Sari Iwasaki

Exclusive expression of proteasome subunit β5t in the human thymic cortex

The ubiquitin-proteasome pathway, which degrades intracellular proteins, is involved in numerous cellular processes, including the supply of immunocompetent peptides to the antigen presenting machinery. Proteolysis by proteasomes is conducted by three beta subunits, beta1, beta2, and beta5, of the 20S proteasome. Recently, a novel beta subunit expressed exclusively in cortical thymic epithelial cells was discovered in mice. This subunit, designated beta5t, is a component of the thymoproteasome, a specialized type of proteasomes implicated in thymic positive selection. In this study, we show that, like its mouse counterpart, human beta5t is expressed exclusively in the thymic cortex. Human beta5t was expressed in approximately 80% of cortical thymic epithelial cells and some cortical dendritic cells. Human beta5t was incorporated into proteasomes with two other catalytically active beta subunits beta1i and beta2i, forming 20S proteasomes with subunit compositions characteristic of thymoproteasomes. The present study demonstrates, for the first time, the existence of thymoproteasomes in the human thymic cortex, indicating that thymoproteasome function is likely conserved between humans and mice.

Rat CD4 + CD8 + Macrophages Kill Tumor Cells through an NKG2D- and Granzyme/Perforin-Dependent Mechanism

We previously identified a subpopulation of monocyte/macrophage lineage cells expressing both CD4 and CD8. This subpopulation was expanded in rat peripheral blood and spleen after immunization with adjuvants containing killed tuberculosis germs. CD4+CD8+ monocytes/macrophages obtained from preimmunized rats exhibited a Th1-type cytokine/chemokine profile, expressed high levels of Fas ligand, perforin, granzyme B, and NKR-P2 (rat ortholog of human NKG2D), and killed certain tumor cells. In the present study, we confirmed that CD4+CD8+ monocytes/macrophages are distinct from splenic dendritic cells (DCs) or IFN-producing killer DCs. In vitro cytotoxic assays revealed that CD4+CD8+ macrophages killed tumor cells in a cell-cell contact-dependent manner and that expression of the retinoic acid early transcript 1 (a ligand for NKG2D) made tumor cells susceptible to killing by CD4+CD8+ macrophages. Furthermore, inhibitors of granzyme and perforin significantly decreased cytotoxic activities of CD4+CD8+ macrophages. Consistent with these in vitro findings, preimmunization with adjuvants containing killed tuberculosis germs elevated the expression of granzyme B in tumor-infiltrating CD4+CD8+ macrophages and significantly inhibited the growth of inoculated tumor cells. Our current work demonstrates that CD4+CD8+ macrophages are a unique subpopulation of monocyte/macrophage lineage cells that kill tumor cells in an NKG2D- and granzyme/perforin-dependent mechanism.

Plasma‐dependent, antibody‐ and Fcγ receptor‐mediated translocation of CD8 molecules from T cells to monocytes

CD8αβ heterodimers are mainly expressed on cytotoxic T lymphocytes. This study demonstrated the detection of CD8αβ heterodimers on human monocytes by whole blood erythrocyte lysis method in flow cytometry. Results revealed that CD8αβ heterodimers were not produced by monocytes themselves, but were transferred from T cells to monocytes when these cells were coincubated in plasma and with anti‐CD8 monoclonal antibody (mAb). For completion of CD8 translocation from T cells to monocytes, cell‐to‐cell contact between T cells and monocytes, as well as binding of the Fc portion of the anti‐CD8 mAb and Fcγ receptor II (FcγRII) on monocytes were required. Furthermore, the dynamism of cell membrane and cytoskeleton were involved in the mechanism of CD8 translocation. Interestingly, CD3 and αβT cell receptor (TCR) were also transferred from T cells to monocytes accompanied by CD8. These phenomena are consistent with Ab‐dependent and FcγR‐mediated trogocytosis, which is recently recognized as one of the intercellular communication processes of the immune system. Trogocytosis means exchange of plasma membrane including cell surface molecules in conjugates formed between immune cells. Results of this study could provide another model of trogocytosis and clearly indicated that putative plasma factors were critically implicated in the mechanism of Ab‐dependent and FcγR‐mediated trogocytosis.

