Yasuo Fukumori

Anti-CCR4 mAb selectively depletes effector-type FoxP3+CD4+ regulatory T cells, evoking antitumor immune responses in humans

Significance Regulatory T (Treg) cells expressing the transcription factor FOXP3 play a critical role in suppressing antitumor immune responses. Here we found that, compared with peripheral blood T cells, tumor-infiltrating T cells contained a higher frequency of effector Tregs, which are defined as FOXP3hi and CD45RA−, terminally differentiated, and most suppressive. Effector Treg cells, but not FOXP3lo and CD45RA+ naïve Treg cells, predominantly expressed C-C chemokine receptor 4 (CCR4) in both cancer tissues and peripheral blood. In vivo or in vitro anti-CCR4 mAb treatment selectively depleted effector Treg cells and efficiently induced tumor-antigen-specific CD4+ and CD8+ T cells. Thus, cell-depleting anti-CCR4 mAb therapy is instrumental for evoking and enhancing tumor immunity in humans via selectively removing effector-type FOXP3+ Treg cells. CD4+ Treg cells expressing the transcription factor FOXP3 (forkhead box P3) are abundant in tumor tissues and appear to hinder the induction of effective antitumor immunity. A substantial number of T cells, including Treg cells, in tumor tissues and peripheral blood express C-C chemokine receptor 4 (CCR4). Here we show that CCR4 was specifically expressed by a subset of terminally differentiated and most suppressive CD45RA−FOXP3hiCD4+ Treg cells [designated effector Treg (eTreg) cells], but not by CD45RA+FOXP3loCD4+ naive Treg cells, in peripheral blood of healthy individuals and cancer patients. In melanoma tissues, CCR4+ eTreg cells were predominant among tumor-infiltrating FOXP3+ T cells and much higher in frequency compared with those in peripheral blood. With peripheral blood lymphocytes from healthy individuals and melanoma patients, ex vivo depletion of CCR4+ T cells and subsequent in vitro stimulation of the depleted cell population with the cancer/testis antigen NY-ESO-1 efficiently induced NY-ESO-1–specific CD4+ T cells. Nondepletion failed in the induction. The magnitude of the responses was comparable with total removal of FOXP3+ Treg cells by CD25+ T-cell depletion. CCR4+ T-cell depletion also augmented in vitro induction of NY-ESO-1–specific CD8+ T cells in melanoma patients. Furthermore, in vivo administration of anti-CCR4 mAb markedly reduced the eTreg-cell fraction and augmented NY-ESO-1–specific CD8+ T-cell responses in an adult T-cell leukemia-lymphoma patient whose leukemic cells expressed NY-ESO-1. Collectively, these findings indicate that anti-CCR4 mAb treatment is instrumental for evoking and augmenting antitumor immunity in cancer patients by selectively depleting eTreg cells.

Mycobacterium bovis BCG Cell Wall-Specific Differentially Expressed Genes Identified by Differential Display and cDNA Subtraction in Human Macrophages

ABSTRACT We have analyzed the gene expression profile of monocytes in response to a highly purified cell wall fraction of Mycobacterium bovis BCG, a clinically approved adjuvant known as BCG cell wall skeleton (BCG-CWS). It is composed of mycolic acid, arabinogalactan, and peptidoglycan and confers Toll-like receptor 2 (TLR2)- and TLR4-dependent signaling that induces monocytes to differentiate into antigen-presenting cells (APCs). Here we report differential gene expression analysis with BCG-CWS-stimulated versus nonstimulated monocytes. BCG-CWS exerted massive induction of genes regulated by TLR signaling. Marked gene regulatory characteristics in BCG-CWS-stimulated cells compared to lipopolysaccharide (LPS)-stimulated cells follow. (i) Spliced mRNAs encoding soluble forms of TREM-1 and TREM-2 (recently discovered inflammatory-signal-amplifying receptors) were regulated by BCG-CWS, resulting in their differential expression. (ii) The genes for zinc-iron transporter protein (ZIP)-like family proteins HKE-1 and LIV-1 were induced exclusively by BCG-CWS. (iii) Interleukin-23 (IL-23), rather than IL-12p70, was induced by BCG-CWS, while interferon-inducible genes were induced only by LPS. By Northern and reverse transcription-PCR analyses, we confirmed the differential expression of more than 30 BCG-CWS regulatory genes, and their expression was compared with that of LPS and other known TLR ligands. A battery of genes responded rapidly and for a short time to LPS but for a long time to BCG-CWS. Structural analysis of the identified novel or hypothetical proteins revealed that some are potential candidates as signaling mediators or transcriptional regulators. Hence, BCG-CWS may profoundly modulate APC responses in a way distinct from that of LPS, leading to possible advantages for its adjuvant-active therapeutic potential.

