CD39 identifies a specific CD8 + T cell population in lung adenocarcinoma-related metastatic pleural effusion.

Malignant pleural effusion (MPE), which is a complex microenvironment that contains numerous immune and tumour signals, is common in lung cancer. Gene alterations, such as driver gene mutations, are believed to affect the components of tumour immunity in the microenvironment (TIME) of non-small-cell lung cancer. In this study, we have shown that pleural CD39 + CD8 + T cells are selectively elevated in lung adenocarcinoma (LUAD) with wild-type epidermal growth factor receptor (EGFRwt) compared to those with newly diagnosed mutant EGFR (EGFRmu). Furthermore, these CD39 + CD8 + T cells are more prevalent in MPE with acquired resistance to EGFR-tyrosine kinase inhibitors (AR-EGFR-TKIs). Our analysis reveals that pleural CD39 + CD8 + T cells exhibit an exhausted phenotype while still retaining cytolytic function. Additionally, they have a higher T cell receptor (TCR) repertoire clonality compared to CD39-CD8 + T cells, which is a unique characteristic of LUAD-related MPE. Further investigation has shown that TCR-Vß clonality tends to be more enhanced in pleural CD39 + CD8 + T cells from MPE with AR-EGFR-TKIs. In summary, we have identified a subset of CD8 + T cells expressing CD39 in MPE, which may potentially be tumour-reactive CD8 + T cells. This study provides new insights into the dynamic immune composition of the EGFRmu tumour microenvironment.

Combining local cryoablation with PD-L1 blockade synergistically eradicates established murine lung cancer by modulating mitochondrial in PD-1+CD8+ T cell.

Immune checkpoint blockade (ICB) has shown improvement in overall survival for lung cancer in clinical trials. However, monotherapies have limited efficacy in improving outcomes and benefit only a subset of patients. Combination therapies targeting multiple pathways can augment an immune response to improve survival further. Here, we demonstrate that combinatorial anti-PD-L1/cryoablation therapy generated a synergistic antitumor activity in the established lung cancer model. Importantly, it was observed that this favorable antitumor immune response comes predominantly from the PD-1+CD8+ T cells generated after the combination therapy, referred as improvement of IFN-γ production and mitochondrial metabolism, which resembled highly functional effectors CD8+ T cells. Notably, the cellular levels of mitochondrial reactive oxygen and mitochondria mass excessively coincided with alteration of IFN-γ secretion in PD-1+CD8+T cell subset. So far, anti-PD-L1/cryoablation therapy selectively derived the improvement of depolarized mitochondria in PD-1+CD8+T cell subset, subsequently rebuild the anti-tumor function of the exhausted CD8+ T cells. Collectively, there is considerable interest in anti-PD-L1 plus cryoablation combination therapy for patients with lung cancer, and defining the underlying mechanisms of the observed synergy.

Imbalance of Circulating Monocyte Subsets in Subjects with Newly Emerged and Recurrent Hospital-Acquired Pneumonia.

OBJECTIVE: Hospital-acquired pneumonia (HAP) is one of the most common diseases in the intensive care unit, where the development of disease is closely related with the host immune response. Monocytes play an important role in both innate and adaptive immune system. We aimed to investigate the changes of circulating monocyte subsets in subjects with HAP to explore its value in monitoring HAP. METHODS: In total, 60 HAP patients and 18 healthy individuals were enrolled in this study. Human monocyte subsets are classified into 3 groups: nonclassical (NC), intermediate (ITM), and classical (CL). Also, programmed death ligand 1 (PD-L1) expression on circulating monocyte subsets was measured by flow cytometry. RESULTS: Data showed that the ratio of NC, ITM, and CL among monocytes was comparable between HAP patients and healthy controls (P > .05). There was a remarkable imbalance of NC and CL in newly emerged HAP compared to healthy controls (P < .05), subsequently reaching normalization in recurrent HAP (P > .05). Furthermore, although PD-L1 was seemly constitutively expressed by NC, ITM, and CL groups regardless of disease status, it was noted that PD-L1 was dominantly expressed in the CL group (P < .05). CONCLUSION: Given distinct PD-L1 expression, a shift of CL/NC in newly emerged HAP would constitute an inhibitory anti-pathogen immune response. Normalization of circulating monocyte subsets on recurrence of HAP might be the consequence of immune memory of bacterial infection.

