Age-related Epstein-Barr virus-associated B-cell lymphoproliferative disorders: special references to lymphomas surrounding this newly recognized clinicopathologic disease

Epstein–Barr virus (EBV) is associated with some disease entities of malignant lymphomas, including Burkitt lymphoma, Hodgkin lymphoma, immunodeficiency-associated lymphoproliferative disorders (LPD), and a part of diffuse large B-cell lymphoma. We have recently identified a series of elderly patients with EBV-associated (or EBV⁺) B-cell LPD (B-LPD) showing similarities in many respects to immunodeficiency-associated LPD, although no evidence of underlying immunodeficiency was found. Therefore, the nosological category of senile or age-related EBV⁺ B-LPD has been proposed for those patients. A larger series of patients with this disease revealed that the relative ratios of such EBV⁺ B-LPD to all diffuse large B-cell lymphoma cases were higher with increasing with age, reaching a peak (20–30%) at ≥90 years of age, with a median of 71 years, providing additional evidence for our assertion that this disease may be related to immunological deterioration as a result of the aging process. This new disease entity is characterized pathologically by centroblasts, immunoblasts, and Hodgkin and Reed–Sternberg-like giant cells with a varying degree of reactive components, often posing therapeutic and diagnostic problems for hematologists and pathologists, respectively. The aim of the present review is to briefly summarize the overall clinicopathological profile of this newly recognized age-related (also called 'senile') EBV⁺ B-LPD and EBV⁺ Hodgkin lymphoma for a practical diagnostic approach. ( Cancer Sci 2008; 99: 1085–1091)     

The Characteristics of Epstein-Barr Virus (EBV)-positive Diffuse Large B-Cell Lymphoma: Comparison between EBV+ and EBV- Cases in Japanese Population

We have investigated 114 cases with diffuse large B-cell lymphoma (DLBCL) to clarify the characteristics of DLBCL with Epstein-Barr virus (EBV) infection. Thirteen cases (11.4%) showed EBVencoded RNA 1 (EBER1) signals by RNA in situ hybridization. EBV-encoded latent membrane protein 1 (LMP1) and EBV-encoded nuclear antigen 2 (EBNA2) were expressed in 11 and 4 cases, respectively. Expression of CD30, Bcl-6 and immunoglobulin (Ig) was found in 92%, 31% and 23% with EBV⁺ DLBCL, and in 15%, 79% and 82% with EBV^- DLBCL, respectively. The sequence of rearranged Ig heavy chain (IgH) variable (V) region gene was analyzed in 5 cases with EBV⁺ DLBCL and 61 cases with EBV^- DLBCL. Somatic mutation was found in all cases except one with EBV^- DLBCL. Average mutation frequency was 9.6% in EBV⁺ DLBCL vs. 11.5% in EBV^- DLBCL. The rates of replacement mutation vs. silent mutation (R/S values) in complementarity determining region II and framework region III were 2.7 and 1.5 in EBV⁺ DLBCL, 2.6 and 1.4 in EBV^- DLBCL. Crippling mutation generating a stop codon was found in 2 of 5 cases (40%) with EBV⁺ DLBCL, but none of 61 cases (0%) with EBV^- DLBCL. These findings suggest that EBV⁺ DLBCL and EBV^- DLBCL were both derived from germinal center (GC) or post-GCB cells, and EBV⁺ DLBCL frequently have a non-functional IgH gene owing to crippling mutation.     

Somatic Hypermutations in the VH Segment of Immunoglobulin Genes of CDS-positive Diffuse Large B-Cell Lymphomas

