Dysfunctional BLK in common variable immunodeficiency perturbs B-cell proliferation and ability to elicit antigen-specific CD4+ T-cell help

Common Variable Immunodeficiency (CVID) is the most prevalent primary antibody deficiency, and characterized by defective generation of high-affinity antibodies. Patients have therefore increased risk to recurrent infections of the respiratory and intestinal tract. Development of high-affinity antigen-specific antibodies involves two key actions of B-cell receptors (BCR): transmembrane signaling through BCR-complexes to induce B-cell differentiation and proliferation, and BCR-mediated antigen internalization for class-II MHC-mediated presentation to acquire antigen-specific CD4⁺ T-cell help.We identified a variant (L3P) in the B-lymphoid tyrosine kinase (BLK) gene of 2 related CVID-patients, which was absent in healthy relatives. BLK belongs to the Src-kinases family and involved in BCR-signaling. Here, we sought to clarify BLK function in healthy human B-cells and its association to CVID.BLK expression was comparable in patient and healthy B-cells. Functional analysis of L3P-BLK showed reduced BCR crosslinking-induced Syk phosphorylation and proliferation, in both primary B-cells and B-LCLs. B-cells expressing L3P-BLK showed accelerated destruction of BCR-internalized antigen and reduced ability to elicit CD40L-expression on antigen-specific CD4⁺ T-cells.In conclusion, we found a novel BLK gene variant in CVID-patients that causes suppressed B-cell proliferation and reduced ability of B-cells to elicit antigen-specific CD4⁺ T-cell responses. Both these mechanisms may contribute to hypogammaglobulinemia in CVID-patients.     

Cancer Cell-Specific Major Histocompatibility Complex II Expression as a Determinant of the Immune Infiltrate Organization and Function in the NSCLC Tumor Microenvironment

IntroductionIn patients with NSCLC, the prognostic significance of the tumor microenvironment (TME) immune composition has been revealed using single- or dual-marker staining on sequential tissue sections. Although these studies reveal that relative abundance and localization of immune cells are important parameters, deeper analyses of the NSCLC TME are necessary to refine the potential application of these findings to clinical care. Currently, the complex spatial relationships between cells of the NSCLC TME and potential drivers contributing to its immunologic composition remain unknown.MethodsWe used multispectral quantitative imaging on the lung adenocarcinoma TME in 153 patients with resected tumors. On a single slide per patient, we evaluated the TME with markers for CD3, CD8, CD14, CD19, major histocompatibility complex II (MHCII), cytokeratin, and 4′,6-diamidino-2-phenylindole (DAPI). Image analysis, including tissue segmentation, phenotyping, and spatial localization, was performed.ResultsSpecimens wherein greater than or equal to 5% of lung cancer cells expressed MHCII (MHCII^hi TME) had increased levels of CD4⁺ and CD8⁺ T cells and CD14⁺ cell infiltration. In the MHCII^hi TME, the immune infiltrate was closer to cancer cells and expressed an activated phenotype. Morphologic image analysis revealed cancer cells in the MHCII^hi TME more frequently interfaced with CD4⁺ and CD8⁺ T cells. Patients with an MHCII^hi TME experienced improved overall survival (p = 0.046).ConclusionsLung cancer cell-specific expression of MHCII associates with levels of immune cell infiltration, spatial localization, and activation status within the TME. This suggests that cancer cell-specific expression of MHCII may represent a biomarker for the immune system’s recognition and activation against the tumor.     

Celecoxib promotes the efficacy of STING-targeted therapy by increasing antitumor CD8+ T-cell functions via modulating glucose metabolism of CD11b+Ly6G+ cells

