JOURNAL OF VIROLOGY, Dec. 2009, p. 12907–12916
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 24
High Frequencies of Virus-Specific CD8?T-Cell Precursors?
Mina O. Seedhom, Evan R. Jellison,† Keith A. Daniels, and Raymond M. Welsh*
Department of Pathology and Program in Immunology and Virology, University of Massachusetts Medical School,
Received 17 August 2009/Accepted 26 September 2009
A productive CD8?T-cell response to a viral infection requires rapid division and proliferation of virus-
specific CD8?T cells. Tetramer-based enrichment assays have recently given estimates of the numbers of
peptide-major histocompatibility complex-specific CD8?T cells in naïve mice, but precursor frequencies for
entire viruses have been examined only by using in vitro limiting-dilution assays (LDAs). To examine CD8?
T-cell precursor frequencies for whole viruses, we developed an in vivo LDA and found frequencies of naïve
CD8?T-cell precursors of 1 in 1,444 for vaccinia virus (VV) (?13,850 VV-specific CD8?T cells per mouse) and
1 in 2,958 for lymphocytic choriomeningitis virus (LCMV) (?6,761 LCMV-specific CD8?T cells per mouse)
in C57BL/6J mice. In mice immune to VV, the number of VV-specific precursors, not surprisingly, dramatically
increased to 1 in 13 (?1,538,462 VV-specific CD8?T cells per mouse), consistent with estimates of VV-specific
memory T cells. In contrast, precursor numbers for LCMV did not increase in VV-immune mice (1 in 4,562,
with ?4,384 LCMV-specific CD8?T cells per VV-immune mouse). Using H-2Db-restricted LCMV GP33-
specific P14-transgenic T cells, we found that, after donor T-cell take was accounted for, approximately every
T cell transferred underwent a full proliferative expansion in response to LCMV infection. This high efficiency
was also seen with memory populations, suggesting that most antigen-specific T cells will proliferate exten-
sively at a limiting dilution in response to infections. These results show that frequencies of naïve and memory
CD8?T cell precursors for whole viruses can be remarkably high.
The immune response to a viral infection often involves the
rapid proliferation of CD8?effector T cells that recognize
virus-infected targets expressing 8- to 11-amino-acid-long pep-
tides on class I major histocompatibility complex (MHC) mol-
ecules. This recognition is mediated by membrane-bound T-cell
receptors (TCRs) that are generated through largely random
DNA recombination events of the many TCR? and -? genes,
encoding polypeptide chains that heterodimerize to form the
recognition structure of T cells. The recombination of the
segments also involves addition or deletion of nucleotides dur-
ing the joining process, causing even greater diversity, and
these processes allow for a very broad range of T-cell specific-
ities, with a calculated theoretical diversity of ?1015TCRs in
the mouse (7). By use of PCR, CDR3 spectratyping, and se-
quencing techniques, it was estimated that there are approxi-
mately 2 ? 106distinct TCR specificities in a mouse spleen (1,
5). This is far below the theoretical level of T-cell diversity, but
considering estimates of T-cell degeneracy that propose that
a single TCR can recognize up to 106peptide-MHC (pMHC)
complexes (17, 36), it is likely that the functional diversity is
much greater than the number of individual TCRs.
It has been of interest to calculate the number of T cells that
would either recognize or respond to a pathogen or to a spe-
cific pMHC complex. Early estimates of numbers of CD8?T
cells that are specific to a single virus, i.e., precursor frequen-
cies, took advantage of an in vitro limiting-dilution assay
(LDA) and calculated CD8?T-cell virus-specific precursor
frequencies to be on the order of 1 in 100,000 in naïve mice and
predicted that these cells needed to undergo about 15 divisions
to reach the higher precursor frequencies found at day 8
postinfection (29, 30). The efficiency of such assays, however, is
relatively poor. Later studies estimated the number of pMHC-
specific CD8?T cells in a naïve mouse by CDR3 sequencing.
H-2Kd-restricted T cells specific to HLA residues 170 to 179
(HLA 170-179) were sorted by tetramer from human tumor-
immunized mice, and their V? CDR3 regions were sequenced.
