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cytolytic activity. It IS also important to include negative controls m which
irrelevant peptides are added to target cells This rules out the possibilty of
nonspecific peptide-mediated cytolysis Finally, wells should be set up dur-
mg the CTL assay contammg only target cells and peptide (no effecters
added) If lys˜s occurs m these wells this indicates that the peptide itself is
toxic to the target cells.
3. Synthetic peptides of defined lengths (e.g., 15 mers with lo-mer overlap), span-
rung the length of the protein or mdividual peptides representing motif-predicted
epitopes. Peptides should be stored at -20°C.
4 All reagents necessary to perform CTL assay

2.5. Murine Chalenge Model
1 Inbred mouse strains Mice should be 5-6 wk old at the beginning of the experi-
ment. Each group of mice needs to be genetically identical, the same age, and the
same sex; httermates are ideal.
2. Immunogen to be tested (e.g., recombinant vaccinia vu-us expressing a herpes-
virus protein, or other vector system as described earlier)
3. Virulent strain of HSV-1 (McKrae strain).
333
Cell-Mediated Immune Responses to HSV

3. Materials
3.1. Cytotoxic T-cell Assay
3.1.1. Immunizing Mice with HSV
1 Inject mice mtrapernoneally (tp) with approx 5 x lo6 plaque-forming units (PFU)
of HSV-l-KOS. This is a commonly used lab-adapted strain well-suited for immu-
nization. If possible, immunize groups of 10 or more mice at one time m order to be
able to perform several CTL assays using mice from the same immunized “stock.”
2. Virus for immunization should be diluted m sterile PBS and drawn into a 3-mL
syringe, using an 1&gage needle. A 26gage needle is then used to administer 0.5
mL of virus ip.
3. Mice are boosted ip 2 wk later with the same amount of virus and then rested for
l-2 wk before collectmg their spleens for testing in the CTL assay
4. Footpad injection represents an alternative route of immunization. Specifically, a
30-gage needle is used to inject both rear footpads of the mouse with 5 x lo6 PFU
of virus per footpad. Five days postimmunization the drammg pophteal lymph
nodes are collected for testing m the CTL assay
3.1.2. Immunizing Mice with Recombinant Vaccinia Wuses,
Recombinant Adenoviruses, Recombinant Retroviruses, and Plasmid DNA
Although the immunization of animals with whole HSV remains an excel-
lent way to induce CMI, viral and nonviral vectors expressing herpesvnus an-
tigens can also be successfully used to induce HSV-specific CMI. In parttcular,
this latter approach allows one to identify mdividual HSV proteins capable of
inducing CMI. Moreover, once an immunogenic viral protein is identified, a
variety of techniques, several of which will be detailed in later sections of this
chapter, can be used to map the exact location of the mmtmal epitope wtthm
the viral protein.
What follows here are mnnunizatron recommendations for several viral and
nonviral vector systems.
1. For immunization with recombinant vaccinia viruses, mice can be injected IP or in
the footpads exactly as described above for HSV, with the exception that the viral
doses given should be increased to l-10 x lo7 PFU IP and 1 x lo7 PFU/footpad
2. Similar doses to those used for vaccinia immunization are also recommended for
recombinant adenoviruses, except that 5 x lo7 PFU IS injected per footpad.
3. Although immunization with retrovectors expressing HSV antigens has not, to our
knowledge, been reported, retrovectors expressing other antigens have been used
to induce CMI. For example, a retrovector expressing the human immunodeficiency
vnus (HIV) env and rev proteins has been shown to successfully induce CM1 m
mice (I 7) Intramuscular injection of the vector mto two sites (1-5 x 1O5 colony
forming units [CFU] per site) 1s followed by a booster injection 1 wk later. One
week followmg the booster, spleens can be harvested for m vitro testing.
Banks, Hariharan, and Rouse
334
4 Regarding the mjectlon of plasmld DNA encodmg HSV antigens, mtramuscular
Injection in at least two sites using l-l 00 pg of DNA/site is recommended; boost-
mg 1salso advisable. However, it should be noted that the amount of DNA needed
for either the primary or booster mjectlon appears highly dependent on the par-
tlcular expressed antigen Thus, the optimal doses need to be empn-lcally deter-
mmed It IS also possible to use a “gene gun” to lmmumze ammals with DNA
(19) In this procedure, gold beads coated with DNA are propelled from a hehum-
driven gun and dehvered directly mto cells (usually mto cells of the skm and mto
cells beneath the skin™s surface) In this system, the optimal amounts of DNA to
dehver must also be determined for each antigen In general, smaller amounts of
DNA (e g , nanograms) can be delivered using the gene gun as compared to other
methods of delivery
3.1.3. Collecting Spleens or Lymph Nodes from Immunized Mice
1. Sacrifice each mouse by cervical dislocation, saturate with 70% ethanol, and lay
the animal with its left side facing up Cut the epidermis and carefully tear the
skm away from the underlaymg spleen
