are not essential for coping with a primary BCG infection.