Melan-A specific CD8+ T lymphocytes after hyperthermic isolated limb perfusion: A pilot study in patients with in-transit metastases of malignant melanoma

Abstract

Purpose: Isolated limb perfusion (ILP) with hyperthermia is an effective treatment for in-transit metastases of malignant melanoma in the extremities. Preclinical studies have shown that hyperthermia may induce an immunogenic death of tumour cells. We therefore decided to study whether ILP may induce tumour-specific immune responses in the clinical setting.

Method: The number of Melan-A/Mart-1 specific CD8+ T cells, as well as other phenotypically different immune cells, was recorded in peripheral blood in 12 HLA-A2+ patients with in-transit metastases undergoing hyperthermic ILP with melphalan.

Results: All patients underwent ILP without any complication and with an overall response rate of 83%. No substantial changes in the number of circulating T-cells, B-cells, NK-cells or monocytes were observed during follow-up. Four out of 12 patients showed an elevation of Melan-A+ CD8+ T-cells 4 weeks after ILP.

Conclusion: We here report our preliminary observations that a small increase in tumour-specific T-cells could be seen in a subpopulation of patients after ILP. However, much more work is necessary to fully delineate the systemic immune response to hyperthermic ILP.     

肺癌患者外周血调节性T细胞和IL-10的检测及其意义

  目的  探讨肺癌患者外周血调节性T细胞和IL-10检测的诊断意义及与临床特征相关性。  方法  采用流式细胞仪检测60例肺癌患者外周血中CD4+CD25+T细胞的表达水平, 同时采用ELISA法检测IL-10的表达水平。并随机选30例健康体检者作为对照组。  结果  肺癌患者外周血中CD4+CD25+T细胞和IL-10的表达水平明显高于健康对照组（P < 0. 05）; CD4+CD25+T细胞与IL-10的检测结果呈正相关; 肺癌患者外周血中CD4+CD25+T细胞和IL-10的表达水平与临床分期有关（P < 0. 05）, 与病理类型无关（P>0. 05）。  结论  检测肺癌患者外周血中CD4+CD25+T细胞和IL-10的表达水平, 有助于肺癌患者病情的评估和治疗。      

Changes in mucosal immune cells of bladder tumor patient after BCG intravesical immunotherapy

The Bacillus Calmette-Guerin (BCG) is considered to be at least as effective, and perhaps superior to chemotherapy in the prophylaxis of recurrent superficial tumors. However, the mechanism of the antitumor effect of BCG is still not exactly known. We have conducted investigations to examine changes in bladder mucosal immune cells in patients with superficial bladder carcinoma treated with a first cycle of BCG. The study group included 15 BCG and 5 doxorubicin instillation patients, most in the intermediate or high risk group for recurrent tumor. Grossly normal bladder mucosal cold cup biopsies were performed at initial TUR and one week after six consecutive weekly instillations of BCG or doxorubicin. All specimens underwent immunohistochemical staining, both pre-treatment and post-treatment, including CD20, CD45RO, CD8, CD4 and CD57. Immunoreactive cell counts were evaluated from three different microscopic fields (x400) under the grid. The mean duration of follow-up was 52.8 months. The post-treatment bladder mucosal B-cells (CD20) and T-cells (CD45RO, CD4, CD8) were significantly increased compared to pre-treatment in patients treated with BCG instillation, but NK-cells (CD57) were not changed. However, there was no change in B-cells or T-cells in patient treated with doxorubicin. The CD20 cells in pre-treatment specimens did not correlate with any other cells. However, it was a statistically significant correlation with CD45RO in post-treatment specimens. The CD4 correlated with CD45RO and CD8 in pre-treatment, but it was correlated with CD45RO and CD57 in post-treatment specimens. There was no tumor recurrence in cases with significantly increased B-cells after BCG instillation. The results of these studies suggest that intravesical BCG immunotherapy for superficial bladder tumor induces a significant increase in T-cells as well as B-cells and that B-cells have a preventive effect on tumor recurrence. Further studies with a larger number of patients are needed to confirm the value of the B-cell increment after BCG instillation as a clinically independent prognostic factor.     