4-1BB Agonism Combined With PD-L1 Blockade Increases the Number of Tissue-Resident CD8+ T Cells and Facilitates Tumor Abrogation.

Although the milestone discovery of immune checkpoint blockade (ICB) has been translated into clinical practice, only a fraction of patients can benefit from it with durable responses and subsequent long-term survival. Here, we tested the anti-tumor effect of combining PD-L1 blockade with 4-1BB costimulation in 3LL and 4T1.2 murine tumor models. Dual treatment induced further tumor regression and enhanced survival in tumor-bearing mice more so than PD-L1 and 4-1BB mAb alone. It was demonstrated that dual anti-PD-L1/anti-4-1BB immunotherapy increased the number of intratumoral CD103+CD8+ T cells and altered their distribution. Phenotypically, CD103+CD8+ T cells expressed a higher level of 4-1BB and PD-1 than their CD103- counterparts. Administration of PD-L1 mAb and 4-1BB mAb further increased the cytolytic capacity of CD103+CD8+ T cells. In vivo, CD103-CD8+ T cells could differentiate into CD103+CD8+ progeny cells. In a human setting, more CD8+ T cells differentiated into CD103+CD8+ T cells in the peripheral tumor region of lung cancer tissues than in the central tumor region. Collectively, infiltrated CD103+CD8+ T cells served as a potential effector T cell population. Combining 4-1BB agonism with PD-L1 blockade could increase tumor-infiltrated CD103+CD8+T cells, thereby facilitating tumor regression.

Investigating the role of circulating CXCR5-expressing CD8+ T-cells as a biomarker for bacterial infection in subjects with pneumonia.

BACKGROUND: Recently, lymphoid follicle-confined and circulating CD8+ T-cells expressing the C-X-C chemokine receptor type 5 (CXCR5) were described, which was involved in anti-virus immune response. However, the dynamics and role of circulating CXCR5-expressing CD8+ T-cells during bacterial infection is unknown. So, we asked whether CXCR5+ CD8+ T cells were also generated during bacterial infections in lower respiratory tract. METHODS: The clinical data of 65 pneumonia patients were analyzed. The patients were divided into groups as tuberculosis, bronchiectasis and community or hospital acquired pneumonia (CAP, HAP). The sputum/bronchial secretion or bronchoalveolar lavage fluid (BALF) samples were taken for microbiological examination. The procalcitonin (PCT) was used to evaluate disease severity of these groups and compared among patients. We characterized the number and phenotype (PD-1 and CD103) of CXCR5 + CD8+ T cells in the peripheral circulation by flow cytometry in all individuals and analyzed their association with the serum PCT level and disease severity. RESULTS: Patients were mainly infected with Escherichia coli, Acinetobacter baumannii, Klebsiella pneumonia (K.p), Pseudomonas aeruginosa, and Staphylococcus aureus. Of note is the finding that PCT was weakly correlated with severity of respiratory infections. Furthermore, it was revealed an increase of CXCR5-expressing CD8+ T cells in peripheral blood of un-controlled CAP and progressive HAP compared controlled CAP and HAP, respectively (P < 0.05). Strikingly, the circulating CXCR5-expressing CD8+ T-cells in K.p-infected group was higher than that non-K.p-infected group (P < 0.05). Meanwhile, the ratio of CXCR5 + CD8+/CD8 was positively correlated with PCT level (P < 0.05). In clinic, the determination of CXCR5-expressing CD8+ T-cells showed better results compared to PCT and can be useful for the prediction of exacerbation of CAP or HAP. Phenotypically, CXCR5+ CD8 + T cell expressed comparable level of inhibitory molecules PD-1 and lower CD103 compared to their CXCR5- counterparts. CONCLUSION: The circulating CXCR5-expressing CD8+ T-cell has diagnostic value for current pneumonia severity, and could act as a biomarker for identifying a bacteria-associated exacerbation. These cells may provide novel insight for the pathogenesis of pneumonia.