De now CDS-positive (CD5+) diffuse large B-cell lymphoma (DLBL) has recently been identified as constituting a homogeneous subgroup with distinct clinicopathologic and genotypic characteristics, but its origin remains to he elucidated. Previous studies by sequence analysis of the variable region of the immunoglobulin heavy chain (V_H) have shown that CDS⁵ B-cell malignancies such as mantle cell lymphoma (MCL) and B-cell chronic lymphocytic leukemia (B-CLL) cells represent pre-geminal center (pre-GC) stage B cells in contrast with the post-GC stage of most DLBLs, which show somatic hyper mutations in V_H genes. In the present study, we investigated the V_H sequence of de novo CD5⁺ DLBL to clarify whether CD5⁺ DLBL represents the pre-GC stage, as do other CD5⁺ B-cell malignancies, or the post-GC stage, as is typical of DLBL. All eight cases (four CDS⁴ DLBL and four CDS-negative (CD5⁻) DLBL) examined by us showed somatic hypermutatiohs in the V_H segment and two of the CD5⁻ DLBL cases showed intra-clonal diversity, suggesting that CD5⁺ DLBLs were derived from the same maturation stage as CD5⁻ DLBL, but were distinct from the other indolent CD5⁺ B-cell lymphomas of B-CLL and MCL. These data suggest that de novo CD5⁺ DLBLs do not merely lie within a continuous spectrum with B-CLL and MCL, but represent a biologically distinct variant within the diagnostic framework of diffuse large B-cell lymphoma.     

A B-CELL LINE HAVING CHROMOSOME 14 ABERRATION AT BREAK BAND qll DERIVED FROM AN ADULT T-CELL LEUKEMIA PATIENT

A B-cell line having translocations of chromosome 14 at break band q11 (the assigned locus of the α-chain gene of the T-cell antigen receptor) and chromosome 3 at break band p25 (the assigned locus of the c- raf -1 oncogene) was established from peripheral blood leukocytes of an adult T-cell leukemia (ATL) patient. The same chromosome 14 aberration at break band q11 and chromosome 3 aberration at break band p25 were also found in fresh T-cell leukemia cells. The B-cell line is surface immunoglobulin (sIg)⁺, immunoglobulin gene rearrangement⁺, ATL-specific antigen (ATLA)⁺, HTLV-1 proviral genome⁺, Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)⁺ and the EBV DNA genome⁺. The fresh T-leukemic cells were T-cell receptor gene rearrangement⁺, the HTLV-1 proviral genome⁺ and EBV DNA genome⁻.     

Selective Suppression of the Generation of Anti-tumor L3T4+ but Not of Lyt-2+ T Cell-mediated Immunity in the Tumor-hearing State

C3H/He mice hyperimmune against syngeneic MH134 hepatoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated id challenges with viable tumor cells. Winn assays performed utilizing spleen cells from these mice have revealed that both Lyt-2⁺ and L3T4⁺ T cell subsets from MH134-hyperimmune mice produced complete tumor protection. The in vivo tumor-neutralizing activity was also found in spleen cells from tumor-bearing mice at various times after id implantation of MH134 tumor cells. However, in contrast to comparable tumor-neutralization by Lyt-2⁺ and L3T4⁺ T subsets from hyperimmune mice, only the Lyt-2⁺ T cell subset from tumor-bearing mice was capable of mediating the in vivo protective immunity. L3T4⁺ T cell-mediated immunity was not detectable in the tumor-bearing state irrespective of the length of the sensitization period with a primary growing tumor, but emerged in the mice which resisted the first tumor challenge after the resection of the primary tumor. These results indicate that the emergence of L3T4⁺ T cell-mediated anti-tumor immunity is stage-dependent and the Lyt-2⁺ T cells represent the main functional subset in the tumor-bearing state, although both subsets of T cells are potentially capable of effecting anti-tumor in vivo immunity. The results are discussed in relation to the selective suppression of the L3T4⁺ but not of Lyt-2⁺ T cell function in the tumor-hearing state.     

