Yukiko Kiniwa

Tumor-Specific Human CD4+ Regulatory T Cells and Their Ligands: Implications for Immunotherapy

Regulatory T cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Little is known, however, about the nature of the physiological target antigens for CD4(+) regulatory T (Treg) cells. Here we report the identification of the LAGE1 protein as a ligand for tumor-specific CD4(+) Treg cell clones generated from the tumor-infiltrating lymphocytes (TILs) of cancer patients. Phenotypic and functional analyses demonstrated that they were antigen-specific CD4(+) Treg cells expressing CD25 and GITR molecules and possessing suppressive activity on the proliferative response of naive CD4(+) T cells to anti-CD3 antibody stimulation. Ligand-specific activation and cell-cell contact were required for TIL102 Treg cells to exert suppressive activity on CD4(+) effector cells. These findings suggest that the presence of tumor-specific CD4(+) Treg cells at tumor sites may have a profound effect on the inhibition of T cell responses against cancer.

CD8+ Foxp3+ regulatory T cells mediate immunosuppression in prostate cancer.

Purpose: Although elevated proportions of CD4+CD25+ regulatory T (Treg) cells have been shown in several types of cancers, very little is known about the existence and function of CD8+ Treg cells in prostate cancer. In this study, we investigated prostate tumor–derived CD8+ Treg cells and their function. Experimental Design: Tumor-infiltrating lymphocytes (TIL) from fresh tumor specimens of patients with prostate cancer were generated and subjected to phenotypic and suppressive function analyses. In particular, we investigated the role and function CD8+ Treg cells in prostate cancer. Results: We show that high percentages of CD4+CD25+ T cells are probably present in the majority (70%) of prostate TILs. Remarkably, both CD4+ and CD8+ T-cell subpopulations possessed potent suppressive activity. T-cell cloning and fluorescence-activated cell sorting analyses showed the presence of CD8+CD25+ Treg cell clones that expressed FoxP3 and suppressed naïve T-cell proliferation, in addition to the previously known CD4+CD25+ Treg cells. These CD8+ Treg cells suppressed naïve T-cell proliferation mainly through a cell contact–dependent mechanism. Importantly, the suppressive function of CD8+ Treg cells could be reversed by human Toll-like receptor 8 (TLR8) signaling. Conclusion: Our study shows that like CD4+CD25+ Treg cells, CD8+ Foxp3+ Treg cells present in prostate tumor–derived TILs suppress immune responses and that their suppressive function can be regulated by TLR8 ligands, raising the possibility that the manipulation of Treg cell function by TLR8 ligands could improve the efficacy of immunotherapy for prostate cancer patients.

NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.

Generation of reactive oxygen species (ROS) has been implicated in carcinogenic development of melanoma, but the underlying molecular mechanism has not been fully elucidated. We studied the expression and function of the superoxide-generating NADPH oxidase (Nox)4 in human melanoma cells. Nox4 was up-regulated in 13 of 20 melanoma cell lines tested. Silencing of Nox4 expression in melanoma MM-BP cells by small interfering RNAs decreased ROS production and thereby inhibited anchorage-independent cell growth and tumorigenecity in nude mice. Consistently, a general Nox inhibitor, diphenylene iodonium, and antioxidants vitamine E and pyrrolidine dithiocarbamate blocked cell proliferation of MM-BP cells. Flow cytometric analysis indicated that Nox4 small interfering RNAs and diphenylene iodonium induced G(2)-M cell cycle arrest, which was also observed with another melanoma cell line, 928mel. This was accompanied by induction of the Tyr-15 phosphorylated, inactive form of cyclin-dependent kinase 1 (a hallmark of G(2)-M checkpoint) and hyperphosphorylation of cdc25c leading to its increased binding to 14-3-3 proteins. Ectopic expression of catalase, a scavenger of ROS, also caused accumulation of cells in G(2)-M phase. Immunohistochemistry revealed that expression of Nox4 was detected in 31.0% of 13 melanoma patients samples, suggesting the association of Nox4 expression with some steps of melanoma development. The findings suggest that Nox4-generated ROS are required for transformation phenotype of melanoma cells and contribute to melanoma growth through regulation of G(2)-M cell cycle progression.

Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level

Background:The availability of molecular-targeted therapies for the treatment of melanoma has emphasised the need to identify mutations in target genes such as BRAF and KIT. Circulating tumour cells (CTC) are present in the peripheral blood of a significant proportion of cancer patients.Methods:High molecular weight melanoma-associated antigen (HMW-MAA) was used to isolate melanoma cells from peripheral blood as it is selectively expressed at high levels on melanomas. The HMW-MAA-positive cells were isolated using immunomagnetic beads. After removing CD45+ cells, CTC were identified by staining with MART-1- and gp100-specific antibodies (HMW-MAA+, CD45−, MART-1/gp100+). Single, isolated CTC were then subjected to BRAF and KIT mutational analysis.Results:CTC (HMW-MAA+, CD45−, MART-1/gp100+) were isolated from the blood of 11 patients and BRAF and KIT were sequenced in nine and four patients, respectively. The BRAF sequences identified in the CTC were inconsistent with those identified in autologous melanoma tumours in three patients and the KIT sequences were inconsistent in three patients. In addition, polyclonal BRAF mutations were identified in one patient and concomitant mutations in BRAF and KIT were identified in another patient.Conclusion:Melanoma cells show clonal heterogeneity. Therefore, CTC genotyping may be crucial for successful molecular-targeted therapy.

Intratumoral expression levels of PD-L1, GZMA, and HLA-A along with oligoclonal T cell expansion associate with response to nivolumab in metastatic melanoma.

ABSTRACT Immune checkpoint inhibitors blocking the interaction between programmed death-1 (PD-1) and PD-1 ligand-1 (PD-L1) are revolutionizing the cancer immunotherapies with durable clinical responses. Although high expression of PD-L1 in tumor tissues has been implicated to correlate with the better response to the anti-PD-1 therapies, this association has been controversial. In this study, to characterize immune microenvironment in tumors, we examined mRNA levels of immune-related genes and characterized T cell repertoire in the tumors of 13 melanoma patients before and after nivolumab treatment. We found that, in addition to the PD-L1 (p = 0.03), expression levels of PD-1 ligand-2 (PD-L2), granzyme A (GZMA) and human leukocyte antigen-A (HLA-A) in the pre-treatment tumors were significantly higher (p = 0.04, p = 0.01 and p = 0.006, respectively) in responders (n = 5) than in non-responders (n = 8). With nivolumab treatment, tumors in responders exhibited a substantial increase of CD8, GZMA and perforin 1 (PRF1) expression levels as well as increased ratio of TBX21/GATA3, suggesting dominancy of helper T cell type 1 (Th1) response to type 2 (Th2) response. T cell receptor β (TCR-β) repertoire analysis revealed oligoclonal expansion of tumor-infiltrating T lymphocytes (TILs) in the tumor tissues of the responders. Our findings suggest that melanoma harboring high PD-1 ligands (PD-L1 and PD-L2), GZMA and HLA-A expression may respond preferentially to nivolumab treatment, which can enhance Th1-skewed cellular immunity with oligoclonal expansion of TILs.

Identification of bladder cancer antigens recognized by IgG antibodies of a patient with metastatic bladder cancer