Stromal CD4/CD25 positive T-cells are a strong and independent prognostic factor in non-small cell lung cancer patients, especially with adenocarcinomas

Within the concert of immune reactions against tumour cells cytotoxic and regulatory T-cells are of utmost importance. Several studies revealed contradictory results on this issue. We therefore focused on functional expression patterns and localization of tumour-infiltrating T-lymphocytes in non-small cell lung cancer (NSCLC) and their impact on patient's survival. 232 curatively operated NSCLC patients were included. After histological reevaluation and construction of tissue-multi-arrays immunohistochemical doublestains for CD3/CD8 and CD4/CD25 were performed to evaluate the total number of T-cells and their subsets of cytotoxic and activated T-cells. Additionally, the localization of the lymphocytes was included in the analysis. Hereby, T-cells within the tumour stroma were regarded as stromal, those among cancer cells as intraepithelial. The number of lymphocytes differed significantly between the histological subtypes being most prominent in large cell carcinomas. Survival analysis showed that high numbers of stromal T-lymphocytes are of beneficial prognostic influence in NSCLC patients. This also proved to be an independent prognostic factor in adenocarcinomas. Thus, in a large and well characterized cohort of NSCLC this is the first study to determine the prognostic value of stromal T-lymphocytes, as these are an independent prognosticator in NSCLC especially in adenocarcinomas whereas intraepithelial T-cells are not.     

Two cases of complete response to combination chemotherapy of gemcitabine and docetaxel for recurrent ovarian cancer

The established standard treatment for advanced ovarian cancer is carboplatin and paclitaxel. However, more than 70% of patients have recurrent disease. The standard therapy for recurrent ovarian cancer has not been confirmed. It was reported that docetaxel had a 30-40% of response rate in patients with recurrent ovarian cancer, and that gemcitabine had a 13-22% response rate. The combination chemotherapy of gemcitabine and docetaxel is also applied to non-small cell lung cancer. We use a regimen of 800 mg/m2 of gemcitabine on day 1 and day 8 in combination with 70 mg/m2 of docetaxel on day 8 with a 3-week interval. We treated 2 patients with recurrent ovarian cancer who responded completely to combination chemotherapy with gemcitabine and docetaxel.     

Reduced numbers of IL-7 receptor (CD127) expressing immune cells and IL-7-signaling defects in peripheral blood from patients with breast cancer

Interleukin-7-receptor-signaling plays a pivotal role in T-cell development and maintenance of T-cell memory. We studied IL-7Ralpha (CD127) expression in PBMCs obtained from patients with breast cancer and examined IL-7 receptor-mediated downstream effects defined by STAT5 phosphorylation (p-STAT5). Reduced numbers of IL-7Ralpha-positive cells were identified in CD4+ T-cells as well as in a CD8+ T-cell subset defined by CD8alpha/alpha homodimer expression in patients with breast cancer. PBMCs obtained from healthy donors (n = 19) and from patients with breast cancer (n = 19) exhibited constitutive p-STAT5 expression in the range of 0-6.4% in CD4+ T-cells and 0-4% in CD8+ T-cells. Stimulation with recombinant human IL-7 for 15 min increased p-STAT5 expression up to 36-97% in CD4+T-cells and to 26-90% in CD8+T-cells obtained from healthy control donors (n = 19). In contrast, PBMCs obtained from 13/19 patients with breast cancer did not respond to IL-7 as defined by STAT5 phosphorylation, despite expression of IL-7Ralpha on T-lymphocytes. T-cells were further characterized for IL- 2 and IFN-gamma production induced by PMA/Ionomycin. PBMCs from 9/19 patients with breast cancer showed decreased IL-2 and IFN-gamma production combined with IL-7-signaling defects; PBMCs from 4 patients with breast cancer exhibited deficient IL-7-signaling, yet intact cytokine production. Reduced numbers of IL-7Ralpha-positive cells and nonresponsiveness to IL-7, defined by lack of STAT5 phosphorylation, characterizes the immunological profile in T-cells from patients with breast cancer.     

The hypereosinophilic syndrome associated with CD4+CD3- helper type 2 (Th2) lymphocytes.

