Tomoki Kikuchi

Arachidonate 5-Lipoxygenase Establishes Adaptive Humoral Immunity by Controlling Primary B Cells and Their Cognate T-Cell Help

In this study, we report the unique role of arachidonate 5-lipoxygenase (Alox5) in the regulation of specific humoral immune responses. We previously reported an L22 monoclonal antibody with which human primary resting B cells in the mantle zones of lymphoid follicles are well-defined. Proteomics analyses enabled identification of an L22 antigen as Alox5, which was highly expressed by naive and memory B cells surrounding germinal centers. Cellular growth of mantle cell lymphoma cells also seemed to depend on Alox5. Alox5(-/-) mice exhibited weak antibody responses specific to foreign antigens at the initial and recall phases. This was probably attributable to the low number of follicular and memory B cells and the functional loss of interleukin-21-mediated responses of follicular B cells. Moreover, Alox5(-/-) mice could not fully foster the development of follicular B helper T (Tfh) cells even after immunization with foreign antigens. Further experiments indicated that Alox5 affected mortality in experimentally induced enterocolitis in germ-prone circumstances, indicating that Alox5 would endow immunologic milieu. Our results illustrate the novel role of Alox5 in adaptive humoral immunity by managing primary B cells and Tfh cells in vivo.

Tonsillar crypt epithelium of palmoplantar pustulosis secretes interleukin-6 to support B-cell development via p63/p73 transcription factors†

Palmoplantar pustulosis (PPP) is an autoimmune disease characterized by psoriasis‐like erythematous lesions on palms and/or soles due to an abnormal humoral immune response. Tonsillectomy is effectively employed for the treatment of PPP; however, how tonsils are involved in the aetiology of PPP remains unclear. Here we analysed surgically resected palatine tonsils from 36 cases of PPP as well as usual recurrent tonsillitis (RT) as a control. Histological examination revealed that a unique lesion, with lymphoid follicles surrounded by reticular crypt epithelial cells, was more frequently observed in tonsils of patients with PPP than in those with RT (p < 0.0001; PPP vs RT). Interestingly, crypt epithelial cells in primary cultures derived from PPP tonsils showed marked production of interleukin‐6 (IL‐6). Moreover, these epithelial cells from PPP tonsils expressed p53‐related transcription factors in their nuclei that were found to contribute to the up‐regulation of IL‐6 gene expression. These findings suggest that, at least in part, the specialized lymphoepithelial symbiosis of PPP tonsils, under the control of p53‐related factors, may be relevant to the generation of the impaired micro‐environment underlying the aberrant production of autoantibodies. Copyright

Immunohistochemical analysis of the p53 family members in human craniopharyngiomas

Craniopharyngiomas are intracranial tumors that usually arise in the site around the sella turcica. They are composed of distinctive sheets of epithelial cells showing adamantinomatous or squamous-papillary histologic type. Because little is known about the tumorigenesis of cranio-pharyngiomas, we retrieved samples from 15 tumor cases to investigate the functional significance of the p53 family of transcription factors, which are known to be expressed in various human epithelia. Immunohistochemical analysis of these cases demonstrated similar expression profiles of p53 family members in the two histologic types of the tumor; i.e., strong nuclear expression of p63 was observed in all cell layers, and moderate to intense nuclear expression of p73 was observed in the basal cell layers. In contrast to p63 and p73, the reactivity of an archetypal tumor suppressor, p53, was occasional and weak in the two histologic types. Because p63 was widely expressed in the tumors, reverse transcription-polymerase chain reaction (RT-PCR) analysis was conducted to elucidate which spliced variant of p63 was expressed. The results showed that †Np63, lacking a terminal transactivation domain of p63, was the dominant isoform. Together with the reported evidence that the †Np63 isoform is highly expressed in human squamous-cell carcinomas, these data suggest that the cellular architecture characteristic of the expression of p53 family members may be required for the histogenesis of craniopharyngiomas, where †Np63 has a possible role in maintaining proliferative activity of the tumor cells, like squamous-cell carcinomas in other tissues.