Recent studies have shown that activation of the cGAS-STING pathway is a key process in antitumor immune responses and various kinds of STING agonists have been developed for cancer immunotherapy. Despite promising preclinical studies, preliminary clinical results have shown only a modest effect of STING agonists. There is therefore a need to develop more effective treatment strategies. Based on previous observations that COX-2 is frequently overexpressed not only in a variety of cancers but also in tumor myeloid cells and that it suppresses antitumor immunity and promotes tumor survival by producing PGE2, we investigated the antitumor effects of combination therapy with a STING agonist cGAMP and the selective COX-2 inhibitor celecoxib in mouse models. Combination treatment with cGAMP and celecoxib inhibited tumor growth compared with either monotherapy, and the combination therapy induced both local and systemic antitumor immunity. cGAMP treatment decreased PD-1 expression on tumor-infiltrating T-cells and enhanced T-cell activation in tumor-draining lymph nodes regardless of the presence of celecoxib. Meanwhile, although celecoxib treatment did not alter the frequency of CD4⁺CD25⁺Foxp3⁺ regulatory T-cells, it enhanced the expression of costimulatory molecules and glycolysis-associated genes in tumor-infiltrating CD11b⁺Ly6G⁺ cells. Moreover, we also found that celecoxib decreased lactate efflux and increased the frequency of IFN-γ- and TNF-α-producing CD8⁺ T-cells in the tumor microenvironment. Taken together, our findings suggest that combined treatment with celecoxib may be an effective strategy to improve the antitumor efficacy of STING agonists.     

Tumor-infiltrating gamma delta T-cells reveal exhausted subsets with remarkable heterogeneity in colorectal cancer

The γδT-cells recognize infected or transformed cells. However, unlike αβT-cells, γδT-cells are innate-like immune cells, with no major histocompatibility complex restriction requirements. γδT-cells are the main population of intestinal intraepithelial lymphocytes (IELs) and are associated with the antitumor immune response, particularly in colorectal cancer (CRC). Although CD8⁺T-cells exhibit dysfunction and even exhaustion in the tumor microenvironment (TME), which contributes to tumor immune escape, whether the same applies to tumor-infiltrating (TI)-γδT-cells is not completely understood. Here, we sought to investigate the expression pattern of inhibitory receptors and functional state of TI-γδT-cells, and reveal the features of exhausted TI-γδT-cells in the CRC TME. We demonstrated that TI-γδT-cells exhibited exhaustion phenotypes and displayed more severe functional exhaustion than TI-CD8⁺T-cells or NK-cells in the TME of CRC. In addition, scRNA-seq analysis of TI-γδT-cells revealed three exhausted subsets with remarkable heterogeneity. The presence of three heterogeneous exhausted γδT-cell (Tex) populations, including Tex^prog, Tex^tran and Tex^term were further confirmed by flow cytometry, on the basis of PD-1 and TIM-3 expression. Finally, we revealed that c-Maf not only contributed to γδT-cell exhaustion via upregulation of inhibitory receptors, but also involved in the exhaustion of CD8⁺T and NK-cells. c-Maf may also be an important contributor to γδT-cell exhaustion in CRC patients. These findings indicated that TI-γδT-cells exhibit phenotypic and functional exhaustion in the CRC TME. The revealed features of exhausted TI-γδT-cells may provide help for understanding the mechanisms and the association of γδT-cell exhaustion with tumor development and pathogenesis.     

Suppression by sorted CD8+CD11b− cells from T-cell growth factor-activated peripheral blood lymphocytes on cytolytic activity against tumour in patients with gastric carcinoma

To confirm the phenotypic characteristics of lymphokine-activated suppressor (LAS)effector cells, we isolated CD8⁺CD11b⁻ and CD8⁻CD11b⁻ cells from T-cell growth factor (TCGF)-activated peripheral blood lymphocytes (PBL) in 7 patients with gastric carcinoma (4 non-resectable and 3 resectable carcinoma) and 3 healthy controls. Sorted CD8⁺CD11b⁻ cells from 3 of the patients with non-resectable carcinoma and from 1 of the patients with resectable carcinoma showed LAS cell activity. However, the LAS cell activity could not be observed in CD8⁺CD11b⁻ cells from healthy controls. In addition, a sorted CD8⁻CD11b⁻ subset of cells from both cancer patients and control did not express any suppressive activity. These facts clearly show that the cell populations responsible for suppression of cell-mediated antitumour immunity reside within CD8⁺CD11b⁻ T-cells, at least in patients with advanced carcinoma.     