Levels of complement regulatory proteins, CD35 (CR1), CD46 (MCP) and CD55 (DAF) in human haematological malignancies

Summary. Levels of the membrane complement regulatory proteins. C3b/C4b receptor (CR1, CD35), membrane cofactor protein (MCP, CD46), and decay‐accelerating factor (DAF, CD55), expressed on cells from patients with haematological malignancies and normal subjects were assessed by flowcytometry using the respective monoclonal antibodies (mAbs). All myeloid and most lymphoid leukaemia samples tested were CR1‐negative: two of the 42 leukaemia samples expressed minute amounts of CR1. Lack of CR1 in leukaemia cells was confirmed with two mAbs raised against CR1, 31R, and 243R, which recognized different epitopes and induced different degrees of CR1‐mediated fluorescent shift on flow‐cytometry in granulocytes and erythrocytes. MCP was increased in most chronic myelogenous leukaemia (CML) and chronic lymphocytic leukaemia (CLL), and was also increased in majority of acute nonlymphocytic leukaemia (ANLL), acute lymphocytic leukaemia (ALL) and non‐Hodgkins lymphoma (NHL). Levels of DAF were also high in CML and CLL, and were variable in other types of leukaemia: some were DAF‐negative while others expressed extremely high levels of DAF. In CML patients, the high level of MCP and the lack of CR1 were normalized after medical treatment. These results are in agreement with the data obtained with human leukaemia cell lines, and support the hypothesis that CR1 is essentially a differentiated cell antigen and that a high level of MCP reflects some malignant transformation or an immature stage in blood cells.

Cancer/testis antigens are novel targets of immunotherapy for adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an intractable hematologic malignancy caused by human T-lymphotropic virus type 1 (HTLV-1), which infects approximately 20 million people worldwide. Here, we have explored the possible expression of cancer/testis (CT) antigens by ATLL cells, as CT antigens are widely recognized as ideal targets of cancer immunotherapy against solid tumors. A high percentage (87.7%) of ATLL cases (n = 57) expressed CT antigens at the mRNA level: NY-ESO-1 (61.4%), MAGE-A3 (31.6%), and MAGE-A4 (61.4%). CT antigen expression was confirmed by immunohistochemistry. This contrasts with other types of lymphoma or leukemia, which scarcely express these CT antigens. Humoral immune responses, particularly against NY-ESO-1, were detected in 11.6% (5 of 43) and NY-ESO-1-specific CD8(+) T-cell responses were observed in 55.6% (5 of 9) of ATLL patients. NY-ESO-1-specific CD8(+) T cells recognized autologous ATLL cells and produced effector cytokines. Thus, ATLL cells characteristically express CT antigens and therefore vaccination with CT antigens can be an effective immunotherapy of ATLL.

Inherited Deficiencies of the Late-Acting Complement Components Other than C9 Found among Healthy Blood Donors

Among sera from 145,640 healthy blood donors in Osaka, 16 were found to have abnormalities in late-acting complement components other than C9. It was found that of these 16 sera, 2 were deficient in C5, 4 in C6, 6 in C7 and 4 in C8 alpha-gamma-subunit. The incidence of deficiency of each component among the Osaka blood donors was calculated as follows: C5 deficiency, 0.0014%; C6 deficiency, 0.0027%; C7 deficiency, 0.0041%; C8 alpha-gamma-subunit deficiency, 0.0027%. We confirmed that 13 donors were healthy and 12 had no past history related to a complement component deficiency. From these results, not only C9 deficiency but also deficiencies of the other late-acting complement components were found among the healthy blood donors, but no early-acting component deficiencies were noted.

Genotyping of the ABO blood group system: analysis of nucleotide position 802 by PCR-RFLP and the distribution of ABO genotypes in a German population

Genotypes of the ABO blood group system were studied by PCR-RFLP analysis of the eight polymorphic nucleotide positions (ups) 261, 467, 526, 646, 703, 796, 802 and 803 of the cDNA from A transferase. In 169 unrelated German individuals, 17 genotypes were found and the calculated allele frequencies of A(Pro), A(Leu), B, O(T), O(A) and O2 were 0.2130, 0.0770, 0.0473, 0.4260, 0.2160 and 0.0207, respectively. These frequency data may provide useful additional information for disputed paternity and stain testing. A variant O allele, O2, was fout at a polymorphic frequency. As the nucleotide (np 261) of the O2 allele is the same as that of A and B alleles, the analysis of at least three nucleotide positions, i.e. ups 261, 526 and 802, is necessary to avoid mistyping of the ABO genotype.