2 Lift the mesentary, which covers the spleen, then cut and peel it back before
removmg the spleen with sterile forceps Finally, trim away any remammg fat
and connective tissue Avoid letting the spleen touch nonsterlle areas Place the
spleen mto a 50-mL tube contammg 10 mL of CTL media.
3 Drammg pophteal lymph nodes are removed 5 d followmg footpad lmmuniza-
tion Mice are placed on their abdomens, and a scalpel 1s used to shave the hair
from the back of both rear legs A vertical mclslon IS then made down the length
of the back of each leg, and the pophteal lymph nodes found at the rear of the
knee joint are removed using sterile forceps and placed into an appropriate tube
contamg CTL media
3.1.4. In Vitro Bulk Culture of CTL Effector Cells
(Splenocytes or Lymph Node Cells)
1 Pour spleen(s) or lymph nodes from the 50-mL tube mto a sterile 6- or IO-cm
Petri dish.
2. Tease the spleen(s) or lymph nodes mto single-cell suspensions by usmg the end
of the plunger from a 3-mL syringe to gently press the cells through a sterile wire
screen. This procedure allows the lymphocytes to pass through the screen while
trapping tissue remnants m the screen
3 Use a plpet to rinse the screen several times with media This ensures that very
few lymphocytes are left behind m the screen. After disposing of the screeqthe
lymph node suspension IS spun at 1600 rpm for 5 mm and resuspended at 2 x 10™
cells/ml. Each well of a 6-well plate 1sthen seeded with 1 mL of the suspension,
and CTL media IS added to bring the volume of each well to a 5-mL total. Plates
are incubated for 3 d at 37°C m 5% CO,
4 Unlike drammg lymph node cells, which can be removed from the ammal and
directly cultured m vitro, splenocytes must be restimulated m vitro with HSV
335
Cell-MedIated Immune Responses to HSV
antigen before bulk CTL cultures can be set up. This is because drammg lymph
nodes presumably still contain viral antigen trapped wlthm the node itself In
contrast, splenocytes from immunized mice are comprised of memory T cells,
which need to be re-exposed to antigen in order to become activated UV-lrradla-
tion of HSV reduces the viral titer to a low enough level such that splenocytes are
exposed to viral antigens without being lysed as the result of a productive viral
Infection UV-inactlvation of HSV is achieved by exposing 2 x 10™ PFU of virus
to a germicidial lamp for 2 mm at a distance of 3 cm and generally reduces viral
titer to approx 100 PFU or less. Thus, splenocytes are restimulated m vitro with
UV-irradiated HSV-1 KOS at a multiplicity of infection (MOI) of 2 for 1 h at
37°C For example, if 1 x lo8 splenocytes are to be restimulated at a MO1 of 2
with HSV- 1 KOS, whose titer is 1 x 1O9PFU/mL, then the neccessary calcuatlon
1s as follows. (1 x I O8splenocytes)(MOI of 2) = 2 x lo8 PFU of virus needed,
thus, 2 x lo8 PFU needed/l x lo9 PFU (actual viral titer before UV-irradiation) =
0.2 mL of UV-lrradlated virus needed to restimulate 1 x lo8 splenocytes at an
MO1 of 2. Cultures are set up in &well plates at 1 x 10™ cells/well in a total
volume of 5 mL/well for 5 d at 37°C in 5% CO,.
Radiolabeling Target Cells for CTL Assay
3.1.5.
1 Cells to be used as targets should be in log-phase growth. Thus, it is usually best to split
cells the day before such that they will be 7@-80% confluent on the day of the assay
2. On the morning of the assay, wash cells with phosphate-buffered salme (PBS)
and add enough of the 0 02% EDTA solution to cover the cells Incubate flasks at
37°C until cells easily detatch when plpetted with a 10 mL plpet
3 Harvest cells by gently pipetmg up and down m the flask The cells must be
treated gently and should not at any point m time become clumped.
4. Cells are washed once by centrlfugatlon using CTL media and then resuspended at
1 x 1O6cells/ml Two milliliters of cells (2 x 1O6cells total) are placed m a 15-mL
tube and labeled with 200 pCl of 51Cr. At the same time that the cells are bemg
labeled they are also Infected with either HSV (MO1 of 5) or a recombinant vac-
cinia vnus expressing an HSV antigen (MO1 of 10). The total time for labeling and
viral infection IS approx 3-4 h Although labeling with 5™Cr only requires 1 h, viral
antigen expression by HSV and the recombinant vaccmia virus takes approx 3-4 h.
5. Both the 5™Cr labeling and the viral mfection occurs optimally when a 37°C water bath
is used, and the tubes are gently mixed every 10 mm during the first hour of incubation
6. Following the incubation period, cells are washed three to four times by centnfu-
gation with CTL media.
7. The washed target cells are then resuspended in CTL media at 1 x IO5 cells/ml and
placed on ice until the splenocyte or lymph node cultures have been harvested.
3.7.6. Harvesting CTL Effector Cells
(Cultured Splenocytes or Lymph Node Cells)
1, Harvest cultured cells from the wells of the plate by gently pipetting up and down
with a 10-mL pipet
Banks, Hariharan, and Rouse
336
2. Pool the contents of all wells together, wash once with CTL media, and resus-
pend pellet at 1 x lo7 cells/ml. Keep cells on me until they are added to the 96-
well microtiter plate.