PD‐1 expression on peripheral blood T‐cell subsets correlates with prognosis in non‐small cell lung cancer

PD-1 expression in peripheral blood T-cells has been reported in several kinds of cancers, including lung cancer. However, the relationship between PD-1 expression in peripheral blood T-cells and prognosis after treatment with a cancer vaccine has not been reported. To elucidate this relationship, we analyzed PD-1 expression in the peripheral blood T-cells of patients with non-small cell lung cancer. The blood samples used in this study were obtained from patients enrolled in phase II clinical trials of a personalized peptide vaccine. Seventy-eight samples obtained before and after a single vaccination cycle (consisting of six or eight doses) were subjected to the analysis. PD-1 was expressed on lymphocytes in the majority of samples. The relative contents of PD1⁺CD4⁺ T-cells against total lymphocytes before and after the vaccination cycle correlated with overall survival (OS) with a high degree of statistical significance (P < 0.0001 and P = 0.0014). A decrease in PD-1⁺CD8⁺ T-cells after one cycle of vaccination also correlated with longer OS (P = 0.032). The IgG response to the non-vaccinated peptides suggested that the epitope spreading seemed to occur more frequently in high-PD-1⁺CD4⁺ T-cell groups. Enrichment of CD45RA⁻CCR7⁻ effector-memory phenotype cells in PD-1⁺ T-cells in PBMCs was also shown. These results suggest that PD-1 expression on the peripheral blood T-cell subsets can become a new prognostic marker in non-small cell lung cancer patients treated with personalized peptide vaccination.     

Clinical relevance of PD-1 positive CD8 T-cells in gastric cancer

Background

We evaluated the relevance of PD-1⁺CD8⁺ T-cells in gastric cancer (GC) including prognostic significance, association with chemotherapy and immunotherapy sensitivity and correlations with the tumor microenvironment (TME).

Methods

Discovery cohort: GC samples were evaluated for AE1/3, CD8, PD-1, Ki-67 and Granzyme-B expression with fluorescence-based multiplex immunohistochemistry (mIHC). Validation cohorts: we analyzed bulk RNAseq GC datasets from TCGA, the “3G” chemotherapy trial and an immunotherapy phase 2 trial. The cox proportional hazards model was used to identify factors that influenced overall survival (OS). To study the TME, we analyzed single-cell RNAseq performed on GCs.

Results

In the discovery cohort of 350 GCs, increased PD-1 expression of CD8 T-cells was prognostic for OS (HR 0.822, p = 0.042). PD-1 expression in CD8 T-cells highly correlated with cytolytic [Granzyme-B⁺] (r = 0.714, p < 0.001) and proliferative [Ki-67⁺] (r = 0.798, p < 0.001) activity. Analysis of bulk RNAseq datasets showed tumors with high PD-1 and CD8A expression levels had improved OS when treated with immunotherapy (HR 0.117, p = 0.036) and chemotherapy (HR 0.475, p = 0.017). Analysis of an scRNAseq dataset of 152,423 cells from 40 GCs revealed that T-cell and NK-cell proportions were higher (24% vs 18% and 19% vs 15%, p < 0.0001), while macrophage proportions were lower (7% vs 11%, p < 0.0001) in CD8PD-1_high compared to CD8PD-1_low tumors.

Conclusion

This is one of the largest GC cohorts of mIHC combined with analysis of multiple datasets providing orthogonal validation of the clinical relevance of PD-1⁺CD8⁺ T-cells being associated with improved OS. CD8PD-1_high tumors have distinct features of an immunologically active, T-cell inflamed TME.

     

Enhancement of adoptive T cell transfer with single low dose pretreatment of doxorubicin or paclitaxel in mice

Ex vivo expansion of CD8⁺ T-cells has been a hindrance for the success of adoptive T cell transfer in clinic. Currently, preconditioning with chemotherapy is used to modulate the patient immunity before ACT, however, the tumor microenvironment beneficial for transferring T cells may also be damaged. Here preconditioning with single low dose of doxorubicin or paclitaxel combined with fewer CD8⁺ T-cells was investigated to verify whether the same therapeutic efficacy of ACT could be achieved. An E.G7/OT1 animal model that involved adoptive transfer of OVA-specific CD8⁺ T-cells transduced with a granzyme B promoter-driven firefly luciferase and tomato fluorescent fusion reporter gene was used to evaluate this strategy. The result showed that CD8⁺ T-cells were activated and sustained longer in mice pretreated with one low-dose Dox or Tax. Enhanced therapeutic efficacy was found in Dox or Tax combined with 2×10⁶ CD8⁺ T-cells and achieved the same level of tumor growth inhibition as that of 5×10⁶ CD8⁺ T-cells group. Notably, reduced numbers of Tregs and myeloid derived suppressor cells were shown in combination groups. By contrast, the number of tumor-infiltrating cytotoxic T lymphocytes and IL-12 were increased. The NF-κB activity and immunosuppressive factors such as TGF-β, IDO, CCL2, VEGF, CCL22, COX-2 and IL-10 were suppressed. This study demonstrates that preconditioning with single low dose Dox or Tax and combined with two fifth of the original CD8⁺ T-cells could improve the tumor microenvironment via suppression of NF-κB and its related immunosuppressors, and activate more CD8⁺ T-cells which also stay longer.     