We describe herein the clinical and laboratory manifestations of a unique group of patients (pts) presenting with hypereosinophilic syndrome (HES) who were treated in our medical centers for 4-13 years. Skin biopsies, flow cytometry of peripheral blood mononuclear cells (PBMC), assays for cytokines and immunoglobulin (Ig) production in vitro, and Southern blots of T-cell receptor (TCR) genes were performed. All four pts had a persistent hypereosinophilia (> 1.9 x 10(9)/L) and chronic skin rash. Three of four had elevated IgE, thrombotic manifestations and lung involvement (asthma and/or infiltrates), and one had deforming sero-negative arthritis of the hands. 66-95% of their peripheral T-cells expressed CD4 but not CD3 or TCR molecules on the cell surface membrane. Activated CD4+CD3- cells secreted interleukin (IL)-4 and/or 5, and were required for maximal IgE secretion by autologous B-cells. Two pts had evidence of rearrangement of TCR genes of the CD4+CD3- cells, one of whom died of anaplastic lymphoma. In conclusion, HES with CD4+CD3- lymphocytosis may be associated with high serum IgE, dermatological, pulmonary, thrombotic and rheumatic manifestations which may be due to Th2 effects of CD4+CD3- cells migrating to end organs. Fatal systemic lymphoid malignancy may also develop in some pts with monoclonal expansion of the CD4+CD3- T-cells.     

Reduced growth, increased vascular area, and reduced response to cisplatin in CD13-overexpressing human ovarian cancer xenografts.

PURPOSE: Expression of aminopeptidase N/CD13 can be detected in several solid tumor types. Thus far, the role of CD13 in ovarian cancer has not been studied. We have investigated the expression pattern and biological function of CD13 in ovarian cancer. EXPERIMENTAL DESIGN: First, we studied the expression of CD13 in ovarian cancer tissue of 15 patients representing three different histological types (5 patients each) by immunohistochemistry. We then stably transfected the IGROV-1 human ovarian cancer cell line with a CD13 expression vector and examined the biological effect of CD13 in vitro and in vivo. RESULTS: The expression of CD13 in ovarian cancer was associated with the histological subtype: CD13 expression in tumor cells was observed in 80-100% of the patients with a serous or mucinous carcinoma and in only 20% of the clear cell carcinoma patients. In all patients' tumor samples, CD13-positive blood vessels were present. CD13 overexpression in IGROV-1 cells did not affect in vitro cell growth and sensitivity to doxorubicin, cisplatin, or gemcitabine. CD13 overexpression reduced invasion in Matrigel, which appeared to be independent of the aminopeptidase activity of CD13. Furthermore, the growth rate of IGROV-1/CD13 xenografts was reduced. The area of the vessel lumens was enlarged in a small percentage of vessels in the CD13-overexpressing xenografts. In addition, the CD13-overexpressing tumors were less sensitive to cisplatin. CONCLUSIONS: CD13 is expressed in tumor as well as endothelial cells in human ovarian cancer. Our results suggest that CD13 overexpression affects ovarian cancer growth, vascular architecture, and response to chemotherapy. Further elucidation of the mechanism of the observed effects of CD13 is warranted to better understand its role in the pathophysiology of ovarian cancer.     

Combined Blockade Of PD-1 and TIGIT is not Sufficient to Improve the Function Of CD8+ T-Cells in Chronic Lymphocytic Leukemia

Background and Objective: Blockade of immune checkpoint receptors in the treatment of cancers has been mentioned in several studies. Here, we investigated the efficacy of combined blockade of two inhibitory receptors, PD-1 and TIGIT, in restoring functional features of CD8+ T-cells in CLL. Methods: CD8+ T-cells were separated from the peripheral blood of 11 CLL patients and targeted with malignant B-cells isolated from the same patients. Cells were then stimulated with anti-CD3/CD28 and PMA/ionomycin to assess their proliferative response and cytotoxic activity using MTT and CD107a degranulation assays, respectively. Cytokine production of isolated CD8+ T-cells was also determined using ELISA. Results: There were no significant differences in proliferation and cytotoxic activity of CD8+ T-cells co-blocked with anti-PD-1/TIGIT compared to those single blocked with anti-PD-1, anti-TIGIT, or the control antibody. There was no significant difference in cytokine production of mentioned groups, either. Conclusions: Collectively, combined blockade of PD-1 and TIGIT failed to restore the proliferation and function of CD8+ T-cells isolated from CLL patients.     