Mechanism of Fcγ Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

The whole blood erythrocyte lysis method is the most common protocol of sample preparation for flow cytometry (FCM). Although this method has many virtues, our recent study has demonstrated false-positive results when surface markers of monocytes were examined by this method due to the phenomenon called Fcγ receptor (FcγR)-mediated trogocytosis. In the present study, similar FcγR-mediated trogocytosis-based false-positive results have been demonstrated when granulocytes were focused on instead of monocytes. These findings indicated that not only monocytes but also granulocytes, the largest population with FcγR expression in peripheral blood, could perform FcγR-mediated trogocytosis. Since the capacity of FcγR-mediated trogocytosis was different among blood samples, identification of factors that could regulate the occurrence of FcγR-mediated trogocytosis should be important for the quality control of FCM. Our studies have suggested that such factors are present in the serum. In order to identify the serum factors, we employed the in vitro model of FcγR-mediated trogocytosis using granulocytes. Investigation with this model determined the serum factors as heat-labile molecules with molecular weight of more than 100 kDa. Complements in the classical pathway were initially assumed as candidates; however, the C1 inhibitor did not yield an obvious influence on FcγR-mediated trogocytosis. On the other hand, although immunoglobulin ought to be resistant to heat inactivation, the inhibitor of human anti-mouse antibodies (HAMA) effectively blocked FcγR-mediated trogocytosis. Moreover, the inhibition rates were significantly higher in HAMAhigh serum than HAMAlow serum. The collective findings suggested the involvement of heterophilic antibodies such as HAMA in the mechanism of false-positive results in FCM due to FcγR-mediated trogocytosis.

Establishment of a vascular endothelial cell-reactive type II NKT cell clone from a rat model of autoimmune vasculitis

We previously generated a rat model that spontaneously developed small vessel vasculitis (SVV). In this study, a T cell clone reactive with rat vascular endothelial cells (REC) was established and named VASC-1. Intravenous injection of VASC-1 induced SVV in normal recipients. VASC-1 was a TCRαβ/CD3-positive CD4/CD8 double-negative T cell clone with expression of NKG2D. The cytokine mRNA profile under unstimulated condition was positive for IL-4 and IFN-γ but negative for IL-2 and IL-10. After interaction with REC, the mRNA expression of IL-2, IL-5 and IL-6 was induced in VASC-1, which was inhibited by blocking of CD1d on the REC surface. Although the protein levels of these cytokines seemed to be lower than the detection limit in the culture medium, IFN-γ was detectable. The production of IFN-γ from the VASC-1 stimulated with LPS-pre-treated REC was inhibited by the CD1d blockade on the REC. These findings indicated VASC-1 as an NKT cell clone. The NKT cell pool includes two major subsets, namely types I and II. Type I NKT cells are characterized by expression of semi-invariant TCRs and the potential to bind to marine sponge-derived α-galactosylceramide (α-GalCer) loaded on CD1d; whereas, type II NKT cells do not manifest these characteristics. VASC-1 exhibited a usage of TCR other than the type I invariant TCR α chain and did not bind to α-GalCer-loaded CD1d; therefore, it was determined as a type II NKT cell clone. The collective evidence suggested that REC-reactive type II NKT cells could be involved in the pathogenesis of SVV in rats.