Membranous and Cytoplasmic Expression of PD-L1 in Ovarian Cancer Cells.

BACKGROUND: Expression of programmed death-ligand 1 (PD-L1) on tumor cells represents a powerful immune evasion pathway, but the role of intracellular or cytoplasmic PD-L1 has not been investigated in ovarian cancer cells. METHODS: Flow cytometry (FCM), Real-time PCR (qPCR), immunohistochemistry (IHC) and western blot were used to determine the expression of PD-L1 in ovarian cancer cells. The cytokines detected in the tumor or tumor associated macrophage (TAM) were used to treat cancer cells. PD-L1 blockade and silencing were used to elucidate the functional significance of cancer-related PD-L1 expression. RESULTS: Based on the results presented, PD-L1 was found variably expressed in the cytoplasm and the cell surface of both HO8910 and SKOV3 cells. TAM or IFN-γ, TNF-α, IL-10 and IL-6 released from TAM stimulated the expression of PD-L1 at the surface of the cancer cells. The IHC results were consistent with the data in vitro showing infiltration of TAM correlated with membranous PD-L1. The increases of PD-L1 at the surface were not due to a shift in the proportion of surface versus intracellular protein, but the contribution of extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide 3-kinase (PI3K) pathway activation. As a consequence, inducible membranous PD-L1 expression on SKOV3 inhibited CD8+ T cell function, and cytoplasmic PD-L1 promoted cancer cell growth. Additionally, in mouse models, both PD-L1 and PD-1 mAb resulted in tumor growth inhibition and demonstrated a potential to decrease the number of PD-1+CD8+T cells. CONCLUSION: We conclude that TAM induced PD-L1 on the cancer cells represents an immune evasion mechanism. The observations confirm the therapeutic potential of PD-L1/PD-1 mAb to reactivate anti-tumor immunity in ovarian cancer.

Analysis of B7-H4 expression in metastatic pleural adenocarcinoma and therapeutic potential of its antagonists.

BACKGROUND: The increasing incidence and poor outcome associated with malignant pleural effusion (MPE) requires finding an effective treatment for this disease. Inhibitory B7-H4 is expressed in many different human cancers but its role in malignant pleural tissue has yet to be established. METHODS: Here, patients with metastatic pleural adenocarcinoma (MPA) or with early-stage lung adenocarcinoma were clinically and statistically analyzed. Immunohistochemistry and confocal microscopy were used to determinate the expression of B7-H4 in the cancer cells. By using MPE model, we sought to a potential immunotherapy for MPE with anti-B7-H4 mAb. RESULTS: When compared to early-stage lung adenocarcinoma, MPA possessed higher level of nuclei membranous B7-H4 and lower cytoplasmic B7-H4 expression. Also, nuclei membranous B7-H4 expression was found to be positively correlated to Ki-67 expression, and indicated a possible poor prognosis of MPA. In mouse MPE model, intra-pleurally injection of anti-B7-H4 mAb effectively suppressed MPE formation. CONCLUSIONS: Taken together, our data was in support of the significance of B7-H4 expression in MPA, which also suggest it warrants further exploration for potential immunotherapy of MPE.

The increase of circulating PD-L1-expressing CD68(+) macrophage in ovarian cancer.