Allogeneic transplantation of CD34+ selected hematopoietic cells--clinical problems and current challenges

Since G-CSF mobilized peripheral blood has become the major source of hematopoietic cells (PBSC) for allogeneic transplantation of patients with h'ematological malignancies, new technologies of T-cell depletion were developed to reduce the risk of graft-versus-host disease. CD34+ selection by immunomagnetic separation has been shown to be the most effective method to achieve a 3 - 4 log depletion of T-cells while preserving the high number of hematopoietic progenitors in the graft. The high number of CD34+ cells contained in the positive fraction have allowed to facilitate engraftment from sibling donors matched for only one haplotype. In addition, studies in pediatric recipients have shown that high numbers of hematopoiteic progenitors infused can lead to timely recovery of T- and B-cells in the first year after transplantation. Positive results have also been reported in patients with chronic myelogenous leukemia who had received CD34+ selected PBSC from HLA-identical sibling donors and subsequent infusions of donor T-cells to establish graft-versus-leukemia effects. On the other hand, studies performed in adults with advanced leukemia receiving CD34+ selected PBSC from haploidentical or unrelated donors demonstrated an increased risk for graft-rejection, relapse and late opportunistic infections. Future efforts will have to concentrate on the restoration of cellular immunity by either adaptively transferring antigen specific effector cells or by studying the use of cytokines like interleukin-7. Although these question have not been resolved yet, allogeneic transplantation of highly purified CD34+ PBSC has become an therapeutic option for patients who are at high-risk for severe GvHD     

盐酸吉西他滨对非小细胞肺癌患者骨髓CD34+造血细胞的毒性研究

目的 探讨非小细胞肺癌患者骨髓CD34+前体造血细胞在细胞毒药物盐酸吉西他滨诱导下,发生细胞凋亡情况及DNA损伤修复信号通路上相关基因表达的变化.方法 采用免疫磁珠法分选出80例非小细胞肺癌患者骨髓单个核细胞中的CD34+细胞,并应用流式细胞术检测其纯度.用盐酸吉西他滨(终末浓度为10μg/ml)诱导CD34+细胞凋亡,检测诱导不同时间点的细胞凋亡率的差异.提取细胞RNA,利用基因芯片技术,将药物诱导前后的CD34+细胞在DNA损伤修复信号通路的相关基因表达情况进行检测,并分析比较.结果 80份骨髓样本CD34阳性率为(6.06±1.61)%;TNM分期不同的患者样本间CD34的表达无统计学差异,P>0.05.免疫磁珠法分选后,流式细胞术检测CD34+细胞的纯度为(88.63±7.29)%;CD34+细胞在经过盐酸吉西他滨诱导0 h、4 h、8 h、12 h后,凋亡率分别为(5±1.37)%、(18±4.76)%、(39±8.15)%、(56±9.64)%,各时间点细胞的凋亡率之间有统计学差异,P<0.05.基因芯片结果显示,骨髓CD34+细胞在盐酸吉西他滨诱导凋亡0 h与诱导12 h后相比,表达差异2倍以上的基因有13种,其中9种基因表达是升高的,4种基因表达降低.结论 盐酸吉西他滨可以诱导非小细胞肺癌患者骨髓前体CD34+细胞发生凋亡,随着药物作用时间延长,细胞凋亡率上升;DNA损伤修复系统中,部分基因表达水平出现变化.     

Treatment strategies for Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH)

In Epstein-Barr virus (EBV) infection, the virus immortalizes B lymphocytes and cytotoxic T lymphocytes (CTLs) are directed toward both latent and lytic viral antigens expressed on EBV-infected B-cells. Various EBV-associated diseases occur as a result of this disruption of immune surveillance. In the majority of EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) cases, the major cell types containing EBV DNA are not B-cells, but clonally proliferating T-cells or NK-cells. Proliferation of these cells produces severe immune reactions in the host, and the clinical features related to massive cytokine production at the onset of disease are unique and distinct from other EBV-associated diseases. In the treatment of EBV-HLH, therapeutic infusion of EBV-specific CTLs appears to be ineffective, and eradication of EBV-containing cells is useful but not sufficient to save lives, because of high incidence of acute mortality due to cytokine-induced multiple organ failure and neutropenia-associated opportunistic infections. The optimal treatment strategy for this disease consists of three steps: (1) control of cytokine storm including coagulopathy and multiple organ failure, (2) control of opportunistic infections, and (3) eradication of clonally proliferating EBV-containing T- or NK- cells by immunochemotherapy and, if necessary, hemopoietic stem cell/bone marrow transplantation (SCT/BMT).     

