ATTACHMENT 6_SOW-APPENDIX 5.pdf

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Appendix 5 - Immunomodulation Assays

T Cell, T Cell Subsets, B Cell, NK Cell and Monocyte Enumeration: spleens are teased into single cell preparations and seeded into 96-well microtiter plates. The first well contains spleen cells and 100 ml goat anti-mouse IgG (heavy and light chain specific) conjugated to fluorescein isothiocyanate (FITC) for enumerating B cells. The second well contains spleen cells, 100 ml anti-mouse CD4 monoclonal antibody conjugated to phycoerythrin (PE), and 100 ml anti-mouse CD8a monoclonal antibody conjugated to

FITC. The third well contains anti-mouse NK1.1 conjugated to PE and anti-mouse CD3e conjugated to

FITC. Cells that are NK1.1 and CD3 are considered to be NK cells. This well is also used to enumerate

T cells (CD3 cells). In the fourth well, splenic macrophages are enumerated using Mac-3 antibody conjugated to FITC.

Following the initial staining with antibody and washing with staining buffer, 100 ml propidium iodide

(PI) is added to each well as a viability stain. Following a five-minute incubation with PI, the cells were washed once with staining buffer and then enumerated on a Becton Dickenson FACScan Flow

Cytometer.

Acquisition of Data: The cell preparation is run on the flow cytometer and viewed as a dot plot of forward scatter (FSC) on the X axis, and side scatter (90 degree; SSC) on the Y axis in the linear mode. Gating removes the majority of the red cell population, debris and non-viable cells. Data for all samples are acquired in list mode with no other gating. Photomultiplier tubes for fluorescence operate in the log mode.

Five thousand cells are counted for each sample.

Spleen IgM Antibody Response to the T-dependent Antigen, sRBC. Day 4 Response: The primary

IgM response to sheep erythrocytes (sRBC) is enumerated using a modified hemolytic plaque assay. Mice receiving the appropriate exposure are sensitized with sRBC delivered intravenously four days prior to the last exposure to the test article. One day after the last exposure, animals are sacrificed, and spleen cells prepared. An aliquot of cells was added to a test tube containing guinea pig complement, sheep erythrocytes and warm agar. After thoroughly mixing, the test tube mixture was plated in a Petri dish, covered with a microscope cover slip, and incubated at 37°C for 3 hours. Cell counts are performed on the original sample and the number of cells/spleen, AFC/spleen and AFC/10 spleen cells determined.

The plaques that develop are counted using a Bellco plaque viewer. Each plaque is generated from a single IgM antibody-producing B cell and, thus, the number of antibody-forming cells (AFC) present in the whole spleen can be calculated. The data are expressed as specific activity (AFC/10 spleen cells) and total spleen activity (AFC per spleen).

Serum IgM Antibody Titers to the T-dependent Antigen, sRBC as Measured by ELISA: An ELISA assay system was used to determine the serum titers of the primary IgM response to sheep erythrocytes

(sRBC) in the same animals evaluated with the hemolytic plaque assay.

Mixed Leukocyte Response to DBA/2 Mouse Spleen Cells. The mixed leukocyte response (MLR) is an in vitro assay which analyzes the ability of spleen T cells from xenobiotic-exposed mice to recognize allogeneic cells as "non-self" and proliferate in response to the presence of the allogeneic cells. Mice are exposed in vivo to the test article. On the day following the final exposure, mice are sacrificed and spleen cells are processed into single cell suspensions. Spleen cells are added to each well of a U-bottom microtiter plate. DBA/2 mouse spleen cells are used as the allogeneic cell (stimulator cell) for B6C3F1

(responder) mice. Stimulator cells are treated with Mitomycin C to render them unable to proliferate; the ratio of stimulators to responders has previously been optimized. The cells are cultured for 5 days, during the last 18 hours in the presence of 1 mCi H-thymidine. The cells are collected with a cell harvester and counted in a LKB liquid scintillation counter, and data collected. The incorporation of H-thymidine into the proliferating cells is used as the endpoint of the assay, and the data are expressed as cpm/culture.

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Macrophage Activation Factor (MAF) Assay. The introduction of a foreign body is often recognized and responded to by the macrophage and, therefore, this cell plays an important role in the immune system. Gamma interferon, in the presence of a small amount of LPS, is known to be a lymphokine that activates macrophages so that they possess the ability to inhibit tumor growth and to kill tumor cells.

Resident- or thioglycollate-recruited macrophages can be utilized in the following assay.

Five days prior to the sacrifice day, the day after the last animal exposure, mice are injected intraperitoneally with a 10% preparation of thioglycollate. Animals are sacrificed and peritoneal cells

(PEC) obtained by lavage using Hank's Balanced Salt Solution (HBSS). The cells are centrifuged and resuspended in RPMI media with 10% Fetal Bovine Serum. The cells are counted, adjusted to the appropriate concentration in 200 ml or medium and pipetted into each designated well of a 96 well plate.

The cells are allowed to adhere for 2 hours, wells are rinsed twice with phosphate buffered saline, and treated with recombinant rat gamma interferon 10 units/ml and LPS 10 ng/ml in RPMI media for 4 hours at 37°C 5% CO2. Following this incubation B16F10 melanoma target cells are added to each well with exception of the macrophage background control well, one for each animal.

Incubation continues for 48-60 hours with B16F10 melanoma target cells at a ratio of 10:1. All wells are pulsed with H-thymidine 14-16 hours prior to harvest. Media is pipetted into the wells and rinsed 2 times with HBSS. Trypsin is added to all wells for 45 minutes and all wells harvested using the Cambridge Cell

Harvester. In addition to the harvester rinses, consistent mechanical scraping is used with the probes of the harvester to agitate the bottom of the well to obtain total H-thymidine counts (CPM) for each well.

Data are calculated to represent percent suppression of B16F10 proliferation.

Natural Killer (NK) Cell Activity: Twenty-four hours following the last exposure, the body weights are recorded and the spleens removed and weighed. Single cell suspensions from individual spleens are prepared aseptically. Following centrifugation, the pellet is resuspended in RPMI 1640 supplemented with 10% fetal calf serum. The cell suspension is adjusted to appropriate concentrations to obtain effector-to-target ratios of 200:1, 100:1, 50:1, 25:1, 12.5:1, and 6.25:1. The target cells, YAC-1 cells, are maintained in a stock culture. Target cells are labeled by incubation with 200 mCi of

Cr for 90 minutes in a 37°C 5% CO2 incabator with frequent agitation. Following the incubation, target cells are washed three times in balanced salt solution (BSS) plus 25mM HEPES and counted. The maximum release is determined by adding an appropriate number of YAC-1 cells to 0.1 ml 0.1% triton X-100 in each of 12 replicate wells on a 96-well microtiter plate. The targets and effector cells are added in 0.1 ml to each of four replicate wells of target cells at each effector concentration. Spontaneous release is determined by adding 0.1 ml of medium to 12 replicate cultures containing the targets. The plates are incubated for 4 hours at 37°C at 5% CO2. Following the incubation, the plates are centrifuged and the supernatant is removed from each well and counted. The mean (± SE) percent cytotoxicity at each effector concentration is determined for each exposure group and compared to the comparable values for the vehicle mice.