After a plateau suggesting that the majority of the different
TCRs had been sequenced was reached, exhaustive sequencing
was then used to identify the frequencies of these sequences
in naïve mice. These studies found that there were about 600
CD8?T cells specific for that pMHC complex in naïve mice
(4). A second strategy used an in vivo competition assay with
H-2Db-restricted lymphocytic choriomeningitis virus (LCMV)
GP33-specific P14-transgenic T cells to estimate the number of
GP33-specific CD8 T cells in naïve mice and calculated the
number to be between 100 to 200 cells per mouse (2).
Others estimated numbers of pMHC-specific T cells by se-
quencing the CDR3? regions of antigen-specific T cells that
had expanded during an acute infection. By calculating a mea-
sure of CDR3 diversity and then assuming a logarithmic dis-
tribution of diversity, they extrapolated the number of T-cell
clones that responded to an acute infection. With this tech-
nique, 300 to 500 H-2Db-restricted mouse hepatitis virus
(MHV)-encoded S510 clonotypes were calculated to be in the
central nervous systems of acutely infected mice, with ?100 to
900 clonotypes calculated to be in chronically infected mice
(24). Later studies used a gamma interferon (IFN-?) capture
assay instead of tetramer sorting and estimated 1,100 to 1,500
H-2Db-restricted S510-specific clonotypes and 600 to 900
* Corresponding author. Mailing address: Department of Pathology,
University of Massachusetts Medical School, Worcester, MA 01655.
Phone: (508) 856-5819. Fax: (508) 856-0019. E-mail: Raymond.Welsh
† Present address: Department of Immunology, University of Con-
necticut Health Center, Farmington, CT.
?Published ahead of print on 7 October 2009.
clonotypes of the subdominant H-2Kb-restricted MHV S598
peptide-specific T cells in the spleens of acutely infected mice
(25). Those studies also estimated that there were 1,000 to
1,200 different H-2Db-restricted GP33-specific clonotypes that
could respond to an LCMV infection.
More-recent studies have taken advantage of magnetic tet-
ramer binding enrichment and double tetramer staining of
cells from the spleen and lymph nodes of naïve mice to deter-
mine pMHC precursor frequencies, with the assumption that
most CD8?T cells in a naïve mouse reside in lymphoid organs
and will react with tetramers. This technique was first de-
scribed by Moon et al. for CD4?T cells, and it detected ?190
I-Ab2W1S 52-68-specific T cells, ?20 I-AbSalmonella enterica
serovar Typhimurium FLiC 427-441-specific T cells, and ?16
I-Abchicken ovalbumin (OVA) 323-339-specific T cells per
mouse (19). This same technique was then used to determine
numbers of pMHC-specific CD8?T cells for epitopes derived
from a variety of viruses and found 15 to 1,070 pMHC-specific
CD8?T cells per mouse, depending on the specificity of the
pMHC tetramer (10, 15, 23). Determinations of CD8?T-cell
precursor frequencies in humans are currently not experimen-
tally attainable, but exhaustive sequencing of an HLA-A2.1-
restricted influenza A virus (IAV) M1 58-66-specific T-cell
response has suggested that there are at least 141 different
clonotypes that can grow out in response to an in vitro stimu-
lation with peptide, providing a minimum number of T cells
that can respond to this pMHC complex in humans (22).
Most of the assays estimate the number of T cells specific to
single peptides in individual mice. These assays, therefore, do
not determine the numbers of CD8?T cells that can prolifer-
ate in response to an entire virus, especially if the virus is
known to have many epitopes or if epitopes for the virus have
not been described. By examining the average number of
pMHC-specific CD8?T cells in a naïve mouse and comparing
this to the number of pMHC-specific CD8?T cells that are in
a mouse at the peak of the T-cell response, it can be calculated
that CD8?T cells divide approximately 12 to 14 times after
virus infection (23). Considering that the progeny of one pre-
cursor after only 12 divisions can result in just over 4,000 cells,
and since recent experiments using H-2Kb-restricted chicken
OVA 257-264-specific OT-1-transgenic T cells have confirmed
that the progeny from a single cell can be detected in a mouse
after infection (31), an in vivo LDA was set up to take advan-
tage of the extensive division and proliferation of virus-specific
CD8?T cells in order to determine virus-specific CD8?T-cell
Here, we show that by transferring limiting amounts of car-
boxyfluorescein succinimidyl ester (CFSE)-labeled Thy1.1?