3. I. 7. Setting up 96-well Microtiter Plate for the CT1 Assay
1. To rows B,C, D, and F of a 96-well microtiter plate add 100 pL of CTL media
Add 200 pL of splenocytes or lymph node cells (effector cell population) at 1 x
lo7 cells/ml to row A Using a 12-channel pipettor, remove 100 pL from row A
and sequentially add, mix, and remove 100 uL of cells to the next row continumg
this process through row D
2 Next add 100 uL of labeled target cells (1 x lo5 cells/ml) to triplicate wells m
rows A-D For example, if one has 4 different labeled targets, then target no 1
would be added to wells l-3 of rows A-D, target no 2 would be added to wells
4-6 of rows A-D, target no 3 would be added to wells 7-9 of rows A-D, and
target no. 4 would be added to wells 10-12 of rows A-D.
3 Similarly, 100 uL of each target are added to triplicate wells of row F This row
serves to indicate the amount of spontaneous release of 5iCr, since the wells m
this row only contam targets and media (no effector cells are added). In contrast,
100 uL of target cells are added to triplicate wells m row G along with 100 pL of
1N HCL This row serves to indicate total 51Cr release of target cells, since IN
HCL induces maximal lys˜s of the cells
4. Followmg the addition of all the target cells to the appropriate effector cells, all
wells should contam a total volume of 200 pL. Moreover, row A should contam
an effector to target (E T) ratio of 100, 1, row B, a ratio of 50 1, row C, a ratio of
25:l; and row D, a ratio of 12.5.1
3.7.8. Harvesting Samples and Data Calculation
1 Remove assay plates from the incubator after a 4-h incubation period.
2 Using the multichannel pipettor, carefully remove and transfer 100 uL of super-
natant from each well into the correspondmg y-counter tubes. It is critical that the
cell pellet is not disturbed when performmg this procedure and that fresh tips are
used for each triplicate set of samples.
3 Place tubes m gamma counter for target cell lysis determmation and include empty
tubes for machme background evaluation. Count tubes for I min at settings appro-
priate for 5iCr y emission and tabulate slCr release as counts/mmute (CPM)
4. The percent-specific target-cell lysis is determined by the followmg formula
[(experimental release - spontaneous release)/(maximum release - spontaneous
release)] x 100
3.2. Limiting Dilution Analysis
to Determine CTL Precursor Frequency
1. Remove lymph nodes (for acute effecters) or spleens (for memory effecters) from
immunized mice, press through sterile stainless steel mesh, and prepare smgle-
cell suspensions. Wash once, count the cells, and resuspend m fresh medium.
Cell-Mediated Immune Responses to HSV 337
2. Make serial dilutions of the effector cells titrating from 4 x lo5 cells/ml.. down to 100
cells/ml. Ideally 11 dilution tubes should be set up (4 x 105/mL, 2 x 105/mL, 1 5 x
10s/mL, 1 x 105/mL, 8 x 104/mL, 6 x 104/mL, 4 x 104/mL, 2 x 104/mL, 1 x 104/mL,
5 x 103/mL and 1 x 103/mL), plus a 12th “mock” tube to which no cells are added.
3. Prepare 96-well U-bottom plates (one for each dilutton) by tilling the outer wells
with PBS (200 uL/well) and then add 100 uL of each dilution to the 60 remammg
wells in the center of each plate Leave plates (should be 12 m total) m the 37°C
incubator while preparing feeder spleens
4. For feeder cells, harvest spleens from natve syngenetc mice, make smgle-cell
suspension, lyse RBC with Tris-NH,Cl, wash three ttmes, and resuspend m fresh
medium Irradiate cells (2400 Rads using X-ray machine), wash, and resuspend
at 5 x lo6 cells/ml.
5. Add 100 uL/well feeder cells to all the dilution plates of effector cells prepared
earlier, and incubate the plates for 7-9 d at 37°C with 5% CO, Momtor for out-
growth of pleomorphic-shaped CTL from these wells Usually these cells are
apparent by d 5 or 6 of culture.
6 When LDA plates are ready to be tested (between d 7 and 9), set up target cells
for CTL assay Briefly, add 3 x lo6 cells of each target cell m a total of 2 mL
medium, add 400 ˜CI slCr, infect with HSV or another vuus and incubate as
detailed previously Wash targets five times, resuspend at 1 x lo4 cells/ml.
Remove 100 yL of each target and count in gamma counter. Ideally 2000-3000
cpm is ideal, but there should be a minimum of 1000 cpm/lOO uL However,
should the cpm be too low, Increase the target cell concentration to 3 x 104/mL m
order to Increase the total cpm/lOO uL-ahquot.
7. Prepare effecters while targets are mcubatmg at 37°C Fhck the effector plates
quickly, add 200 uL PBS/well, and centrifuge briefly to pellet cells Fhck PBS and
add 200 yL fresh medmm/well and make a 4-way split of each well (50 uL/well) mto
96-well, V-bottom plates. Incubate plates at 37™C unttl targets are prepared and ready.
Note that a 4-way spht allows one to test effecters against four targets Two- or three-
way splits can also be done when fewer targets are needed. However, splits greater
than 4 are not recommended because the cell numbers become prohtbittve.
8. Add 100 yL of appropriate target to each well (resulting in a total of 150 uL/
well), spm plates gently, and incubate for 4-5 h at 37°C as in the case of a stan-
dard 51Cr release assay.
9 Harvest supernatant fluids from the wells and count as usual m a y-counter. Calculate
CTL precursor frequency according to the method described by Taswell (4). This
provtdes provides the frequency of precursors withm a confidence limit of 95%