Tumor-associated macrophages (TAMs) have been characterized as a critical population of immunosuppressive cells in a variety of tumor types. PD-L1 (also termed B7-H1) has been described to exert co-inhibitory and immune regulatory functions. Here, in ovarian cancer, PD-L1 is selectively overexpressed on some TAM compared that of benign ovarian disease. When expanding the data in peripheral blood, the proportion of PD-L1(+)CD68(+) cell among CD68(+) cells and the intensity of PD-L1 staining on CD68(+) cell in healthy group were similar to that observed in ovarian cyst group; instead, these two measures were significantly higher in ovarian cancer group, thereafter related to TNM stage. Interestingly, intracellular levels of IL-10, IL-6, TNF-α, and IFN-γ in PD-L1(+)CD68(+) macrophage were higher than those in PD-L1(-)CD68(+) macrophage, especially IL-6 expression. Based on the PD-L1 receptor PD-1 expression on tumor-infiltrating cytotoxic cells, our data supported that expression of PD-L1 on TAM promoted apoptosis of T cells via interaction with PD-1 on CD8(+)T cells. Taken together, these results suggested that PD-L1-expressing macrophage represents a novel suppressor cell population in ovarian cancer, which contributes immune escape of ovarian cancer.

Soluble CD40 in plasma and malignant pleural effusion with non-small cell lung cancer: A potential marker of prognosis.

OBJECTIVE: Soluble CD40 (sCD40) is a potential modulator for both antitumor responses and CD40-based immunotherapy; however the levels and significance of sCD40 in non-small cell lung cancer (NSCLC) patients with malignant pleural effusion are unknown. METHODS: Forty-eight patients with lung cancer were treated in our institutions from January 2008 to January 2010. Peripheral blood and pleural effusion samples were collected from each subject. sCD40 levels in plasma and malignant pleural effusions supernatant were measured. The CD40L expression on CD3t T-cells was confirmed by flow cytometric direct immunofluorescence analysis. All patients were followed up after the study ended on January 1, 2010. RESULTS: Patients with malignant pleural effusion of NSCLC had elevated circulating and pleural effusion levels of sCD40, and these elevated sCD40 levels were associated with advanced diseases and a poor prognosis. CONCLUSIONS: These findings indicate that elevated sCD40 may have a role in modulating antitumor responses and may also be a useful prognostic marker.

[Preliminary study of breast cancer magnetic resonance targeting diagnosis basing on magnetic nanoparticles in vitro].

OBJECTIVE: To develop a magnetic nanoparticles based magnetic resonance (MR) probe targeting CD40 mutant in the imaging of breast cancer cells in vitro. METHODS: For preparing an immunologically competent probe, monoclonal antibody was conjugated with ultrasmall superparamagnetic iron oxide (USPIO) particles basing on chemical cross-linking method.Its bioactivity was analyzed with flow cytometry, confocal microscopy and Prussian blue staining. The probe's cell MR imaging in vitro was conducted on breast cancer cells (M231) high expressing CD40 mutant. The signal data from different groups were collected and analyzed with one-way variance and least significant difference-t test. RESULTS: The molecular probe carrying nanoparticles and CD40 mutant antibody was constructed and separated successfully. The probe had similar magnetic property compared with original USPIO particles.It could recognize CD40 mutant on breast cancer cells (M231) with high specificity. MR cell imaging in vitro shows that T2 and T2(*) obviously shortened after probe binding with M231 cells and T2 weighted imaging become darker than control groups, the time of T2 is 5H6-USPIO (51.66 ± 5.31) , 5C11-USPIO (92.89 ± 4.72), USPIO (64.56 ± 3.85) ms. The T2 and T2(*) relaxation time of experiment group was shorter than control groups with statistical significance (P < 0.01) . CONCLUSION: MR molecular probe targeting CD40 mutant may bind with breast cancer cells (M231) to provide further in vivo animal MR imaging. And CD40 mutant is expected to provide a new target for MR molecular imaging of breast cancer.

CD40 signal regulates CXCR4 mediating ovarian carcinoma cell migration: implications for extrapelvic metastastic factors.