Ly5.2?heterogeneous CD8?T cells into Thy1.2?Ly5.1?
hosts, we are able to calculate CD8?T-cell precursor frequen-
cies for whole viruses. Our calculations are based on finding
the number of donor CD8?T cells that results in low-level-
CFSE (CFSElo) (i.e., proliferated) donor CD8 T cells in 50%
of the hosts. Using probit or Reed and Muench 50% endpoint
calculations (3, 26), we are able to calculate CD8?T-cell
precursor frequencies. We show here that frequencies of naïve
CD8?T-cell precursors for whole viruses are quite high and
that our in vivo LDA calculates whole-virus precursor frequen-
cies in line with determinations using other methods with naïve
and immune mice.
MATERIALS AND METHODS
Mice. B6.SJL (Ly5.1?Thy1.2?host) mice were used between 6 and 20 weeks
of age and were either obtained from Taconic Farms (Germantown, NY) or bred
in our own mouse-breeding colony. B6.Cg-IgHaThy-1aGPi-1a/J (Ly5.2?
Thy1.1?donor) mice were used at 6 to 32 weeks of age and bred in our own
mouse-breeding colony. Transgenic TCR-LCMV-P14 mice were used at 6 to 20
weeks of age and bred in our own mouse-breeding colony. All experiments were
done in compliance with the Institutional Animal Care and Use Committee of
the University of Massachusetts Medical School (Worcester, MA).
Viruses and viral infections. LCMV strain Armstrong was propagated in
BHK21 baby hamster kidney cells (34, 38). Vaccinia virus (VV) strain Western
Reserve was propagated on L929 cells (38). Mice were inoculated intraperito-
neally with 5 ? 104PFU of LCMV or 1 ? 106PFU VV in 0.2 ml for acute viral
infections, and some of these mice were tested for memory T-cell responses 3 to
6 months later.
CFSE label of mouse splenocytes. CFSE labeling of splenocytes was per-
formed as previously described (9, 16). Briefly, a single-cell suspension was
prepared from the spleen, red blood cells were lysed in an 0.84% NH4Cl solution,
and splenocytes were washed in cold Hank’s balanced salt solution (HBSS)
(Gibco ; Invitrogen, Carlsbad, CA) and resuspended in HBSS for count-
ing. Spleen leukocytes were then resuspended in a 2 ?M CFSE solution in HBSS
at 2 ? 107per ml and labeled for 15 min in a 37°C water bath, with mixing every
5 min. After CFSE labeling, cells were again washed twice with cold HBSS and
counted immediately before transfer. An aliquot of splenocytes was used for a
surface stain, and the rest of the splenocytes were diluted in HBSS for adoptive
FACS staining. Single-cell suspensions of the spleen, lymph nodes, bone mar-
row, blood, peritoneal cavity, and lungs (minced finely with a razor blade and
filtered) were prepared; red blood cells were lysed in an 0.84% NH4Cl solution;
and the leukocytes were then washed in RPMI 1640 medium (11875-093; Sigma-
Aldrich, St. Louis, MO). Cells were then counted by a hemacytometer and
resuspended in fluorescence-activated cell sorting (FACS) buffer for staining. Fc
receptors were blocked with antibody to CD16/CD32 (Fc? III/II receptor
; BD Biosciences, San Diego, CA), and cells were then stained in 96-well
plates. For the in vivo LDA, the single-cell suspension from each whole spleen
was divided into 8 to 16 wells of a 96-well plate for staining and later recombined
for analysis on an LSRII flow cytometer. After the surface stain with the indi-
cated antibodies, cells were either fixed using Cytofix (554655; BD) and resus-
pended in FACS buffer for analysis or, for intracellular assays, permeabilized
using Cytofix/Cytoperm (554722; BD) and stained intracellularly with the indi-
cated antibodies per the manufacturer’s instructions.
Antibodies and peptides. CD3ε phycoerythrin (PE)-Cy7 (552774; BD), CD8?