Cytotoxic Activity of CD4+ T Cells against Autologous Tumor Cells

The ⁵¹Cr-release assay is mostly applied to detecting the cytotoxic activity of CD8⁺ T cells, and little is known about the activity of CD4⁺ T cells. Therefore, the correlation between the cytotoxic activity of CD4⁺ or CD8⁺ T cells and the incubation period with autologous tumor cells was analyzed by two methods. The incubation periods were 4 and 20 h (4 h and 20 h assay) for the ⁵¹Cr-release assay. Eight pairs of tumor cells and T cells were assayed. T cells were fractionated into CD4⁺ and CD8⁺ T cells by using magnetic beads and panning methods, and those cells were activated by culture with recombinant interleukin-2 and immobilized anti-CD3 monoclonal antibody. In 6 out of 8 cases, no cytotoxic activity of CD4⁺ T cells was detected by the 4 h assay, whereas cytotoxic activity was detected in all cases in the 20 h assay. The cytotoxic activities in 20 h assay of CD4⁺ T cells were increased 67-fold in comparison with the activities in 4 h assay (range: 5–197). In the case of CD8⁺ T cells, cytotoxic activities were detected in 6 out of 8 cases in the 4 h assay. The lytic unit ratio of CD4⁺ and CD8⁺ T cells was calculated as 1.5 in the 20 h assay (range: 0.2->7.2) versus 0.4 in the 4 h assay (range: < 0.1–1.3). Cytotoxic activities in colorimetric assay using Crystal Violet with a 24 h incubation were similar to those in the 20 h ⁵¹Cr-release assay in all eight cases. These results indicate that CD4⁺ T cells have cytotoxic activity as strong as that of CD8⁺ T cells towards autologous tumor cells.     

The Roles of CD8+ and CD4+ Cells in Tumor Rejection

In vivo administrations of anti-Lyt-2.2 (CDS) mAb and anti-L3T4 (CD4) mAb selectively eliminated CD8⁺ cells amd CD4⁺ cells, respectively. The relative potencies of CD8⁺ cells and CD4⁺ cells and their roles in primary tumor rejections were studied by investigating the effects of these mAbs on tumor growth. CD8⁺ cells were themselves fully capable of mediating rejection in 5 different tumor rejection systems: two radiation leukemia virus (RadLV)-induced leukemias, B6RV2 and BALBRVD, a radiation-induced leukemia BALBRL♂1, and a plasmacytoma BALBMOPC-70A in CB6F₁ mice, and a Friend virus-induced leukemia B6FBL-3 in B6 mice. On the other hand, CD4⁺ cells were capable of resisting tumor growth of B6FBL-3, but not of the other four tumors. Furthermore, for efficient rejection of CB6F₁UV+^˚l sarcoma by CB6F₁ mice, synergy of CDS⁺ and CD4⁺ cells was necessary. Blocking of UV+^˚ 1 rejection was abrogated by delayed administration of anti-L3T4 (CD4) mAb but not anti-Lyt-2.2 (CDS) mAb, indicating the involvement of CD4⁺ cells in only the initial phase of rejection.     

Sequential Involvement of Two Distinct CD4+ Regulatory T Cells during the Course of Transplantable Tumor Growth and Protection from 3–Methylcholanthrene-induced Tumorigenesis by CD25–depletion

The involvement of two phenotypically different regulatory T cells in different stages of tumor growth was investigated. Treatment of BALB/c mice with anti-CD25 monoclonal antibody (mAb) (PC61), but not anti-CD4 mAb (GK1.5) before RL male 1 or Meth A inoculation caused tumor rejection. On the other hand, treatment of BALB/c mice with anti-CD4 mAb (GK1.5) but not anti-CD25 mAb (PC61) on day 6 after inoculation of the same tumors caused rejection. The findings suggest that CD4⁺CD25⁺ T cells downregulated the rejection response in the early stage of tumor growth. On the other hand, putative CD4⁺CD25⁻ T cells downregulated the tumor rejection response in the late stage. Both CD4⁺CD25⁺ and putative CD4⁺CD25-T cells appeared to inhibit the efficient generation of cytotoxic T lymphocytes (CTL). The present study also demonstrated that the treatment of BALB/c mice with anti-CD25 mAb (PC61) at 4 or 6 weeks after 3–methylcholanthrene (3–MC) inoculation retarded tumor occurrence and prolonged survival.     