Understanding of the immunological heterogeneity of canine mammary carcinomas to provide immunophenotypic features of circulating leukocytes as clinically relevant prognostic biomarkers

We have evaluated the phenotypic features of peripheral blood leukocytes as putative novel biomarkers with prognostic values to monitor canine mammary carcinomas. Female dogs were categorized into distinct groups, referred as mammary carcinoma in benign mixed tumor-MC-BMT and mammary carcinoma-MC. Our findings demonstrate that decreased percentage of B-cells along with increased frequency of NK-cells, CD8⁺T-cells, and CD8⁺CD5^Low+T-cells beside higher T/B-cells and lower CD4⁺/CD8⁺ ratio were the hallmarks of MC-BMT. Despite the lower expression of MHCI and MHCII, the lymphocytes from MC-BMT and MC displayed higher migration potential as suggested by enhanced frequency of CD18⁺ events. Although increased levels of macrophage-like cells/(CD14⁺CD16⁺) and decreased levels of MHCII expression were a common phenotypic feature in mammary carcinoma, down-regulation of MHCI was selectively observed in MC. Decreased frequency of CD4⁺ T-cells with increased levels of CD8⁺ T-cells and lower CD4⁺/CD8⁺ T-cell ratio were relevant biomarkers of MC-BTM(−). Although decreased expression of MHCI by monocytes was observed in MC-BTM regardless of the presence of lymph node metastasis, this phenotypic feature was restricted to MC free of metastasis. The CD4⁺/CD8⁺ T-cells ratio lower than 1.8 was elected as a valid parameter with outstanding performance to predict survival in MC-BMT. On the other hand, the MHCI expression by monocytes higher than 10² MFI showed good value to estimate worse outcome in MC. These results should help to improve our understanding of the immunological heterogeneity of canine mammary carcinomas and provide tools for the determination of cut-off scores of clinically relevant immonophenotypic prognostic biomarkers.     

Expression of CD3-zeta on T-cells in primary cervical carcinoma and in metastasis-positive and -negative pelvic lymph nodes

Lymphocytic infiltrate is often present in cervical cancer lesions, possibly reflecting an ongoing, but ineffective, immune response to the tumour. Recently, evidence has accumulated for systemically impaired T-cell functions in cancer patients, associated with decreased expression of signal-transducing zeta (zeta) chain dimer molecules on circulating T-cells and NK-cells. Here, we report on the intralesional down-regulation of zeta chain expression on T-cells in cervical carcinoma. Paraffin-embedded or snap-frozen sections from 24 different cervical cancer specimens were studied. Paraffin-embedded tumour-positive (n = 7) and tumour-negative (n = 15) pelvic lymph nodes were also included in the study. Immunostaining was performed on consecutive sections with antibodies specific for CD3-epsilon or the CD3-associated zeta chain dimer. Antigen retrieval by sodium citrate/microwave treatment was essential for zeta staining of paraffin sections. The amount of zeta positive cells was quantitated and related to the number of CD3-epsilon+ cells in corresponding tumour areas. Of the 24 cervical cancer specimens studied, zeta chain dimer expression was reduced in seven cases and strongly reduced in the other 17 samples. In tonsil control sections, CD3-epsilon and CD3-zeta were always co-expressed in almost equal numbers. Also, both tumour-negative and -positive lymph nodes showed zeta chain expression which equalled that of CD3-epsilon expression. These data indicate that a decreased expression of signal-transducing zeta molecules on tumour-infiltrating T-cells is frequent in cervical cancer. The apparently unimpaired zeta chain expression within draining lymph nodes suggests that local tumour-derived factors at the primary site are instrumental in zeta chain down-regulation.     

T-cell derived cytokines co-stimulate proliferation of CD40-activated germinal centre as well as follicular lymphoma cells

Follicular lymphomas, the malignant counterparts of normal germinal centre (GC) B-cells, grow in vivo in close association with polyclonal T-cells, predominantly from the T-helper cell type. T-cell-derived growth factors are involved in the development of GC B-cells. However, their role in the pathogenesis of follicular lymphomas has not been clearly defined. We investigated the co-stimulatory activity of 14 cytokines (interleukin-1 to -8, IL-10, IL-13, IFN-α, TNF-α, GM-CSF and SCF) on the proliferation of CD40-activated follicular lymphoma cells in comparison to tonsillar GC B-cells. Tonsillar GC B-cells (n=4), malignant cells from diagnostic lymph node biopsies of patients with follicular (n=4) or transformed (n=4) lymphomas were grown on irradiated CD40-ligand transfectants, with and without cytokines. [³H]-thymidine uptake was measured at day 7. IL-10 and IL-4 proved to be the most potent co-stimulators of proliferation of tonsillar GC B-cells, whereas proliferation of follicular lymphoma cells was co-stimulated by IL-4. The fact that IL-4 is a T-cell derived cytokine, suggests that lymphoma infiltrating T-cells play a role in the growth of these malignancies. Moreover, proliferation of both non-neoplastic tonsillar GC B-cells and follicular lymphomas is co-stimulated by T-cell derived cytokines, indicating that responsiveness to paracrine factors may not be a characteristic of the malignant phenotype. © 1997 John Wiley & Sons, Ltd.     