Pacific Blue (558106; BD), Thy1.1 PE (554898; BD), Ly5.2 peridinin chlorophyll
protein (PerCP)-Cy5.5 (552950; BD), and V?2 allophycocyanin (APC) (17-5812-
80; eBioscience) were used for in vivo LDAs. For the intracellular cytokine assay,
monoclonal antibody (MAb) to CD8? Alexa Fluor 700 (557956; BD), CD44
PE-Cy7 (25-0441-82; eBioscience, San Diego, CA), Thy1.1 PE (554898; BD), and
Ly5.2 PerCP-Cy5.5 (552950; BD) were used for the surface stain, and MAb to
IFN-? APC (554413; BD) was used for the intracellular stain. Peptides for
stimulation were purchased from 21st Century Biochemicals (Marlboro, MA).
For comparison of uninfected donor and host T-cell phenotypes, CD3ε PE Cy7
(552774; BD), CD8? Alexa Fluor 700 (557956; BD), Thy1.1 PE (554898; BD),
CD127 APC (17-1271-82; eBioscience), CD62L Pacific Blue (57-0621-82;
eBioscience), and CD44 PerCP-Cy5.5 (45-0441-82; eBioscience) were used.
Peptide-specific stimulations. Peptide stimulations were performed as previ-
ously described (33). Briefly, single-cell suspensions of lymphocytes were cul-
tured for 5 hours in the presence of 3 ?M of the indicated peptides (21st Century
Biochemicals) or purified MAb to CD3ε (1 ?g/ml) (553058; BD) for a polyclonal
stimulation, with human recombinant interleukin-2 (10 U/ml) and GolgiPlug
Determination of donor take. The take of Ly5.2?Thy1.1?donor CD3?CD8?
cells in Ly5.1?Thy1.2?host mice was determined by plotting the log10of the
number of donor CD3?CD8?cells transferred by the log10of the number of
CD3?CD8?donor cells recovered in the spleens of uninfected host mice. Mice
that received fewer than 1.25 ? 105splenocytes were not included in the analysis,
because at this number transferred, donor CD3?CD8?cells were not repro-
ducibly detectable in uninfected hosts. The resulting formula was then used to
calculate a percent take in the spleen. Then, using the assumption that 67% of
all CD3?CD8?cells in a naïve uninfected mouse reside in the spleen (6, 8), we
were able to calculate a total donor take.
In vivo LDA. Splenocytes were labeled with CFSE as described above and
diluted in HBSS to appropriate concentrations. Pilot experiments gave an indi-
12908 SEEDHOM ET AL.J. VIROL.
cation as to the limiting number of T cells that would need to be in host mice to
respond to each viral infection. To determine precursor frequencies, twofold
dilutions of splenocytes were made in HBSS. Each dilution was transferred into
four to six (usually five) host mice. In each experiment, one mouse at each
dilution was left uninfected to serve as a negative control. An infected mouse was
scored as a responder if donor Thy1.1?Ly5.2?CFSElo cells were detected after
FACS analysis to be above a determined threshold as described below. If there
were Thy1.1?Ly5.2?CFSElo cells in any dilution of any of the uninfected
animals, this number of cells was multiplied by three (with a correction for
number of cells collected), and this would serve as a responder cutoff. In in-
stances where there was no background detected by FACS in the uninfected mice
of the individual experiment, a cutoff of 10 CFSElo cells was used. The back-
ground value of 10 CFSElo cells was used because it was three times the average
number of CFSElo cells detected in all uninfected mice in all experiments, and
it was also the average number of CFSElo cells in uninfected mice that had
detectable CFSElo cells plus 1 standard deviation. After determination of re-
sponders versus nonresponders, probit and Reed and Muench 50% endpoint
analyses were performed, and the resulting number was multiplied by two to
determine the limiting number of transferred cells required to result in a re-
sponder ?100% of the time, i.e., the precursor frequency (3, 26). These two
analyses resulted in comparable but not identical precursor numbers. Almost all
individual experiments also included two control host mice that received adop-
tive transfers of a large number of donor splenocytes, with one mouse infected
and one uninfected, serving as positive and negative controls.