Possible Implication of Fcγ Receptor-Mediated Trogocytosis in Susceptibility to Systemic Autoimmune Disease

Leukocytes can “gnaw away” the plasma membrane of other cells. This phenomenon, called trogocytosis, occurs subsequent to cell-to-cell adhesion. Currently, two mechanisms of trogocytosis, adhesion molecule-mediated trogocytosis and Fcγ receptor-(FcγR-) mediated trogocytosis, have been identified. In our earlier study, we established an in vitro model of FcγR-mediated trogocytosis, namely, CD8 translocation model from T cells to neutrophils. By using this model, we demonstrated that the molecules transferred to neutrophils via FcγR-mediated trogocytosis were taken into the cytoplasm immediately. This result suggests that the chance of molecules transferred via FcγR-mediated trogocytosis to play a role on the cell surface could be time-limited. Thus, we consider the physiological role of FcγR-mediated trogocytosis as a means to remove antibodies (Abs) that bind with self-molecules rather than to extract molecules from other cells. This concept means that FcγR-mediated trogocytosis can be a defense mechanism to Ab-mediated autoimmune response. Moreover, the activity of FcγR-mediated trogocytosis was revealed to be parallel to the endocytotic activity of neutrophils, which was critically related to the susceptibility to systemic autoimmune diseases. The collective findings suggest that FcγR-mediated trogocytosis could physiologically play a role in removal of Abs bound to self-antigens and prevent autoimmune diseases.

Focal 18F-FDG uptake in bone marrow on PET/CT in a patient with JAK2 mutation without overt myeloproliferative neoplasm.

A 58-year-old female diagnosed with early stage esophageal carcinoma in our hospital underwent endoscopic resection by endoscopic submucosal dissection (ESD) in April 2013. F-FDG PET/CT performed immediately prior to the ESD showed focal F-FDG accumulations in the vertebral body of Th8, and vertebral body and arch of L4 with SUV max of 3.98–4.42 (Fig. 1a–c). No masses or osteoclastic lesions were observed. MRI findings of the lesions showed low intensity on T1WI and high intensity on STIR image (Fig. 2a–d). To clarify the cause of the FFDG accumulation in the bone, we performed bone biopsy from the vertebral arch of L4. Histopathological findings revealed hypercellular marrow (80 % cellularity) and increases in number and size of megakaryocytes, most of which were in maturated form with hyperlobulated nuclei, which are usually found in cases with myeloproliferative neoplasm (MPN) (Fig. 3a, b). Reticulin fibrosis of marrow was observed minimally by Gitter staining (Fig. 3c), and collagen fibrosis was not observed. In contrast, laboratory workup for peripheral blood showed no abnormality: WBC 6,920/lL (stab 0.0 %, seg 61.0 %, lymph 30.0 %, mono 6.0 %, eosino 1.5 %, and baso 1.5 %), RBC 452 9 10/ lL, Hb 14.1 g/dL, Ht 40.6 %, PLT 33.3 9 10/lL, reticulocytes 1.2 %, LDH 229 U/L, VB12 351 mg/dL, and NAP score 223. Bone marrow biopsy subsequently performed from the left iliac bone showed normocellular marrow, but the number of megakaryocytes was also increased (Fig. 3d). Furthermore, JAK2 V617F mutation was detected in the bone marrow sample by real-time qualitative PCR with a sensitivity of more than 2 % of all alleles. G-banding showed normal diploid karyotype, and BCR–ABL translocation was not detected by FISH analysis. 5 months after the first visit, her laboratory data of peripheral blood was within the normal range, and FFDG PET/CT also showed similar F-FDG accumulation in the bone, without new lesions. We will continue to follow her progress carefully. The JAK2 V617F mutation is present in patients with Philadelphia-negative MPN, including over 90 % of polycythemia vera cases and about half of essential thrombocythemia and primary myelofibrosis cases [1]. The JAK2 V617F mutation may also be detected in healthy individuals without overt MPN [2]. Nielsen et al. [2] reported that the JAK2 V617F mutation was detected in 18 of 10,507 participants (0.2 %) in the general population, and three of these 18 individuals with the JAK2 V617F mutation developed overt myeloproliferative disorder during up to 17.6 years of follow-up. In the present case, focal F-FDG accumulation in bone marrow and histopathological findings, other than the finding of the left iliac bone marrow as positive for JAK2 V617F mutation, suggest that the patient is more likely to develop some form of overt MPN in the A. Fujimi (&) Y. Kanisawa Department of Hematology and Oncology, Oji General Hospital, 3-4-8 Wakakusa-cho, Tomakomai 053-8506, Japan e-mail: Akihito.fujimi@ojihosp.or.jp