Ovarian carcinomas are highly invasive, especially in the peritoneal cavity. SDF-1α and its receptor, CXCR4, play a crucial role in migration of cancer cells. Here, SDF-1α directed HO8910 cell migration, but not SKOV3 cells. After being educated to express CXCR4 in vivo or by treating with sCD40L, SDF-1α reexhibited the ability of directing SKOV3 cell migration, which could be antagonized by CXCR4-neutralizing antibody. Furthermore, concomitant expression of CXCR4/CD40 in ovarian carcinoma tissues had stronger correlation with pelvic metastasis than did each alone. It is suggest that SDF-1α acts through CXCR4 to induce ovarian cancer cell migration, which could be facilitated by CD40 activation. Simultaneously examining the expression of CXCR4 and CD40 will provide valuable diagnosis of pelvic metastasis for ovarian carcinomas.

Induced expression of B7-H3 on the lung cancer cells and macrophages suppresses T-cell mediating anti-tumor immune response.

Macrophages are the prominent components of solid tumors and have complex dual functions in their interaction with cancer cells. Strong evidence suggests that TAM is a part of inflammatory circuits that promote tumor progression. B7-homologue 3 (B7-H3), a recently identified homologue of B7.1/2 (CD80/86), has been described to exert co-stimulatory and immune regulatory functions. Here, we showed that a fraction of macrophages in tumor stroma expressed surface B7-H3 molecule. Normal macrophages, which did not express B7-H3, would be induced expressing B7-H3 molecule when culturing with tumor cell. Although a lung cancer cell line constitutively expressed B7-H3 mRNA and protein in plasma, primary tumor cell isolated from the transplanted lung carcinoma model expressed B7-H3 on the surface. Interestingly, in transplanted lung carcinoma model, the expression of membrane-bound B7-H3 in tumor cells was increased as prolonging of tumor transformation. In support, IL-10 released from TAM could stimulate cancer cell expression of membrane bound B7-H3. Furthermore, Lung cancer and TAM-related B7-H3 was identified as a strong inhibitor of T-cell effect and influenced the outcome of T cell immune response. In conclusion, TAM-tumor cell interaction-induced membrane-bound B7-H3 represents a novel immune escape mechanism which links the pro-inflammatory response to immune tolerance in the tumor milieu.

[Construction of eukaryotic expression vector of muCD40-IgG1Fc fusion protein and its expression in CHO cells].

AIM: To construct an eukaryotic expression vector of human muCD40Ig fusion protein, and to express it stably in Chinese hamster ovary (CHO) cells for obtaining muCD40Ig fusion protein and founding an experimental basis for investigating the soluble muCD40 molecule in vivo. METHODS: Extracellular domain of human muCD40 gene was amplified by RT-PCR from L929/muCD40-transfected cells, and the genes encoding the constant regions of human IgG1 were cloned from human splenocytes. The genes were inserted into eukaryotic expression vector pIRES2-EGFP, respectively. The recombinant vector was transfected into CHO cells by Superfectin. The transfected cells stably secreting muCD40Ig fusion protein was selected with G418 and subcloned. The serum-free culture supernatant of the selected positive clone was subjected to Western blotting and RT-PCR to confirm the expression of the fusion gene. The affinity of muCD40Ig and L929/CD40L was analyzed by flow cytometry (FCM). RESULTS: The eukaryotic expression vector pIRES2-EGFP/muCD40Ig was constructed successfully. PCR and Western blotting showed that the transfected CHO cell strain was able to secret muCD40Ig fusion protein stably. FCM demonstrated a good affinity between muCD40Ig and L929/CD40L. CONCLUSION: A transfected CHO cell strain stably expressing muCD40Ig fusion protein has been obtained, and the muCD40Ig fusion protein can bind to CD40L.

Analysis of CD8+CD28- T-suppressor cells in gastric cancer patients.