Wild-type AIRE cooperates with p63 in HLA class II expression of medullary thymic stromal cells

During T cell development in the thymus, autoreactive T cells are deleted through a mechanism that is actively supported by medullary epithelial cells. These epithelial cells possess particular transcription factors including autoimmune regulator (AIRE), which is responsible for regulating expression of self-antigens, as well as p63, a p53-like molecule. Here we present evidence suggesting interaction of AIRE with p63 through a SAND domain and a transactivation domain, respectively. Interestingly an AIRE molecule with a mutated SAND domain of G228W, whose genetic alteration is inherited in an autosomal dominant manner, could not establish a complex with p63 as indicated by immunoprecipitation and molecular modeling analyses. Further in vitro study indicated that the G228W mutation led to downregulation of the transcription levels of CIITA and, accordingly, the cell surface expression of HLA class II molecules in thymic epithelial cells with p63. This indicates novel involvement of AIRE and p63 in the regulation of HLA class II, and suggests that defects in the AIRE-p63 interaction may lead to malfunction of HLA-based selection of self-reactive helper CD4(+) T cells in the thymus.

Clinicopathologic Analysis of Angioimmunoblastic T-cell Lymphoma With or Without RHOA G17V Mutation Using Formalin-fixed Paraffin-embedded Sections.

Angioimmunoblastic T-cell lymphoma (AITL) is an infrequent subtype of peripheral T-cell lymphoma derived from follicular helper T cells. Recently, a somatic G17V RHOA gene mutation has been reported. In this article, we examined the RHOA G17V mutation in 18 cases of AITL by 3 different techniques of Sanger sequencing, fully automated SNP genotyping, and deep sequencing, using routine diagnostic formalin-fixed paraffin-embedded tissue. The RHOA G17V mutation was detected in 10 cases (56%). Among the 10 mutated cases, 8 cases were detected by all 3 methods. The status of RHOA mutation was subsequently compared with the clinicopathologic characteristics of AITL. RHOA-mutated AITL (10 cases) was clinically characterized by high serum IL-2R and a poor ECOG performance status. By immunohistochemistry, expression of CD10, PD-1, CXCL13, and CCR4 and a wide distribution of CD21(+) follicular dendritic cells were observed in RHOA-mutated cases. Among these, CCR4 expression and the CD21(+) network in RHOA-mutated AITL cases were more extensive than in the RHOA mutation-negative AITL cases (P<0.05). Thus, RHOA-mutated AITL cases are more characteristic of follicular helper T cells, and the presence of such a mutation is an important marker for AITL.

Intratumoral heterogeneity of HER2 protein and amplification of HER2 gene in salivary duct carcinoma

Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands, and accounts for 1–3% of all malignant salivary gland tumors, resembling morphologically invasive ductal carcinoma (IDC) of the breast. In contrast to IDC of the breast and gastric carcinoma (GC), the study of human epidermal growth factor receptor 2 (HER2) in SDC has not progressed. Therefore, we investigated the relationship between HER2 protein expression and amplification of the HER2 gene, and compared them in terms of intratumoral heterogeneity (ITH) in 13 cases of SDC using immunohistochemistry and dual color in situ hybridization. We found seven cases with protein overexpression (53.8%) and five cases with gene amplification (38.5%) in accordance with ASCO/CAP guidelines. ITH of HER2 protein expression was seen in seven cases (53.8%). Interestingly, the ratio of the HER2 gene showed homogenous distribution with or without the presence of ITH of HER2 protein expression. SDC tends to have more ITH of HER2 protein similarly to GC, in contrast to IDC of the breast. ITH of HER2 protein in SDC has no heterogeneity of the HER2 gene amplification. The mechanism of HER2 protein expression in SDC might proceed through a more complex pathway relative to that of IDC of the breast.

Sorting nexin 5 of a new diagnostic marker of papillary thyroid carcinoma regulates Caspase-2.

Papillary thyroid carcinoma (PTC) is a well‐differentiated endocrine malignant tumor that develops from thyroid follicular epithelium. The tumor represents the most common type of endocrine malignancy; however, its tumorigenesis is not fully elucidated. The aim of this study was to address the functional role of the sorting nexin (SNX) family in PTC because of recent experimental evidence suggesting that the SNX family members actively control endocytotic transportation as well as cell fate. Expression profiles of SNX family members of PTC showed a significant quantity of transcripts of SNX5. Further immunohistochemical analysis with an SNX5‐specific monoclonal antibody established in this study consistently demonstrated the preferential expression of SNX5 in PTC (94.2%, 113/120 cases) as indicated by studies on 440 cases of various tumors. In contrast, other major carcinomas originating from the lung (2.6%, 1/38 cases), breast (5.1%, 2/39 cases), and intestine (4.2%, 1/24 cases) scarcely expressed SNX5. When we investigated models of murine thyroid tumors induced by the administration of carcinogens, high expression of Snx5 was also observed in well‐differentiated thyroid tumors, further implying that the tumorigenesis of the thyroid gland was tightly associated with the abundance of SNX5/Snx5. Moreover epithelial cells expressing excess SNX5 showed high levels of Caspase‐2 of an initiator caspase. Collectively these findings suggest that the evaluation of SNX5 expression would support pathological diagnosis of primary and secondary PTC. (Cancer Sci 2012; 103: 1356–1362)