An in vivo LDA was designed to estimate the CD8?T-cell
precursor frequency for entire viruses. This was achieved by
adoptive transfer of donor splenocytes into host mice that
differed by using two congenic markers (Ly5 and Thy1) to
decrease the fluorescent background when donor CD8?T-cell
populations were stained for and by increasing the detection
limit of resultant T-cell progeny by counting only CD8?events
by FACS. By ignoring non-CD8?events, we increase the total
number of CD8?events that we were able to collect, and we
could collect just over 4 ? 106CD8?events, about one-fifth of
the total number of CD8?T cells in an uninfected animal
(assuming 2 ? 107CD8 T cells per mouse) and, because of
T-cell proliferation, approximately 5% of all CD3?CD8?
events in an LCMV- or VV-infected mouse. This allows reli-
able detection of donor CD8?T-cell progeny at limiting dilu-
The phenotype of adoptively transferred donor CD8?T cells
is naïve and shows linear take after transfer. To set up the in
vivo LDA, we verified that the adoptive transfer of donor cells
into host mice did not alter the phenotype of donor cells and
that adoptive transfer of decreasing numbers of T cells resulted
in a linear decrease of donor T cells in host mice. B6.Cg-IgHa
Thy-1aGPi-1a/J (Ly5.2?Thy1.1?donor) splenocytes were la-
beled with CFSE and diluted so that each mouse would receive
?5 ? 107splenocytes. Twofold dilutions of this stock were
made, and these samples were transferred intravenously into
groups of four B6.SJL (Ly5.1?Thy1.2?host) mice. After 3
days, mice were sacrificed and immunophenotyping of the
splenocytes was performed. Donor CD8?T cells had pheno-
types that remained largely naïve, with high levels of CD127
and CD62L and mostly low levels of CD44, and were similar to
the phenotype of host CD3?CD8?T cells (Fig. 1A). As
decreasing numbers of T cells were transferred into host mice,
the number of donor CD8?T cells detected in the spleen
linearly decreased, with an R2value of 0.994 (Fig. 1B).
Adoptively transferred CD8?T cells traffic to lymphoid
organs and peripheral sites at similar frequencies. To test
whether adoptively transferred T cells would traffic normally
throughout the body, Ly5.2?Thy1.1?donor splenocytes were
labeled with CFSE, and 1.35 ? 107of these cells was trans-
ferred into Ly5.1?Thy1.2?hosts. After 5 days, mice were
sacrificed, and FACS analysis of cells from lymphoid organs
FIG. 1. Adoptively transferred donor CD8?T cells dilute linearly in host mice and have a phenotype that is similar to that of host CD8?T cells.
(A) B6.Cg-IgHaThy-1aGPi-1a/J (Ly5.2?Thy1.1?donor) splenocytes were labeled with CFSE, three twofold dilutions were made (mice at the
highest dilution received 5 ? 107splenocytes), and each dilution was adoptively transferred into four B6.SJL (Ly5.1?Thy1.2?host) mice. On day
5, the mice were sacrificed and FACS analysis was performed on splenocytes. Immunophenotyping (CD62L, CD127, and CD44) of donor and host
CD8?T cells is shown. (B) The graph plots the number of donor CD8?T cells transferred into mice against the number of donor CD8?T cells
detected in the spleens of host mice.
VOL. 83, 2009 NUMBERS OF NAI¨VE CD8?T-CELL PRECURSORS 12909
was performed. We found that Ly5.2?Thy1.1?donor CD8?T
cells trafficked at similar frequencies to the mediastinal lymph
nodes, the axillary lymph nodes, the peribronchial lymph
nodes, the spleen, and, to some extent, the bone marrow (Fig.
2A). When total numbers of recovered Ly5.2?Thy1.1?donor
CD8?T cells in these tissues were counted, 65% of the recov-
ered donor CD8?T cells were in the spleen. In a separate,
similar experiment, peripheral sites were examined. There was
a reproducibly detectable number of donor T cells in periph-
eral sites such as the peritoneal cavity and lungs, although the
take in such peripheral sites was lower than that in lymphoid
sites such as the spleen or mediastinal lymph nodes (Fig. 2B).