To identify tumor antigens useful for the diagnosis and treatment of patients with bladder cancer, a lambda phage cDNA library constructed from a high‐grade bladder cancer cell line was screened with autologous serum from a patient with metastatic bladder cancer. Forty‐eight distinct antigens were isolated. By evaluating the immunogenicity and the tissue‐specific expression, KU‐BL‐1 and KU‐BL‐2 were identified as immunogenic antigens with restricted tissue expression. KU‐BL‐1 was found to be a putative human lipoic acid synthetase with a metal‐binding site, CXXXCXXC, that was expressed in bladder cancer cell lines and most bladder cancer tissues, as well as normal bladder mucosa and testis tissues. Immunoglobulin (Ig)G antibody to KU‐BL‐1 was detected in 2 of 28 patients with bladder cancer, but not in 30 healthy individuals. KU‐BL‐2 was found to be a putative human kelch‐like protein that was homologous to Drosophila kelch, with a BTB/POZ domain and kelch repeats. KU‐BL‐2 was expressed in bladder cancer cell lines, most bladder cancer tissues, testis and heart, but not in normal bladder mucosa. IgG antibody to KU‐BL‐2 was detected in 8 of 28 patients with bladder cancer, but not in 16 healthy individuals. Tumor reactive T cells were induced from peripheral blood mononuclear cells (PBMC) by stimulation with one of the HLA–A24 binding KU‐BL‐2 peptides. Therefore, KU‐BL‐1 and KU‐BL‐2, which showed preferential expression in bladder cancer with restricted expression in normal tissues, as well as immunogenicity in multiple patients with bladder cancer, may be useful for the development of diagnostic and therapeutic methods for patients with bladder cancer.

IgA/IgG pemphigus with infiltration of neutrophils and eosinophils in an ulcerative colitis patient.

© 2014 The Authors. doi: 10.2340/00015555-1836 Journal Compilation

Low p53 positivity in verrucous skin lesion in diabetic neuropathy occurring on the dorsum of the foot

Typically, the skin findings will improve with remission of malignancy but may subsequently worsen with recurrence of the primary neoplasm; however, when patients exhibit MAN eruption, the malignancy is usually already in the progressive stage, and it may therefore be difficult to achieve complete resolution of MAN. Although there is a predilection for flexural areas, eruptions may become widespread and generalized in cases of MAN, especially in severe cases. The unique feature of the present case is not only that the eruptions became generalized but also that the major skin folds of the abdomen and preaxillar regions were spared, producing the deck-chair sign. The deck-chair sign defines a peculiar clinical pattern observed in patients with PEO, characterized by selective sparing of major skin folds and flexures. Although the deck-chair sign was originally considered a specific feature of PEO, this sign has also been observed in certain other skin diseases. However, the deck-chair sign has never been reported in association with MAN in the English literature so far. PEO is essentially a clinical diagnosis and its etiology is unclear. Therefore, the reason skin creases is spared and its clinical significance is also obscure. The present case provides another example suggesting that the deck-chair sign is not a specific feature of PEO but rather a pattern of expression in several inflammatory dermatoses. However, the precise pathogenesis and clinical significance of the deck-chair sign remains to be elucidated.

A Case of Epidermolysis Bullosa Acquisita Associated with Laryngeal Stenosis

© 2012 The Authors. doi: 10.2340/00015555-1163 Journal Compilation

A case of systemic anaplastic large cell lymphoma with ‘Hodgkin-like’ appearance and skin involvement mimicking lymphomatoid papulosis

We report a unique case of the CD30+ anaplastic large cell lymphoma (ALCL). A 44‐year‐old Japanese male presented with lymphadenopathy followed by skin involvement. Initially, a swollen cervical lymph node was recognized in 1989 and relapsed in 1991, which was histologically diagnosed as Hodgkin disease of nodular sclerotic type. In 1996, he presented ulcerative cutaneous nodules and swollen lymph nodes in his left inguinal region, which was then diagnosed with CD30+ ALCL. Both the lymphadenopathy and the skin lesion had been completely remitted by combining chemotherapy followed by radiotherapy. Thereafter, he had relapsing and remitting episodes of multiple papules and nodules on his face, trunk and extremities for 10 years. Repeated histopathological examination revealed similar tumor cell proliferation in the papules/nodules of the skin. Essentially similar immunohistochemical features, including CD30 and granzyme B expression, but not anaplastic lymphoma kinase (ALK), strongly suggested that all these tumors were sequential expression of one disease continued for 19 years. This case was finally diagnosed as CD30+ ALCL with unique skin involvement mimicking lymphomatoid papulosis (LyP).