Adult T-cell leukemia/lymphoma cells from blood and skin tumors express cytotoxic T lymphocyte-associated antigen-4 and Foxp3 but lack suppressor activity toward autologous CD8+ T cells

Adult T cell leukemia/lymphoma (ATL) cells share the CD4⁺CD25⁺ phenotype with regulatory T (Treg) cells. However, it is still controversial whether ATL cells are Treg cells. The aim of the present study was to investigate the Treg nature of ATL cells obtained from peripheral blood and skin tumors in terms of their phenotype and function. By flow cytometry and immunohistochemistry, the expression of the Treg-associated molecule cytotoxic T lymphocyte-associated antigen (CTLA)-4 and Foxp3 was examined in freshly isolated circulating and skin-infiltrating tumor cells from 21 ATL patients with skin eruptions. The expression of CTLA-4 on freshly isolated circulating tumor cells was elevated in two of 15 patients, and Foxp3 was expressed intracytoplasmically at high levels in three of nine patients. In five of the patients examined, skin-infiltrating tumor cells bore variously elevated CTLA-4 with high Foxp3 expression. The potentiality of ATL cells as Treg cells was further addressed by stimulating ATL cells with anti-CD3/CD28 monoclonal antibodies and monitoring CTLA-4 expression. With the stimulation, even CTLA-4-low ATL cells expressed higher levels of CTLA-4 than normal CD4⁺CD25⁺ cells. To study function, ATL cells isolated from blood and skin tumors were tested for their ability to suppress the proliferation of autologous CD8⁺ T cells stimulated with allogeneic lymphocytes. Despite the expression of CTLA-4 and Foxp3, these tumors were incapable of suppressing the proliferation of autologous CD8⁺ T cells. ATL cells are phenotypically Treg cells in at least some patients, but lack immunoregulatory functions, at least toward CD8⁺ T cells. ( Cancer Sci 2008; 99: 98–106)     

Sequential T Cell Response Involved in Tumor Rejection of Sarcoma, Meth A, in Syngeneic Mice

We investigated the type of T cell response involved in Meth A tumor rejection in primary immune and hyperimmune syngeneic mice. It was found that a CD4⁺ T cell-mediated delayed-type hypersensitivity (DTH) response activating non-specific killer cells such as macrophages, NK and LAK cells, without a specific CD8⁺ cytotoxic T lymphocyte (CTL) response, was the major immune response leading to Meth A tumor rejection in primary immune mice. In contrast, the specific CD8⁺ CTL response was the major response leading to the tumor rejection, in addition to CD4⁺ T cell-mediated DTH response, in hyperimmune mice. Analysis of CD4⁺ T cell clones established from primary immune and hyperimmune spleen cells indicated that a CD4⁺ T cell clone (C9) of primary immune mice (although only one clone was established) was of Th1 type, and induced cytotoxicity in accessory cells by classic DTH in vitro. Eight CD4⁺ T cell clones were established from hyperimmune spleen cells. Six out of the eight clones were of the Th2 type and two were Th0-like. However, no Th1-type CD4⁺ T cell clone was established from hyperimmune spleen cells. All of these CD4⁺ T cell clones, even the Th2-type clones, were capable of inducing cytotoxicity in vitro in T cell-depleted accessory cells, as in an in vitro DTH response. We postulate on the basis of these results that the T cell response leading to Meth A tumor rejection in vivo sequentially changed from a CD4⁺ T cell-mediated classic DTH response to a CD8⁺ CTL response, in addition to a cellular response mediated probably by Th2-type cells, during the process of repeated immunization.     

