There may also be more direct impacts of ranitidine on B cells. of enhanced antitumor antibody responses. This was not limited XMD 17-109 to the tumor setting since ranitidine-treated mice immunized with ovalbumin also demonstrated increased IgG antibody responses. Analysis of B XMD 17-109 cell populations indicated that while B1 cell populations remained unchanged there was a significant decrease in B2 cells in the tumor-draining inguinal lymph nodes. Notably, ranitidine did not significantly inhibit primary tumor growth XMD 17-109 in B cell-deficient animals. Examination of NK cell populations revealed a significant decrease in the proportion of intermediately functionally mature NK cells populations (CD27+CD11b?) in ranitidine-treated tumor-bearing mice compared with untreated tumor-bearing controls. Conclusion These data demonstrate an important role for B cells in the enhanced antitumor immune response that occurs in response to ranitidine treatment. Our findings are consistent with a model, whereby ranitidine reduces tumor-associated immune suppression allowing for the development of more effective antitumor responses mediated by B cells which may include the participation of NK cells. These data underline the importance of considering widely used histamine receptor antagonists as modulators of antitumor immunity to breast cancer. Cancer Models Histamine antagonists were added to drinking water 1?day prior to tumor cell injection and were refreshed every other day. An adapted protocol was employed for orthotopic models (20). For the E0771-GFP model, 6- to 8-week-old female C57BL/6 mice were anesthetized and 200,000 cells in 100?L of Matrigel? (Corning) were injected subcutaneously into the mammary fat pad near the fourth nipple. The volume of the tumor was determined by caliper measurements every second day using the equation volume?=?length??width2/2. For the 4T1 model, 6- to Rabbit polyclonal to PHF7 8-week-old BALB/c female mice were anesthetized and 100,000 4T1 cells in 50?L PBS were injected subcutaneously into the mammary fat pad near the fourth nipple. For the B16-OVA model, 6- to 8-week-old female mice were anesthetized, and 100,000 B16-OVA cells in 50?L PBS were injected subcutaneously into the back flank. The volumes of the tumors were measured as previously stated above. At day 19 post injection for the E0771-GFP and 4T1 models, and day 21 post injection for the B16-OVA model, the mice were sacrificed, and the primary tumor, spleen, and tumor-draining inguinal lymph node were collected. Blood Smear and Staining On the day of tumor cell implant, and days 7, 14, and 19 after implant, 4T1 and E0771 tumor-bearing mice were restrained and 100?L of blood was isolated by puncturing the submandibular vein with a lancet. Circulating leukocyte concentrations were counted on a hemocytometer XMD 17-109 using 3% acetic acid in methylene blue. Approximately 10?L of blood was used for a blood smear on microscope slides and then allowed to dry overnight. A modified protocol of blood staining was performed using Differential Quik Stain Kit (Electron Microscopy Sciences). The samples were then mounted with DPX mounting medium (Sigma-Aldrich) and viewed under a light microscope at 400. Flow Cytometric Assessment of Tumor-Specific Antibodies Secondary antibodies: rat anti-mouse IgG2a-bio (BioLegend), rat anti-mouse Ig-bio (BD Biosciences), rat anti-mouse Ig-bio (BD Biosciences), and rat anti-mouse Ig-FITC (BD Biosciences). Streptavidin (SA)-conjugated detection proteins: PE-SA (eBioscience) and APC-SA (BioLegend). E0771-GFP cells or SK-BR-3 cells (a Her2-positive cell line) were routinely cultured. Cells were then blocked in FACS buffer containing human IgG (1?L/50?L FACS buffer). Mouse serum, obtained from tumor-bearing or control animals, was added to the cells at dilutions of 1/10 and 1/100, and the cells were incubated on ice for 15?min. Cells were washed and biotinylated secondary anti-mouse-Ig antibodies were added and incubated for 15?min on ice. Again, cells were washed, and SA-conjugated fluorochromes were added, and the cells were fixed with 1% paraformaldehyde. Stained cells were acquired for analysis using a BD FACSCalibur, and results were analyzed using FCS express software. The amount of antibody binding to whole E0771-GFP tumor cells was quantified by relative fluorescence intensity (relative to the average mean fluorescence intensity of control group). Flow Cytometric Analysis of.