Carboplatin plus gemcitabine repeating doublet therapy in recurrent breast cancer

PurposeThe combination of cisplatin plus gemcitabine is active in metastatic breast cancer. Carboplatin plus gemcitabine, widely used in ovarian and non–small-cell lung cancers, has also been used in breast cancer. This trial examined the efficacy and toxicity of split-dose carboplatin plus gemcitabine in advanced breast cancer.Patients and MethodsPatients with measurable disease, recurrent after adjuvant and ≤ 1 previous treatment for systemic disease, received carboplatin area under the curve = 2.0 (Calvert) plus gemcitabine 800 mg/m², both drugs administered days 1 and 8 every 21 days. Of 15 patients accrued, 13 are fully evaluable.ResultsThere were 2 complete (13.3%) and 6 partial (40%) responses, for an overall response rate by intention to treat of 53.3% (95% CI, 28%-82%). The median time to progression was 4.5 months (95% CI, 2.03-6.97 months), and median overall survival was 28.8 months (95% CI, 9.4-48.2 months). There were 2 patients with grade 3 (13.3%) anemia, 7 patients with grade 3 (46.6%) and 4 patients (26.6%) with grade 4 neutropenia, 4 patients with grade 3 (26.6%) and 3 patients (20%) with grade 4 thrombocytopenia.ConclusionThe repeating doublet of split-dose carboplatin plus gemcitabine reveals activity comparable to that of cisplatin plus gemcitabine, is well tolerated, and warrants evaluation in patients with recurrent breast cancer.     

CD3+CD56+NKT细胞及CD3-CD56+NK细胞在恶性肿瘤患者的临床意义研究

背景与目的细胞毒性淋巴细胞在抗肿瘤免疫效应中发挥着重要作用,CD3 CD56 NKT细胞作为一类新的具有细胞毒性的效应细胞,目前关于其抗肿瘤意义的探讨主要集中于血液系统恶性疾病,而在实体肿瘤中的应用和临床价值研究尚少。本研究旨在初步探讨抗肿瘤细胞CD3 CD56 NKT细胞及CD3-CD56 NK细胞在恶性肿瘤患者外周血的表达状态及其临床意义。方法采用流式细胞术分析118例恶性肿瘤患者(55例肺癌患者和63例乳腺癌患者)及46例健康对照组外周血中的T细胞亚群及CD3 CD56 NKT细胞、CD3-CD56 NK细胞表达。结果恶性肿瘤患者组CD3 CD8 T细胞、CD3 CD56 NKT细胞以及CD3-CD56 NK细胞表达率均明显高于健康对照组(P<0.01)。肺癌患者中以CD3 CD8 T细胞和CD3 CD56 NKT细胞明显增加为主;乳腺癌患者中以CD3 CD56 NKT细胞和CD3-CD56 NK细胞明显增加为主。结论CD3 CD56 NKT细胞在肺癌和乳腺癌患者的抗肿瘤效应中占据重要地位,而CD3 CD8 CTL和CD3-CD56 NK细胞在不同类型肿瘤患者中具有不同的重要性。     

Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer.