3.3. T-Helper Cell-Proliferation Assay
1. Prepare antigen presentmg cells by infecting naive, syngenetc spleen cells m vitro
with UV-inactivated HSV at an MO1 of 5 for 3-4 h at 37”C, and then irradiate
these infected cells with 30004500 rads. Wash one or two times in medium and
resuspend cells at 5 x lo6 cells/ml These cells are often referred to as stimulator
cells (antigen presenting cells) in the proliferation assay
Banks, Hariharan, and Rouse

2 Harvest spleens or lymph nodes from HSV or recombmant vaccmla virus-inJected
mice. Collect effector cells as described previously and resuspend at 1 x 10™
cells/ml Effector cells are usually referred to as responder cells m the prohfera-
tion assay
3 Titrate responder cells and stimulator cells by plating the cells at different con-
centratlons m 96-well U-bottom plates to achieve a final volume of 200 pL/well
Responder cells are usually titrated down the plate using serial twofold dllutlons
Stimulator cells (2 x lo5 - 1 x lo4 cells/well) are then added to the responder
cells and, ideally, all wells are set up m trlpllcate and the plates are incubated for
72 h It is important to include appropriate positive and negative controls m this
assay As a posltlve control the mltogen concanavalin A (Con A) is used at a final
concentration of 2-4 pg/mL and naive. unmfected spleen cells serve as negative
controls. Con A control wells need to be harvested wlthm 3 d, smce this IS the
time of maximal mltogen-induced proliferation Therefore, It IS advisable to set
up the Con A controls m their own separate plate Lastly, don™t forget to Include
control wells that contam responder cells alone or stimulator cells alone as part
of the assay
4. At 72 h poststlmulatlon, add 1 &l/well 3H-thymldme to all wells and incubate
for an additional S-18 h Note: For Con A controls, 3H-thymldme should be
added after 54-64 h of mcubatlon, smce harvesting occurs at 72 h
5. Harvest cells onto glass fiber filters using a cell harvester, transfer filters to scm-
tillatlon vials, add 5 mL of appropriate scmtlllatlon fluid per vial, and count for
3H-thymidine incorporation using a liquid scintillation counter
6 Stlmulatlon index (SI) 1scalculated as follows.

SI = CPM m HSV-stimulated wells/CPM in nonstimulated wells

Cytokme assays can be performed most easily by setting up duplicate plates
of responder cells and stimulator cells exactly as you would for a prohferatlon

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