乌司他丁抑制CCL17/CCL22-CCR4介导的小鼠乳腺癌肝转移

背景与目的：越来越多的研究表明趋化因子及其受体在癌症的发生、发展和转移中起关键作用。本文研究了乌司他丁（ulinastatin）对胸腺和活化调节趋化因子（thymus and activation-regulated chemokine,TARC）即CCL17/巨噬细胞来源的趋化因子（macrophage-derived chemokine,MDC）即CCL22-CC族趋化因子受体4（CC chemokine receptor 4,CCR4）信号通路介导的乳腺癌肝转移的影响及其作用机制。方法：通过小鼠乳腺脂肪垫（mfp）接种4T1乳腺癌细胞的方式构建小鼠乳腺癌模型,15 d后取小鼠乳腺肿瘤,并记录乳腺肿瘤的质量;采用免疫组织化学法检测乳腺肿瘤组织中CCR4及肝脏转移瘤中CCL22、CCL17蛋白的表达;采用慢病毒转染的方式抑制4T1乳腺癌细胞CCR4基因,采用蛋白质印迹法（Western blot）检测抑制效果,并以同样方式进行小鼠乳腺成瘤并观察肿瘤生长情况;用三种不同浓度乌司他丁处理4T1细胞荷瘤小鼠,15 d后采用免疫组织化学法检测乳腺癌组织中CCR4及肝脏组织中CCL22及CCL17的表达,采用Westernblot和实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR）分别检测肝脏组织中TGF-β的表达以及microRNA-34a和microRNA-31的含量,并进行TGF-β和microRNA-34a、microRNA-31、CCL22以及CCL17的相关性分析。结果：小鼠乳腺癌组织高表达CCR4,肝脏转移瘤高表达CCL22和CCL17;沉默4T1乳腺癌细胞CCR4的表达,可抑制乳腺癌成瘤性;乌司他丁抑制CCR4、TGF-β、CCL22、microRNA-31和CCL17的表达,促进microRNA-34a的表达。结论：乌司他丁具有抑制乳腺癌肝转移的作用,其具体机制可能与乌司他丁通过TGF-β-microRNA-34a-CCL22轴及microRNA-31-TGF-β-CCL17轴抑制肝乳腺癌;肝转移;乌司他丁;CCR4;CCL22;CCL17脏组织中与乳腺癌肝转移密切相关的CCL17/CCL22-CCR4信号通路有关。     

Murine Asialo GM1+CD8+ T Cells as Novel Interleukin-12-responsive Killer T Cell Precursors

Freshly isolated CD8⁺ T cells, but not CD4⁺ T cells, contained 20–30% of asialo GM1⁺ (ASGM1⁺) T cells which were distinct from ASGM1⁺NK1.1⁺ natural killer cells. This novel ASGM1⁺CD8⁺ T cell subpopulation showed a strong proliferative response to interlenkin-12 (IL-12) in the presence of IL-2. Culture of ASGM1⁺CD8⁺ T cells with IL-12 plus IL-2 allowed the generation of anomalous killer T cells concomitantly with the accumulation of cytolytic molecules. Moreover, ASGM1⁺CD8⁺ T cells produced high levels of interferon-γ (IFN-γ), but not IL-4, upon stimulation with IL-12 plus IL-2. Such immune responses were not observed in ASGM1⁻ CD8⁺ T cell snbpopulations constituting the majority of CD8⁺ T cells. These results demonstrated that ASGM1⁺CD8⁺ T cells are a novel subpopulation of IL-12-responsive and IFN-γ-producing killer T cell precursors.     

Interleukin-12 Augments the Generation of Autologous Tumor-reactive CD8+ Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes

Human tumor-infiltrating lymphocytes (TIL) were obtained from breast cancer, renal cancer or neuroblastoma to investigate the generation of autologous tumor-reactive CD8⁺ cytotoxic T lymphocytes (CTL). When TIL were cultured with interleukin (IL)-2 (100 U/ml), the growth of TIL peaked around 8–10 days after the initiation of culture. In contrast, the proliferation of TIL cultured with IL-2 plus IL-12 peaked around 4–5 days after culture and tumor cells rapidly disappeared from the culture. To determine the generation of autologous tumor-reactive CD8⁺ CTL, TIL-derived CD8⁺ T cells were separated by FACStar. Both IL-2-activated and IL-2 plus IL-12-activated TIL-CD8⁺ T cells showed the same level of lymphokine-activated killer activity against a variety of tumor cells. However, TIL-CD8⁺ T cells activated with IL-2 plus IL-12 revealed greatly augmented cytotoxicity against autologous tumor cells compared with that induced by IL-2 alone. The autologous tumor cell-killing activity of TIL-CD8⁺ CTL was significantly inhibited by the addition of F(ab)₂ anti-CD3 monoclonal antibody, indicating that these CTL recognize autologous tumor antigen through T cell receptor. These results imply that IL-12 is a novel cytokine which facilitates the generation of autologous tumor-reactive CD8⁺ CTL from TIL.     

