Methods

The methods detailed in the following section outline (a) the isolation and preparation of the diabetic donor spleen cells from female diabetic NOD mice, (b) the primary culture for isolation as well as maintenance and expansion of antigen-specific T-cell clones in vitro (5-7,14), (c) the adoptive transfer of the prepared diabetogenic spleen cells or T-cell clones/lines by intraperitoneal injection of young (6- to 10-d-old) NOD and NOD.Rag mice (7,14-18), and (d) urine and blood glucose monitoring of recipient mice for detection of glucosuria and hyperglycemia after adoptive transfer of diabetogenic T cells.

3.1. Sterile Removal of Lymph Nodes and Spleen (see Note 3)

1. Prior to sacrificing mice (by approved animal care protocol), have an ice bucket with 100 mL 95% ethanol in a beaker for maintaining sterility of surgical utensils, 10 mL of DMEM plus 0.3-0.5% supplemented with normal mouse serum (NMS) (DMEM-NMS) in a 15-mL conical tube on ice, sterile tools, and a sterile pad.

2. In a biosafety hood on a sterile blue pad, lay the mouse flat on its back and coat the exposed fur with 70% ethanol. Designate one pair of scissors and forceps as the outside pair and the other set as the inside pair prior to making an incision. Use the outside forceps to lift the skin away from the mouse at the urethral opening and make an incision beginning at the urethral opening to the chin of the mouse (Fig. 1).

3. Next, make an incision on either side of the first incision down to the knee, resulting in an incision that looks like an upside-down Y. This should expose the organs and leave the peritoneal wall intact.

4. Put the designated outside tools back in the beaker of 95% ethanol and use the inside forceps to lift the peritoneal layer and cut it to expose the organs. At this point, you are ready to remove the LNs (see Fig. 2). If the animal was immunized prior to LN harvest, only the draining LN from the site of injection need be removed. Use the forceps to hold the spleen gently while using the scissors to detach it from the pancreas and intestines. Immediately place the spleen in the DMEM-NMS.

5. Pour the spleen(s)/LN and medium into a glass homogenizer and pour off 5 mL of DMEM-NMS, leaving the spleen and 5 mL of DMEM-NMS in the homog-enizer. Grind to a single-cell suspension and pour the cells into a 15-mL conical tube. Pour the remaining 5 mL of media into the base of the homogenizer and repeat the grinding.

6. Spin the cells at 330g for 5 min. Aspirate the supernatant (SN) and gently resuspend the pellet in 3 mL of RBC lysis buffer per spleen. Incubate for 5 min at room temperature. Immediately add an equal volume of DMEM-NMS, mix by gentle pipetting, and spin at 330g for 5 min. Repeat the wash (see Note 4). Resuspend the pellet from one spleen in 5 mL of DMEM-NMS and pour through a cell strainer into a sterile conical tube. Wash tube and strainer with an additional 5 mL per spleen for a total of 10 mL per spleen or LN. The single-cell suspension of spleen/LN is now ready for primary culture.

3.2. Primary Culture of T Cells

1. For primary cultures, cells are normally seeded at 1 x 107 cells in 1-2 mL of medium in 24-well tissue culture plates. The 1 x 107 LN/spleen cells are put into culture with the appropriate antigen at the desired concentration (see Note 5).

2. The cultures are then adjusted to the final volume with medium. No cytokine is added at this time to avoid generating interleukin (IL)-2-dependent T cells.

Fig. 1. Diagram of subcutaneous incision for isolation of LNs and spleen from diabetic and antigen-specific immunized mice. Animals are soaked in 70% ethanol to create an aseptic environment and matt the fur. The first incision is from the urethral opening to the chin, then from the urethral opening to either side of the knee. The incision is made to keep the peritoneal wall intact so the organs remain within the peritoneal cavity. (Obtained with permission from The Virtual Mouse Necropsy, www.geocities.com/virtualbiology/necropsy.html.)