Mapping of susceptibility and protective loci for acute GVHD in unrelated HLA-matched bone marrow transplantation donors and recipients using 155 microsatellite markers on chromosome 22.

Despite matching donors and recipients for the human leukocyte antigens (HLAs) expressed by the major histocompatibility genomic region of the short arm of chromosome 6, several recipients still develop acute graft-versus-host disease (aGVHD) after bone marrow transplantation (BMT). This is possibly due to non-HLA gene polymorphisms, such as minor histocompatibility antigens (mHas) and genes coding for cytokines. However, a detailed genetic background for aGVHD has not yet been established. To find novel susceptibility and/or protective loci for aGVHD, a whole genome-wide association study of donors and recipients needs to be performed. As the first step to such a study, we retrospectively analyzed polymorphisms of 155 microsatellite markers spread across the long arm of chromosome 22 in 70 pairs of HLA-matched unrelated BMT donors and recipients. We performed individual typing and then compared the markers’ allele frequencies (1) between all the aGVHD (grades III and IV GVHD) and GVHD-free (grade 0 GVHD) groups in donors and recipients and (2) between the aGVHD and aGVHD-free groups in donor/recipient pairs that were matched and mismatched for the microsatellite marker’s allele. Screening of the microsatellite markers revealed five loci with a significant difference between the aGVHD and GVHD-free groups and revealed eight loci on chromosome 22, where the microsatellite allele mismatched markers were associated with aGVHD. This screening analysis suggests that several aGVHD-associated susceptible and protective loci exist on chromosome 22, which may encompass novel gene regions that need to be elucidated for their role in aGVHD.

p63 induces CD4+ T-cell chemoattractant TARC/CCL17 in human epithelial cells.

To preserve immunosurveillance, epithelial cells support T-cell trafficking toward inflammatory foci. However, how epithelial cells are enrolled in recruiting T cells has not been fully elucidated. In this study we investigated the function of p63, a p53 family member, in the regulation of the expression of various types of chemokine ligands by focusing on the property of p63 as an epitheliotropic transcription factor. As assessed by experiments using three different human epithelial cell lines with small-interfering RNAs or plasmids of p63, certain CC chemokine ligands were found to be under the control of p63. In these CC chemokine ligands, p63 had the common capacity to upregulate TARC/CCL17 in the different cell lines, whose receptor CCR4 was preferentially presented on CD4(+) T cells such as memory, regulatory, IL-17-producing and type II helper T cells. More interestingly, when cells were stimulated with transforming growth factor-beta (TGF-beta) or epidermal growth factor (EGF) as observed during tissue repair process, the expression of p63 and TARC/CCL17 was concomitantly suppressed. This implies that, in local inflammatory regions with general epithelial tissue remodeling, the p63-TARC/CCL17 axis may participate in the engagement of efficient immune reactions by specified T-cell subsets.

Composite Follicular Lymphoma and CD5-Positive Nodal Marginal Zone Lymphoma.

Composite CD10-positive low-grade B-cell and CD5-positive low-grade B-cell lymphoma is extremely rare. We report a case of a composite follicular lymphoma (FL) and CD5-positive nodal marginal zone lymphoma (NMZL) in a resected inguinal lymph node of a 72-year-old Japanese male. Histologically, multiple follicles had reactive-germinal centers with tingible body macrophages, a thin mantle zone and a wide marginal zone. The wide marginal zone consisted of medium-sized cells having slightly indented nuclei and clear cytoplasm, indicating monocytoid cells with CD5-positive B-cells. Several follicles had germinal centers filled with many centrocytes, with CD10-positive B-cells. Polymerase chain reaction/sequence analysis of the immunoglobulin heavy chain gene obtained from microdissected regions of CD5-positive NMZL and FL showed different sequences within the CDR3 region. To our knowledge, this is the first report of FL and CD5-positive NMZL.