High Reproducibility of ELISPOT Counts from Nine Different Laboratories.

The primary goal of immune monitoring with ELISPOT is to measure the number of T cells, specific for any antigen, accurately and reproducibly between different laboratories. In ELISPOT assays, antigen-specific T cells secrete cytokines, forming spots of different sizes on a membrane with variable background intensities. Due to the subjective nature of judging maximal and minimal spot sizes, different investigators come up with different numbers. This study aims to determine whether statistics-based, automated size-gating can harmonize the number of spot counts calculated between different laboratories. We plated PBMC at four different concentrations, 24 replicates each, in an IFN-γ ELISPOT assay with HCMV pp65 antigen. The ELISPOT plate, and an image file of the plate was counted in nine different laboratories using ImmunoSpot® Analyzers by (A) Basic Count™ relying on subjective counting parameters set by the respective investigators and (B) SmartCount™, an automated counting protocol by the ImmunoSpot® Software that uses statistics-based spot size auto-gating with spot intensity auto-thresholding. The average coefficient of variation (CV) for the mean values between independent laboratories was 26.7% when counting with Basic Count™, and 6.7% when counting with SmartCount™. Our data indicates that SmartCount™ allows harmonization of counting ELISPOT results between different laboratories and investigators.

Ras-induced modulation of CXCL10 and its receptor splice variant CXCR3-B in MDA-MB-435 and MCF-7 cells: relevance for the development of human breast cancer.

Interactions between chemokines and chemokine receptors have been proposed recently to be of importance in the development and progression of cancer. Human breast cancer cells express the chemokine CXCL10 (IP-10) and also its receptor CXCR3. In this study, we have investigated the role of Ras activation in the regulation of CXCL10 and its receptor splice variant CXCR3-B in two human breast cancer cell lines MDA-MB-435 and MCF-7. In cotransfection assays, using a full-length CXCL10 promoter-luciferase construct, we found that the activated form of Ras, Ha-Ras(12V), promoted CXCL10 transcriptional activation. Ras significantly increased CXCL10 mRNA and protein expression as observed by real-time PCR, fluorescence-activated cell sorting analysis, and ELISA. Selective inhibition of Ha-Ras by small interfering RNA (siRNA) decreased CXCL10 mRNA expression in a dose-dependent manner. Further, using effector domain mutants of Ras, we found that Ras-induced overexpression of CXCL10 is mediated primarily through the Raf and phosphatidylinositol 3-kinase signaling pathways. We also observed that the expression of the splice variant CXCR3-B, known to inhibit cell proliferation, was significantly down-regulated by Ras. Selective inhibition of CXCR3-B using siRNA resulted in an increase in CXCL10-mediated breast cancer cell proliferation through G(i) proteins and likely involving CXCR3-A. Finally, we observed intense expression of CXCL10 and CXCR3 in association with human breast cancer in situ, indicating that these observations may be of pathophysiologic significance. Together, these results suggest that activation of Ras plays a critical role in modulating the expression of both CXCL10 and CXCR3-B, which may have important consequences in the development of breast tumors through cancer cell proliferation.

CD40: a mediator of pro- and anti-inflammatory signals in renal tubular epithelial cells.

Infiltration of immune cells into the renal interstitium is characteristic of chronic inflammatory kidney diseases. CD4+ T cells and platelets express CD40 ligand (CD40L) and are reported to mediate proinflammatory events in renal proximal tubular epithelial cells (RPTEC) via interaction with CD40. In other cell types, CD40 signals can also induce protective genes. Here, human RPTEC were treated with sCD40L to ligate CD40, and a significant increase in the generation of proinflammatory reactive oxygen species was found; however, CD40-activated cells did not undergo apoptosis. This suggests that CD40 signals may simultaneously induce antiapoptotic genes for cytoprotection of RPTEC. Heme oxygenase-1 (HO-1) expressed in RPTEC serves as a protective gene, but it is not known whether it is regulated by CD40. Next, RPTEC were transiently transfected with a full-length HO-1 promoter-luciferase construct and were treated with sCD40L. CD40 ligation was found to significantly increase HO-1 promoter activity. By electrophoretic mobility shift assay, it was confirmed that CD40 signaling induced the transcriptional activation of HO-1 through the binding of NF-kappaB to its promoter. By Western blot analysis, a marked increase in HO-1 protein expression following CD40 ligation was also found. These observations are of clinical significance because it was found that CD40 and HO-1 are induced in expression in vivo in inflamed rejecting kidney biopsies and co-expressed in renal tubules. Therefore, ligation of CD40 in RPTEC promotes both inflammatory and anti-inflammatory processes. Regulating the balance between these two events may be of importance in the prevention of tubular injury associated with renal disease.

Vascular endothelial growth factor impairs the functional ability of dendritic cells through Id pathways.

Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that plays an important role in tumor growth and progression. Recent evidence suggests an alternate, albeit indirect, role of VEGF on host immune response to tumors. VEGF appears to diminish host immunity by altering the function of major antigen-presenting cells such as dendritic cells (DCs) [D.I. Gabrilovich, T. Ishida, S. Nadaf, J.E. Ohm, D.P. Carbone, Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function, Clin. Cancer Res. 5 (1999) 2963-2970, D. Gabrilovich, T. Ishida, T. Oyama, S. Ran, V. Kravtsov, S. Nadaf, D.P. Carbone, Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo, Blood 92 (1998) 4150-4166, T. Oyama, S. Ran, T. Ishida, S. Nadaf, L. Kerr, D.P. Carbone, D.I. Gabrilovich, Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-kappa B activation in hemopoietic progenitor cells, J. Immunol. 160 (1998) 1224-1232.]. DCs are prime initiators of host immunity as they are known to activate both primary as well as secondary immune responses [J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque, Y.J. Liu, B. Pulendran, K. Palucka, Immunobiology of dendritic cells, Ann. Rev. Immunol. 18 (2000) 767-811.]. However, the exact nature of how VEGF suppresses DC function is not fully clear. In this report, we show that DCs cultured in the presence of VEGF are less potent in stimulating antigen-specific T-cells. Furthermore, by using DCs derived from Id1(-/-) mice that are defective in Flt-1 signaling, we demonstrated that the inhibitory function of VEGF on DC function is most likely mediated by Flt-1. Thus, the role of VEGF in downregulating host immunity may highlight a unique role of VEGF in the pathogenesis of cancer.