Host anti-tumor immune responses can be attenuated by suppressor T cells of the phenotype CD8(+)CD28(-) (T(s) cells). In the present study, we investigated the presence of CD8(+)CD28(-) (T(s) cells) in the peripheral blood compartment of gastric cancer (GC) patients. Flow cytometry was used to detect the population of CD8(+)CD28(-) T(s) cells present in peripheral blood in therapy naïve patients with gastric cancer (n = 26), postoperative chemotherapy naïve gastric cancer patients (n = 23), and healthy controls (n = 27). Meanwhile, the clinical data of gastric cancer patients were analyzed. A significant difference in the percentage of T(s) cells was observed when comparing peripheral blood samples from cancer patients to healthy volunteers (27.08 ± 1.60% versus 10.86 ± 0.75%). In the patient group, the percentage of CD8(+)CD28(-) cells among lymphocytes was higher in patients with LN metastasis than those without LN metastasis. The percentage of CD8(+)CD28(-) cells was also related to tumor infiltration and size, but not with the degree of differentiation of cancer cells. Moreover, the percentage of CD8(+)CD28(-) cells was higher in preoperative gastric cancer patients (26.24 ± 1.78%) than in those of postoperation patients (15.79 ± 1.11%). These findings may reflect the possibility of tumor-induced immunosuppression, and they should be complemented with further studies.

Induced expression of B7-H4 on the surface of lung cancer cell by the tumor-associated macrophages: a potential mechanism of immune escape.

B7-homolog 4 (B7-H4), a recently identified homolog of B7.1/2 (CD80/86), has been described to exert co-stimulatory and immune regulatory functions. We investigated the expression and the functional activity of B7-H4 in lung cancer in vitro and in vivo. Although a lung cancer cell line constitutively expressed B7-H4 mRNA and protein in plasma, primary tumor cell isolated from the transplanted lung carcinoma model expressed B7-H4 on the surface. Interestingly, in transplanted lung carcinoma model, the expression of membrane-bound B7-H4 in tumor cells was increased as prolonging of tumor transformation. Exposure to tumor-associated macrophages strongly induced membrane-bound B7-H4 expression on the lung cancer cell line. To elucidate the functional significance of lung cancer-related B7-H4 expression, we performed co-culture experiments of lung cancer cell with allo-reactive T cells. Lung cancer-related B7-H4 was identified as a strong inhibitor of T-cell effect. Furthermore, B7-H4 mAb had an ability to inhibit tumor growth in vivo. B7-H4 expression may thus significantly influence the outcome of T-cell tumor cell interactions and TAM induced membrane-bound B7-H4 on the lung cancer cell represents a novel mechanism by which lung cancer cells evade immune recognition and destruction.

Overexpression of B7-H4 in tumor infiltrated dendritic cells.

DCs infiltrated tumors appears to be phenotypically and functionally defective. B7-H4 was highlighted for its inhibitory role in T cell responses. In this study, we showed that B7-H4 was moderately expressed in imDCs, and up-regulated by IL-10, and TNF-α could counteract the up-regulatory effects of IL-10 on expression of B7-H4 in DCs in vitro. Furthermore, tumor infiltrated DCs expressed B7-H4 at high levels. Blockade of B7-H4 expressed in DCs highly resulted in enhanced T cell proliferation and IFN-γ production significantly. Otherwise, the high level of IL-10 and TNF-α was both detected in the tumor, which suggested that TNF-α can not antagonize the effects of IL-10 on expression of B7-H4 in DCs in vivo. These data indicate that tumor environment may condition local DCs to become dysfunctional in the phenotype, and that the high expression of B7-H4 may contribute to the tumor infiltrated DCs to mediate immune invasion.

CD40 is overexpressed by HPV16/18-E6 positive cervical carcinoma and correlated with clinical parameters and vascular density.