Expression of cathepsins V and S in thymic epithelial tumors

Cathepsins are a group of proteolytic enzymes of the endosomal/lysosomal pathway involved in the thymic development of T cells restricted by major histocompatibility complex class II molecules. In the normal thymus, cathepsin V (CTV) and cathepsin S (CTS) are expressed in cortical and medullary epithelial cells, respectively. To investigate whether cathepsins could serve as a diagnostic marker, we performed immunohistochemical analysis for CTV and CTS in 77 cases of thymic epithelial tumors. Almost all cases (59/60) of thymoma expressed CTV, whereas 28 of 60 cases of thymoma expressed CTS. Notably, CTS was expressed in most cases of type A and type AB thymomas, but not in type B thymoma. The expression of cathepsins in type AB thymoma showed a clear correlation with histologic features; CTV was found predominantly in the type B component, and CTS was frequently expressed in the type A component. In thymic carcinoma, CTV was expressed in less than half cases (7/17), and the ratio of CTS-positive cases was equivalent to that of thymoma (8/17). Cases of CTV-negative thymic carcinoma tended to have a higher incidence of recurrence than did CTV-positive cases. Although further studies with a larger number of cases are required to confirm the utility of cathepsin immunostaining, CTV and CTS appear to serve as auxiliary diagnostic and/or prognostic markers in thymic epithelial tumors.

Thrombocytopenia and Anemia with Anti-c-Mpl antibodies Effectively Treated with Cyclosporine in a Patient with Rheumatoid Arthritis and Chronic Renal Failure.

A 61-year-old woman with rheumatoid arthritis who was undergoing hemodialysis for end-stage renal failure was transferred to our hospital due to severe thrombocytopenia and anemia. A bone marrow biopsy showed the complete absence of megakaryocytes and erythroblasts. Cyclosporine treatment resulted in the improvement of her megakaryocyte and erythroblast levels, and a decrease in her serum level of anti-c-Mpl (thrombopoietin receptor) antibodies. After this initial improvement, her anemia progressively worsened, despite the continuous administration of immunosuppressive therapy with cyclosporine. Her platelet and leukocyte counts remained stable. This is the first report of a probable case of anti-c-Mpl antibody-associated pure red cell aplasia and acquired amegakaryocytic thrombocytopenic purpura.

[Successful treatment with dose-adjusted EPOCH-R for triple-hit lymphoma having BCL2, BCL6 and MYC translocations].

Double- and triple-hit lymphomas (DHL/THL), high-grade B-cell lymphomas with an extremely poor prognosis, are defined by a chromosomal breakpoint affecting the MYC/8q24 locus in combination with another recurrent breakpoint. The successful use of dose-adjusted (DA) EPOCH-R in patients with MYC-positive lymphoma and Burkitt lymphoma (BL) was recently reported. A 74-year-old man with acute renal dysfunction and hyperkalemia was transferred to our emergency center by ambulance. PET-CT revealed a left renal hilar mass enveloping the abdominal para-aortic domain and bladder and hydronephrosis. High (18)F-FDG uptake revealed lymph node, peritoneum, and multiple bone metastases. Analysis of the bone marrow aspirate revealed abnormal lymphoid cells with deeply basophilic cytoplasm and numerous vacuoles resembling Burkitt cells. Chromosomal analysis revealed a complex chromosomal karyotype, including t(14;18)(q32;q21), and FISH analysis confirmed split BCL2, BCL6, and MYC signals. Bone marrow biopsy revealed diffusely infiltrating large abnormal lymphoid cells with a CD10⁺, CD20⁺, BCL2⁺, BCL6⁺, c-MYC⁺ and MUM1(-) immunophenotype. B-cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma and BL, was diagnosed. The patient achieved a partial response after eight courses of DA-EPOCH-R chemotherapy. Our experience suggests that DA-EPOCH-R may be an effective treatment for DHL/THL.