Fig. 1. Diagram of subcutaneous incision for isolation of LNs and spleen from diabetic and antigen-specific immunized mice. Animals are soaked in 70% ethanol to create an aseptic environment and matt the fur. The first incision is from the urethral opening to the chin, then from the urethral opening to either side of the knee. The incision is made to keep the peritoneal wall intact so the organs remain within the peritoneal cavity. (Obtained with permission from The Virtual Mouse Necropsy, www.geocities.com/virtualbiology/necropsy.html.)

3. The cells are cultured for 5 d at 37°C and 5% CO2. After the 5-d incubation, the cells are ready to be restimulated according to the standard restimulation protocol (see Subheading 3.3.). This protocol is carried out every 2 wk (see Note 6). At this point, the primary culture contents can be switched into complete medium (CM).

3.3. Restimulation of Th1 T-Cell Clones

3.3.1. Responder T Cells

1. Responder T cells from a 2-wk restimulation flask are considered resting T cells and are used to continue the propagation of the antigen-specific T cells in a new 2-wk restimulation cycle. After resuspending the cells thoroughly, remove 100 ^L to determine cell count and viability.

2. Add 20 mL trypan blue to identify dead cells, and count viable cells using a hemacytometer.

Fig. 2. Diagram of LNs located subcutaneously or in the connective tissues between muscles. If the animals have been immunized prior to harvesting the LNs, it will not be difficult to locate the nodes. If the animal has not been immunized, the nodes may be difficult to see. Look for bead-shape structures, which can range in color from a yellowish white to tan, depending on the strain of the mouse. Clearly, if the mouse is pigmented, the nodes will be darker than those of an albino mouse.

Fig. 2. Diagram of LNs located subcutaneously or in the connective tissues between muscles. If the animals have been immunized prior to harvesting the LNs, it will not be difficult to locate the nodes. If the animal has not been immunized, the nodes may be difficult to see. Look for bead-shape structures, which can range in color from a yellowish white to tan, depending on the strain of the mouse. Clearly, if the mouse is pigmented, the nodes will be darker than those of an albino mouse.

3.3.2. Preparation of APCs

1. Aseptically remove spleens from nondiabetic NOD mice not younger than age 6 wk and place in snap-cap tubes containing 2 mL CM.

2. In a homogenizer, break up organ to the point at which a single-cell suspension is achieved as described in Subheading 3.2.

3. Prepare a 1:50 dilution by adding 20 ^L of spleen cells to 980 mL CM. Pipet 100 ^L of cells into a 0.6-mL centrifuge tube containing 20 mL trypan blue. Count viable cells (not RBCs) using a hemacytometer (see Note 7).

4. APCs must be irradiated at 3500 rad or treated with mitomycin C (25 ^g/mL) prior to addition to restimulation cultures. These treatments block lymphocyte proliferation, but allow processing and presentation of antigen by APCs. In addition, peritoneal macrophages may be used as a source of APCs without the need for irradiation (see Note 8).

3.3.3. Antigen for Whole Islet/Islet-Antigen Reactive T Cells

Irradiated (3500 rad) islet cells, islet fractions, or specific islet-associated antigen(s) may be used (see Note 8).

3.3.4. Components of Restimulation Culture

1. In a 25-cm2 tissue culture flask, combine 1 x 106 responder T cells (5 mL at 2 x 105 cells/mL) and 2.5 x 107 irradiated or mitomycin C-treated APCs (25 ^g/mL) (2.5 mL at 1 x 107 cells/mL).

2. If carrying T cells on islet cells use 5 x 104 islet cells (2.5 x 103 cells/mL) or other desired antigen and 7 U/mL recombinant IL-2 (rIL-2).

3. Bring to a volume of 20 mL with CM, loosen caps slightly to allow for gassing (if not vented), and tighten after 24 h.

4. Incubate flasks upright at 37°C and 5% CO2 for 2 wk.

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