Further analysis of Del (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers

Del (D‐elute) in the Rh blood group system is a variant with very weak D antigen and no agglutination is found by the indirect antiglobulin test. This variant is characterized by the presence of anti‐D eluate obtained after an adsorption‐elution test using anti‐D antibodies. We studied here the molecular genetic status of Del by using polymerase chain reaction with sequence‐specific primers (PCR‐SSP).

Genotyping of ABO blood groups by PCR and RFLP analysis of 5 nucleotide positions

The genotyping of ABO blood groups was performed using the polymerase chain reaction (PCR) method. The 4 DNA fragments containing the nucleotide position 261, 526, 703 and 796 of cDNA from A-transferase were amplified by PCR, and the amplified DNA subjected to restriction fragment length polymorphism (RFLP) analysis. The different nucleotide at position 803 was clearly distinguished by electrophoresis of the PCR products amplified with allele-specific primers. By analyzing the electrophoresis patterns, ABO genotyping was conclusively accomplished. The frequencies of ABO genotypes found in Japanese blood donors with A and B phenotypes were as follows: in the phenotype A group, AA =19.8 % and AO = 80.2%; and in the phenotype B group, BB =12.8% and BO=87.2%.ZusammenfassungDie Bestimmung der ABO-Blutgruppen wurde mittels Polymerase-Kettenreaktion (PCR) durchgeführt. Die vier DNA-Fragmente, die Nukleotidpositionen 261, 526, 703 und 796 von der cDNA der A-Transferase enthielten, wurden mittels PCR amplifiziert. Die amplifizierte DNA wurde einer Restriktionsfragmentl ängen-Analyse (RFLP) unterzogen. Nach Amplifikation mit allelspezifischen Primern konnte der Nukleotidunterschied an Position 803 durch Elektrophorese der PCR-Produkte klar getrennt werden. Die Bestimmung der ABO-Genotypen war durch die Analyse der eletrophoretischen Muster eindeutig durchführbar. Folgende Frequenzen der ABO-Genotypen von japanischen Blutspendern wurden für die Phänotypen A und B gefunden: In der Phänotypgruppe A, AA =19,8% und AO=80,2%, Phänotypgruppe B, BB=12,8% und BO= 87,2%.

Distribution of abo genotypes and allele frequencies in a korean population

SummaryThe genotypes of the ABO blood group system were investigated in Korean living in Kangwon-Do area by PCR-RFLP analysis of the seven polymorphic nucleotide positions 261, 467, 526, 646, 703, 796 and 803 of the cDNA from A1 transferase. In 253 unrelated Korean individuals, 15 genotypes were found and the allele frequencies of A(Pro), A(Leu), B, O(T) and O(A) were 0.022, 0.209, 0.209, 0.360 and 0.200, respectively, with no deviation from Hardy-Weinberg expectations (Χ 2=2.145, d.f.=6, 0.90<p<0.95). As for the distribution of allele frequencies, a significant difference was noticed between the Korean and a Japanese (Χ 2=30.87, d.f.=4, p<0.001) and a German (Χ 2=127.76, d.f.=4, p<0.001) populations.

Application of the basophil activation test in the analysis of allergic transfusion reactions

Allergic reactions, which are mediated by mast cells and/or basophils, are the most common adverse effects associated with transfusion, particularly with use of platelet concentrates (PCs). In general, the activation of mast cells/basophils and their subsequent histamine release are mediated via two pathways: the allergendependent and allergen-independent pathways. In the latter, biologic response modifiers (BRMs), such as bacterial components, activated complement components, cytokines and chemokines directly activate mast cells/basophils. Although allergens in blood components may include food or medications ingested by the donor immediately before blood collection, such specific allergens have not been identified. Indeed, the only allergens identified in blood components are plasma proteins including IgA, haptoglobin and C4; transfusion to patients who are deficient in those plasma proteins reportedly causes serious allergic reactions. Besides such cases with plasma protein deficiency, allergic reactions after PC transfusion are likely to be elicited mainly via the allergen-independent pathway by BRMs that are present or accumulated in PCs, although the roles of BRMs in this pathway remain largely unknown (Cognasse et al., 2006; Blumberg et al., 2006; Wakamoto et al., 2003). In addition, because allergic transfusion reactions are diagnosed based on symptoms, it is often difficult to determine whether the transfused PCs are causative or whether the reactions are, in actuality, allergic in nature. These difficulties are partly because of the lack of an assay system with which activation of mast cells/basophils can be assessed. Fortunately, however, a simple test called the basophil activation test (BAT) has recently been developed for managing allergic diseases. In the test, whole blood is incubated with an allergen, and subsequent basophil activation is assessed using flow cytometry based on upregulation of cell degranulation/activation markers, CD63 or CD203c (Boumiza et al., 2005; Bühring et al., 1999). In this study, using