Boosting immune surveillance by low-dose PI3K inhibitor facilitates early intervention of breast cancer

Prevention of estrogen receptor-negative (ER-) breast cancer is an unmet challenge, although tamoxifen and aromatase inhibitors can successfully decrease the incidence of ER-positive (ER+) breast cancer. PI3K pathway activation has been detected in tamoxifen-resistant ER- breast lesions of patients. Here, we further ratified that the PI3K pathway is significantly activated in premalignant ER- breast lesions compared with paired normal tissues of patients, which prompted our assessment of targeting PI3K on inhibition of ER- mammary tumor initiation and progression. Both genetic knockdown of PIK3CA or intervention with low-doses of a PI3K inhibitor (GDC-0941) prevented the dysplasia phenotype of semi-transformed human ER- mammary epithelial cells in 3-dimensional culture in vitro. Importantly, low-dose GDC-0941 treatment significantly delayed mammary tumor initiation in the MMTV-neu mouse model without exhibiting discernable adverse effects. Interestingly, increased CD8⁺/GZMB⁺ T-cells were detected in mammary tissue after GDC-0941 treatment, suggesting enhanced immune surveillance. Mechanistically, elevated expression of potent T-cell chemo-attractants, including CCL5 and CXCL10, were detected both in vitro and in vivo after GDC-0941 treatment. Furthermore, inhibition of PI3K significantly increased T-cell recruitment in a CCL5/CXCL10-dependent manner. In human ER- breast cancer, PI3K activation is correlated with significantly reduced CCL5, CXCL10 and CD8A expression, suggesting that the decreased CD8⁺ T-cell recruitment and escape of immune surveillance may contribute to ER- breast cancer development. In summary, our study indicates that low-dose PI3K inhibitor treatment may intervene early stage ER- breast cancer development by enhancing immune surveillance via CCL5/CXCL10.     

In vitro Induction of Cytotoxic T Lymphocytes against HTLV-I-infected T-Cells from Adult T-Cell Leukemia Patients,Asymptomatic HTLV-I Carriers and Seronegative Healthy Donors

We investigated an in vitro method to produce cytotoxic T lymphocytes (CTLs) against HTLV-I-infected T-cells using peripheral blood mononuclear cells (PBMC) of adult T-cell leukemia (ATL) patients, asymptomatic HTLV-I carriers (AC) and seronegative healthy donors. The PBMC were restimulated repeatedly for 4 weeks with HLA-matched HTLV-I-infected T-cells which had been pretreated at 56deg;C for 30 min to inactivate infectious HTLV-I. The culture medium included 10–100 units/ml of recombinant lymphokines (rIL-1, rIL-2, rIL-4, rIL-6 and rIL-7) and 10% fetal calf serum in RPMI-1640 medium. The cytotoxic activity was measured against HLA-matched HTLV-I-infected T-cell lines after CD4⁺ or CD8⁺ cells were positively panned from the cultured PBMC. The PBMC of ATL, AC and healthy donors were able to produce either CD4⁺ or CD8⁺ CTLs against HTLV-I-related antigens (env, gag, p21^x, p27^rex and p40^tax) as well as the antigen(s) of as-yet unknown specificity expressed on HTLV-I-infected T-cells. All the CTLs recognized the specific antigens in the context of either class I or class II HLA types. These results indicated that ATL patients, AC and healthy donors were immunocompetent to generate CTLs against HTLV-I-infected T-cells and probably against HTLV-I-transformed T-cells.     

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.     