Expression of Tumor-rejection Antigens in Gynecologic Cancers

We recently reported the four tumor-rejection antigens (SART1₂₅₉ SART2, SART3, and ART4) that possess tumor epitopes capable of inducing HLA-A2402-restricted cytotoxic T lymphocytes (CTLs) in cancer patients. This study investigated the expression of these tumor antigens in gynecologic cancers, including 33 ovarian cancers, 38 cervical cancers, and 40 endometrial cancers. SART1₂₅₉ antigen was detected in 56%, 35%, and 30% of ovarian, cervical and endometrial cancers, while SART2 antigen was detected in 46%, 66%, and 30% of these cancers, respectively. Both SART3 and ART4 antigens were detectable in the majority of these gynecologic cancers tested. In contrast, none of these antigens was detectable in any of the normal ovarian and uterine tissues tested. Peripheral blood mononuclear cells (PBMCs) of HLA-A24⁺ patients with gynecologic cancers were found to produce significant levels of interferon-γ in response to HLA-A24⁺ SART3⁺ gynecologic cancer cells after having been stimulated three times in vitro with either SART3_109–118 or SART3_315–323 peptide. These PBMCs lysed HLA-A24⁺ SART3⁺ gynecologic cancer cells, but not HLA-A24^- SART3⁺ gynecologic cancer cells or HLA-A24⁺ normal cells. Therefore, these four antigens and their peptides, including SART3 peptides, would be appropriate molecules for use in specific immunotherapy of HLA-A24⁺ gynecologic cancer patients.     

Maintenance and Characterization of an Epstein Barr Virus-infected CD56-negative T Cell Lymphoma

T cell lymphoma carrying Epstein Barr virus (EBV⁺TL) is very rare among Western countries while it is much more common among Japanese. Here we report an EBV⁺TL which has been maintained for years by the use of mice with severe combined immune deficiency (SCID) mice. Lymphoma was obtained from a 55-year-old male suffering from oculomotor nerve palsy and lymphadenopathy. A small piece of biopsied tumor was transplanted into SCID mice and the lymphoma has been maintained for over 3 years with passages every 2–3 weeks. The maintained lymphoma, termed as TMS24, and the original lymphoma cells showed identical phenotype and genotype, including diffuse medium-sized cell morphology lacking granules, suppressor/cytotoxic immunophenotype and identical T cell receptor β-chain gene rearrangement mode. Further, both were shown to carry an identical EBV clone in terms of the number of terminal repeats and the latency II-type restricted gene expression profile. Cytogenetically, TMS24 retained two characteristic chromosomal translocations of t(l;18)(q32;q21) and t(6;12)(p21;q24). Since only one cell line with such characters has been reported previously, TMS24 should be useful for detailed analysis of EBV⁺TL.     

Japanese phase II study of 90Y-ibritumomab tiuxetan in patients with relapsed or refractory indolent B-cell lymphoma

There is no data about the efficacy and safety of radioimmunotherapy with ⁹⁰Y-ibritumomab tiuxetan in patients with relapsed or refractory indolent B-cell lymphoma pretreated with rituximab-containing chemotherapy. We focused on this in a Japanese phase II study. Radioimmunotherapy with ⁹⁰Y-ibritumomab tiuxetan (11.1 and 14.8 MBq) was evaluated in patients with 100–149 × 10⁹ and >150 × 10⁹ platelets/L, respectively. The primary endpoint was the overall response rate. Forty patients were treated with ⁹⁰Y-ibritumomab tiuxetan (18 with 11.1 MBq/kg and 22 with 14.8 MBq/kg). Thirty-five patients (88%) had been pretreated with rituximab, including 27 (68%) pretreated with rituximab-containing chemotherapy. The overall response rate was 83% (33/40; 95% confidence interval, 67–93%), and the complete response rate was 68% (27/40; 95% confidence interval, 51–81%). The overall response rates in patients pretreated with rituximab-containing chemotherapy and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) were 83% (19/23) and 94% (17/18), respectively. The median progression-free survival time of the 40 patients who received ⁹⁰Y-ibritumomab tiuxetan was 9.6 months. Toxicity was primarily hematological and mostly transient. No grade 4 non-hematological toxicity was observed. In conclusion, radioimmunotherapy with ⁹⁰Y-ibritumomab tiuxetan is safe and highly effective in patients with relapsed or refractory indolent B-cell lymphoma, including those pretreated with rituximab-containing chemotherapy. (ClinicalTrials.gov number NCT00220285) ( Cancer Sci 2009; 100: 158–164)     