CD40 is expressed in many tumor cells, however, its role in tumor biology is yet to be demonstrated. In the present study, we investigated the role of CD40 in cervical carcinoma. In vivo, we evaluated CD40 expression in 56 cervical carcinoma tissues, 43 cervicitis and 38 normal cervix, and investigated the relationship between CD40 and HPV antigen, histopathological parameters, vascular density, and vascular endothelial growth factor (VEGF) expressions. The results clearly demonstrated that CD40 expression, including membranous and cytoplasmic staining, was significantly higher in cervical carcinoma than in the cervicitis and normal cervix. The expression of CD40 was significantly correlated with HPV and VEGF expressions and microvessel density (MVD). These observations provide evidence that CD40 may be involved in neovascularization of cervical carcinoma, they also suggest that CD40 and VEGF may be useful biomarkers for evaluating the risk of developing cervical carcinoma, and may also be used as a target for therapy.

Sensitization of SiHa cell to gemcitabine by CD40 activation and its overexpression in cervical carcinoma.

CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed on various cell types. Some studies show that CD40 expression is related to several carcinomas, where its function remains largely unknown. This study investigated the expression of CD40 on cervical carcinoma and evaluated the effect of agnostic anti-CD40 mAb (5C11) on tumor cell line (SiHa). CD40 expression on the primary cervical carcinoma samples was detected by immunohistochemistry. Results showed that CD40 is commonly expressed in human cervical carcinoma, which is higher than that of normal cervix, cervical precancerous tissue and chronic cervicitis. Treatment of the SiHa cell with the agonistic anti-CD40 monoclonal antibody or Gemcitabine alone did not inhibit the proliferation of the SiHa cell in vitro, but the activation of CD40 on SiHa could enhance its sensitivity to Gemcitabine. Furthermore, CD40 activation blocked SiHa in the S phase, stimulated proapoptotic Bax and inhibited antiapoptotic Bcl-XL mRNA synthesis in the SiHa cell. Apoptosis in SiHa was associated with an increasing ratio of Bax to Bcl-XL in mRNA levels. It is concluded that use of the anti-CD40 mAb 5C11 in combination with chemotherapy may have significant therapeutic potential.

Preparation and characterization of a novel chimeric antibody against human CD40 with the potential to inhibit Daudi cell proliferation.

5C11, a murine monoclonal antibody with a high specificity for human CD40 molecule, is a promising candidate for cancer targeting therapy. We have therefore attempted to construct a humanized antibody of 5C11 to minimize its immunogenicity for potential clinical use. A chimeric version of 5C11 (ch-5C11) was generated by transferring these mouse variable regions onto a human framework. This chimeric antibody retained reactivity to human CD40. In vitro, ch-5C11 could effectively inhibit B lymphoma Daudi cell proliferation, suggesting that it might have the potential to be developed for future clinical use.

CD40-activated apoptotic tumor cell-pulsed dendritic cell could potentially elicit antitumor immune response: involvement of up-regulation of B7-H3 expression.

Dendritic cells (DCs) initiate and direct immune responses. Previous in vitro and in vivo studies have showed that DCs matured with CD40 linking signal could potentially elicit and boost antitumor immunity, however, its molecular mechanism remain elusive. This study demonstrates that expression of B7-H3 on apoptotic cell-loading DCs is up-regulated markedly by CD40 activation but not by tumor necrosis factor-alpha stimulation. There was no significant difference found with CD40, CD80, or CD86 expressions when activated by CD40 or tumor necrosis factor-alpha stimulation. In tumor-bearing mice, T cells conditioned with B7-H3-blocked on CD40-activated apoptotic tumor cell-pulsed DCs had a decreased ability to inhibit tumor growth. Therefore, it is hypothesized that high levels of B7-H3 expression contributes to the ability of CD40-activated tumor associated DCs in eliciting efficient antitumor immune response, given this fact the potentially significant clinical implications, CD40-activated DCs merit further considerations when preparing DCs for clinical application.