Colibactin-positive Escherichia coli induce a procarcinogenic immune environment leading to immunotherapy resistance in colorectal cancer

Colibactin-producing E. coli (CoPEC) are frequently detected in colorectal cancer (CRC) and exhibit procarcinogenic properties. Because increasing evidence show the role of immune environment and especially of antitumor T-cells in CRC development, we investigated the impact of CoPEC on these cells in human CRC and in the APC^Min/+ mice colon. T-cell density was evaluated by immunohistochemistry in human tumors known for their CoPEC status. APC^min/+ mice were chronically infected with a CoPEC strain (11G5). Immune cells (neutrophils and T-cell populations) were then quantified by immunofluorescent staining of the colon. The quantification of lymphoid populations was also performed in the mesenteric lymph nodes (MLNs). Here, we show that the colonization of CRC patients by CoPEC is associated with a decrease of tumor-infiltrating T lymphocytes (CD3⁺ T-cells). Similarly, we demonstrated, in mice, that CoPEC chronic infection decreases CD3⁺ and CD8⁺ T-cells and increases colonic inflammation. In addition, we noticed a significant decrease in antitumor T-cells in the MLNs of CoPEC-infected mice compared to that of controls. Moreover, we show that CoPEC infection decreases the antimouse PD-1 immunotherapy efficacy in MC38 tumor model. Our findings suggest that CoPEC could promote a procarcinogenic immune environment through impairment of antitumor T-cell response, leading to tumoral resistance to immunotherapy. CoPEC could thus be a new biomarker predicting the anti-PD-1 response in CRC.     

Mechanisms of immune suppression in patients with head and neck cancer: Influence on the immune infiltrate of the cancer

Freshly excised human head and neck cancers (219 primary cancers; 64 metastatic lymph node cancers) were analyzed for the immune inhibitory mediators released from the cancer tissues and the immune infiltrate within the tumor. Significant levels of the immune inhibitory mediators transforming growth factor-β (TGF-β), prostaglandin E₂ (PGE₂) and interleukin-10 (IL-10) were released from these cancers. Also released was granulocyte-macrophage colony-stimulating factor (GM-CSF), whose secretion was associated with an intratumoral presence of CD34⁺ cells. We have previously shown that CD34⁺ cells within human head and neck cancers are immune inhibitory granulocyte-macrophage progenitor cells. The presence of TGF-β, PGE₂ and IL-10 was associated with a reduced content of CD8⁺ T-cells within the cancers. The CD4⁺ cell content appeared to be less affected by these immune inhibitory mediators. Instead, parameters indicative of CD4⁺ cell function (p55 IL-2 receptor expression, release of IL-2 and IFN-γ) were diminished in cancers that released higher levels of TGF-β, IL-10 and GM-CSF and had a higher CD34⁺ cell content. Furthermore, metastatic cancers released higher levels of the soluble immune inhibitory mediators and lower levels of IFN-γ and IL-2 than did primary cancers, although CD34⁺ cells were similarly present in both primary and metastatic cancers. Our results show that human head and neck cancers have a multiplicity of non-mutually exclusive mechanisms of immune suppression that are most prominently associated with reduced CD8⁺ cell influx and reduced influx and altered function of intratumoral CD4⁺ cells. © 1996 Wiley-Liss, Inc.     

The clinical significance of memory T cells and its subsets in gastric cancer

Background

Long life of memory T cell (Tm) determines its crucial role in the carcinogenesis and carcinogenic progression which usually take long time. The Tm compartment contains two populations, central memory T cells (Tcm) and effector memory T cells (Tem), based on their phenotypic markers, functional attributes, and migratory properties.

Methods

We investigated the subsets of the Tm in peripheral blood and tumor microenvironments in patients with gastric cancer by flow cytometry, and aimed to explore the correlation between the Tm and clinicopathologic features of gastric cancer.

Results

The percentages of CD4⁺/CD8⁺ Tm and CD4⁺/CD8⁺ Tcm in peripheral blood from gastric cancer patients were statistically lower, whereas the percentages of CD4⁺/CD8⁺ Tem were significantly higher than healthy controls. The proportion of CD4⁺/CD8⁺ Tcm increased after tumor resection, while the percentage of the CD4⁺/CD8⁺ Tem decreased significantly. Significant associations were detected between the peripheral CD4⁺/CD8⁺ Tm and clinical stage, as well as the CD8⁺ Tcm and clinical stage and nodal involvement. Tumor infiltrating CD8⁺ Tm expressed both central and effector memory phenotypes, whereas CD4⁺ Tm displayed predominantly an effector memory phenotype. Higher percentages of tumor infiltrating CD4⁺/CD8⁺ Tm were significantly associated with the early disease stage.