Immunosuppression may contribute to the progression of cancer. In this study we assessed the structural and functional status of T cells from tumor specimens obtained from patients with early stage non-small cell lung cancer and late-stage ovarian cancer. Although some groups have described structural alterations in the TCR in patients with other malignancies, we did not observe decreased expression of the CD3zeta subunit in the tumor-associated T cells. However, increased percentages of CD4(+)CD25(+) T cells were observed in the non-small cell lung cancer tumor-infiltrating lymphocytes and ovarian cancer tumor-associated lymphocytes. Furthermore, these CD4(+)CD25(+) T cells were found to secrete transforming growth factor-beta, consistent with the phenotype of regulatory T cells. Despite a generalized expression of lymphocyte activation markers in the tumor-associated T-cell populations, the CD8(+) T cells expressed low levels of CD25. To determine whether expression of CD25 could be restored on the CD8 cells, tumor-associated T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. After stimulation, nearly all of the CD8 T cells expressed CD25. Furthermore, despite the low levels of interleukin 2, IFN-gamma, and tumor necrosis factor-alpha secretion by the tumor-associated and peripheral blood T cells at baseline, stimulation with anti-CD3 and anti-CD28 monoclonal antibodies significantly increased the fraction of cells producing these cytokines. Thus, tumor-associated T cells from patients with early and late-stage epithelial tumors contain increased proportions of CD4(+)CD25(+) T cells that secrete the immunosuppressive cytokine transforming growth factor-beta. Furthermore, our results are consistent with previous reports showing impaired expression of CD25 on CD8(+) T cells in cancer patients. Finally, increased lymphocyte costimulation provided by triggering the CD28 receptor is able to increase CD25 expression and cytokine secretion in tumor-associated T cells. These observations provide evidence for the contribution of regulatory T cells to immune dysfunction in cancer patients.     

Characterization of BIS20x3, a bi-specific antibody activating and retargeting T-cells to CD20-positive B-cells

This paper describes a bi-specific antibody, which was called BIS20x3. It retargets CD3varepsilon-positive cells (T-cells) to CD20-positive cells and was obtained by hybrid-hybridoma fusion. BIS20x3 could be isolated readily from quadroma culture supernatant and retained all the signalling characteristics associated with both of its chains. Cross-linking of BIS20x3 on Ramos cells leads to DNA fragmentation percentages similar to those obtained after Rituximab-cross-linking. Cross-linking of BIS20x3 on T-cells using cross-linking F(ab')2-fragments induced T-cell activation. Indirect cross-linking of T-cell-bound BIS20x3 via Ramos cells hyper-activated the T-cells. Furthermore, it was demonstrated that BIS20x3 effectively re-targets T-cells to B-cells, leading to high B-cell cytotoxicity. The results presented in this paper show that BIS20x3 is fully functional in retargeting T-cells to B-cells and suggest that B-cell lymphomas may represent ideal targets for T-cell retargeting bi-specific antibodies, because the retargeted T-cell is maximally stimulated in the presence of B-cells. Additionally, since B-cells may up-regulate CD95/ Fas expression upon binding of CD20-directed antibodies, B-cells will become even more sensitive for T-cell mediated killing via CD95L/ Fas L, and therefore supports the intention to use T-cell retargeting bi-specific antibodies recognizing CD20 on B-cell malignancies as a treatment modality for these diseases.     

Increased proteinase inhibitor-9 (PI-9) and reduced granzyme B in lung cancer: mechanism for immune evasion?

Cytotoxic CD8(+) T-cells mount immune responses to cancer via cytotoxic pathways including granzyme B. Cancer cells are also known to develop immune evasion mechanisms. We hypothesised that lung cancer cells would over-express the granzyme B-inhibitor, proteinase inhibitor-9 (PI-9) and down-regulate granzyme B expression by neighbouring CD8(+) T-cells. We investigated PI-9 expression in lung cancer cell lines, and primary lung cancer cells obtained at curative lung resection from cancer patients with/without chronic obstructive pulmonary disease (COPD). Granzyme B and PI-9 expression was also determined in CD8(+) T-cells from the cancer and non-cancer areas of resected lung tissue and from bronchoalveolar lavage (BAL). We then evaluated the effects of conditioned media from lung cancer cell lines on granzyme B expression and the cytotoxic activity of CD8(+) T-cells. PI-9 was highly expressed in lung cancer cell lines. Increased PI-9 expression was also observed in primary cancer cells vs. epithelial cells from non-cancer tissue or bronchial brushing-derived normal primary large airway epithelial cells. Expression significantly correlated with cancer stage. Significantly reduced granzyme B was noted in CD8(+) T-cells from cancer vs. non-cancer tissue. Granzyme B production by CD8(+) T-cells was reduced in the presence of conditioned media from lung cancer cell lines. Our data suggest that lung cancer cells utilise their increased PI-9 expression to protect from granzyme B-mediated cytotoxicity as an immune evasion mechanism, a function that increases with lung cancer stage.