Both CD133+ and CD133− subpopulations of A549 and H446 cells contain cancer-initiating cells

Tumors have been known to contain a small population of cancer stem cells that initiate tumor growth and promote tumor spreading. CD133 alone or in combination with other markers is currently being used for identification and isolation of the putative cancer stem cell population from malignant tumors. To determine whether the CD133⁺ cells constitute the stem cell populations of lung cancer cells A549 and H446, CD133⁺ and CD133⁻ subpopulations were sorted from A549 and H446 cells by magnetic cell separation and characterized for their in vitro stem cell-like properties. Interestingly, both the CD133⁺ and CD133⁻ cells displayed similar abilities of colony formation, self-renewal, proliferation, differentiation, and invasion, as well as resistance to chemotherapy drugs. Furthermore, colony formation assays showed that more than 40% of cells in both the CD133⁺ cells and CD133⁻ subpopulations could form large colonies capable of regenerating the unsorted populations and forming tumors in nude mice. These results suggest that CD133 alone cannot be used as a stem cell marker for the lung cancer cells A549 and H446, and both the CD133⁺ and CD133⁻ subpopulations contain similar numbers of cancer stem cells. ( Cancer Sci 2009; 100: 1040–1046)     

Multipotent and Committed CD34+ Cells in Bone Marrow Transplantation

In order to study the role of CD34⁺ cells in hematological recovery following bone marrow transplantation (BMT), bone marrow cells stained with HPCA-1 (CD34) and MY-9 (CD33) monoclonal antibodies were analyzed by using a fluorescence-activated cell sorter on or about days 14 and 28, as well as at later times, following BMT in 6 recipients. Single cell cultures of CD34⁺ cells were also performed to evaluate their in vitro hematopoietic function. CD34⁺ cells were detectable in bone marrow cells on day 14. More than 80% of CD34⁺ cells co-expressed the CD33 antigen, and macrophage (Mac) colony-forming cells predominated among total colony-forming cells of CD34⁺ cells. In normal bone marrow cells, CD34⁺, CD33⁺ cells amounted to about 40% of CD34⁺ cells, and the incidences of erythroid bursts, granulocyte/macrophage (GM) colonies, and Mac colonies were similar to each other. After more than 10 weeks, CD34⁺, CD33⁻ cells gradually recovered, as erythroid burst colony-forming cells increased following GM colony-forming cells. This phenomenon was well-correlated with the time course of peripheral blood cell recovery. CD34⁺, CD33⁺ cells as committed progenitors and CD34⁺, CD33⁻ cells as multipotent stem cells have distinctive biological behaviors in BMT.     

Regulatory (FOXP3+) T cells as target for immune therapy of cervical intraepithelial neoplasia and cervical cancer

Regulatory (FOXP3⁺) T cells (T_regs) comprise a subpopulation of CD4⁺ T cells that suppress autoreactive immune cells, thereby protecting organs and tissues from autoimmunity. T_regs have also been detected in human malignancies and their depletion or inactivation substantially improves cellular antitumor immunity in preclinical studies. Novel therapeutic strategies for cervical cancer and precancerous cervical intraepithelial neoplasia (CIN) focus on immune-modulatory and cancer vaccination approaches. In this context, the frequency of T_regs in cervical cancer and precancerous CIN could influence therapeutic strategies. We determined the frequency of infiltrating CD4⁺ and CD8⁺ T cells as well as FOXP3⁺ T_regs in high-grade CIN lesions (CIN III) and cervical carcinoma compared to colon carcinoma, skin melanoma, and bronchial carcinoma. We show that human papilloma virus-derived lesions have a significantly higher number of infiltrating lymphocytes and FOXP3⁺ T_regs compared to three other common tumor entities. In addition we explored the therapeutic effect of agonistic anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein antibodies that, by single systemic application, inactivate T_regs and induce strong intratumoral invasion of CD8⁺ T cells and complete tumor eradication in 70% of treated animals. The large number of T_regs in human papilloma virus-derived lesions suggests a pivotal role of T_regs for counteracting the host immune response. We therefore regard CIN and cervical cancer as prime targets for new immune-based non-invasive therapies. ( Cancer Sci 2009; 100: 1112–1117)