Conclusions

Tm and its subsets were good immune indicators for the disease stage of gastric cancer. The proportion of Tm subsets may reflect the immune suppressive and immune response to the tumor associated antigen.     

Frequency of Immune Cell Subtypes in Peripheral Blood Correlates With Outcome for Patients With Metastatic Breast Cancer Treated With High-Dose Chemotherapy

BackgroundThe frequency of circulating leukocytes has been shown to be a prognostic factor in patients being treated for different types of cancer. In breast cancer, tumor-infiltrating leukocytes may predict patient outcome, but few studies have investigated such associations for circulating leukocytes.Patients and MethodsMultiparametric flow cytometry was used to examine the immunophenotypes of circulating peripheral blood mononuclear cells for 88 patients with metastatic breast cancer, which was then correlated to breast cancer–specific survival. Patients had been treated either with high-dose cyclophosphamide-containing regimens (group 1, n = 51 patients) or high-dose paclitaxel-containing regimens (group 2, n = 37 patients).ResultsThe frequency of peripheral blood CD14⁺ monocytes indicated prognosis for patients in group 1 (but not group 2), while higher levels of CD11c⁺ dendritic cells indicated a better prognosis for patients in group 2 (but not group 1). The frequency of a number of different CD4⁺ or CD8⁺ T cell subtypes also predicted prognosis for patients in group 2. For example, patients in group 2 with a higher frequency of circulating CD4⁺ or CD8⁺ naive T cells (CD45RA⁺CD95−CD27⁺CD28⁺) showed a poorer prognosis. In contrast, T cells were not associated with prognosis for patients in group 1.ConclusionCirculating leukocytes can predict clinical outcome for patients with breast cancer. Prediction of clinical outcome in this cohort of metastatic breast cancer patients was specific to the type of chemotherapy, and this finding is likely to apply to other therapies.     

Exosomal NAMPT from chronic lymphocytic leukemia cells orchestrate monocyte survival and phenotype under endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress has been reported to be transmitted from tumor cells to immune cells via exosome and implicated in immune escape. However, the influence of ER stress on monocytes in chronic lymphocytic leukemia (CLL) cells is largely unknown. Here, we observed the expression of ER stress markers (GRP78, ATF6, PERK, IRE1a, and XBP1s) in CLL cells. The increasing mRNA expression of these ER stress response components was positively correlated with more aggressive disease. Exosome from ER stress inducer tunicamycin (TM)-primed CLL cells (ERS-exo) up-regulated the expression of ER stress marker on monocytes, indicating ER stress is transmissible in vitro via exosome. Treatment with ERS-exo promoted the survival of monocytes and induced phenotypic changes with a significantly larger percentage of CD14⁺CD16⁺monocytes. Finally, we identified exosome-mediated transfer of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) from ER stressed CLL cells into monocytes as a novel mechanism through which ERS-exo regulated monocytes. Exosomal eNAMPT up-regulated nicotinamide adenine dinucleotide (NAD⁺) production which subsequently activated SIRT1-C/EBPβ signaling pathway in monocytes. Our results suggest the role of ER stress in mediating immunological dysfunction in CLL.     

Adenovirus-mediated Gene Transfer of B7.1 Induces Immunological Anti-tumor Effects in a Murine Brain Tumor

The purpose of the present study was to determine if adenovirus-mediated transfection of a syngeneic mouse brain tumor with the gene encoding B7.1 enhances immunogenicity against tumor. Malignant astrocytoma cells were transfected with adenoviral vectors carrying the B7.1 gene (AdB7). Immunocytochemical analysis confirmed the expression of B7.1 in vitro and in vivo. To investigate the effects of B7.1 expression on tumorigenicity of the malignant astrocytoma, mice were implanted intracerebrally with B7.1-transfected glioma cells. There was no significant difference in proliferation between B7.1-transfected cells and controls in vitro. Nevertheless, mice implanted with B7.1-transfected cells survived significantly longer than those in the control groups. Immunocytochemical analysis of the tumors showed that there was infiltration of a number of CD8⁺ T-cells and CD25⁺ activated T-cells in the brain implanted with B7.1-transfected glioma cells. The results showed the possibility that adenovirus-mediated B7.1 gene transfection to a brain tumor induced activation of CD8⁺ cytotoxic T-lymphocytes.