Immunodominance hierarchies of host and donor CD8?T
cells are similar after LCMV or VV infection. To ensure that
transferred T cells had immunodominance hierarchies compa-
rable to the ones observed in normal C57BL/6J mice, we trans-
ferred large numbers (?5 ? 107) of CFSE-labeled Ly5.2?
Thy1.1?donor splenocytes into Ly5.1?Thy1.2?host mice,
waited 3 days, and infected the mice with LCMV. On day 7 of
LCMV infection, intracellular IFN-? assays were performed by
stimulating spleen cells with LCMV peptides or by polyclonal
stimulation with MAb to CD3ε. The percentages of host and
donor CD8?T cells that produced IFN-? when stimulated
with MAb to CD3ε or with LCMV-specific peptides GP33,
NP396, GP276, GP118, and NP205 were similar in donor and
host CD8?T cells (Fig. 3). Comparable experiments were
performed using VV, and the hierarchies of CD8?T cells that
produced IFN-? after polyclonal or VV-specific peptide stim-
ulation were also similar in donor and host CD8?T cells
(CD3ε ? B8R ? A47L) (data not shown).
In vivo LDA for virus-specific T cells. To determine virus-
specific CD8?T-cell precursor frequencies, graded amounts of
splenocytes were transferred into host mice at limiting-dilution
numbers that result in donor proliferated CD8?T cells in
?50% of hosts. Ly5.2?Thy1.1?donor splenocytes from unin-
fected mice were labeled with CFSE and transferred at de-
creasing numbers (5 ? 106, 2.5 ? 105, 1.25 ? 105, and 0.625 ?
105) into Ly5.1?Thy1.2?hosts. Pilot experiments had demon-
strated that in all host mice at the 5 ? 106dose, some donor
CD8?T cells proliferated, as shown by a CFSElo cell peak in
FIG. 2. Adoptively transferred donor CD8?T cells traffic to similar
frequencies to lymph organs and to peripheral sites. (A) Ly5.2?Thy1.1?
donor splenocytes were labeled with CFSE, and 1.35 ? 107splenocytes
were adoptively transferred into Ly5.1?Thy1.2?host mice. Five days
later, FACS analysis was performed on lymphocytes from the bone mar-
row, spleen, peribronchial lymph nodes (LN), axillary lymph nodes, and
mediastinal lymph nodes to examine donor CD8?T-cell take in lymph
nodes of host mice. (B) Ly5.2?Thy1.1?donor splenocytes were labeled
FACS analysis was performed on lymphocytes isolated from the spleen,
blood, peritoneal cavity, and lungs to examine donor CD8?T-cell take in
peripheral sites of host mice.
FIG. 3. Transferred donor and host CD8?T cells have similar
immunodominance hierarchies for LCMV and VV infections. Ly5.2?
Thy1.1?donor splenocytes (5 ? 107) were labeled with CFSE and
adoptively transferred into Ly5.1?Thy1.2?host mice, which were
subsequently infected with LCMV. Seven days later, mice were sacri-
ficed, splenocytes isolated, and peptide and polyclonal stimulations
performed, followed by an intracellular-cytokine stain for IFN-?.
FACS analysis was then performed. Results are plotted as percentages
of donor or host CD8?T cells that are IFN-? positive (IFNg?).
12910SEEDHOM ET AL.J. VIROL.
response to a VV infection, so this dilution was used in sub-
sequent experiments as a positive control. The 2.5 ? 105-,
1.25 ? 105-, and 0.625 ? 105-splenocyte dilutions resulted in
responders and nonresponders, so these dilutions were used
for in vivo LDA calculations. A small aliquot of the transferred
splenocytes was stained, and FACS analysis was performed to
determine the exact number of CD8?T cells transferred into
host mice. Previous experiments (data not shown) indicated
that VV-specific CD8?T-cell responses peaked at day 6, so on
day 6, mice were sacrificed and their splenocytes analyzed by
FACS under two conditions. For each spleen, a small aliquot
was run, analyzing all events to determine the CD3?CD8?
percentage, and then, to allow detection of the small number
of donor CD3?CD8?progeny at limiting dilutions, the rest of
the spleen was analyzed, with the threshold set to collect only
CD8?events. Figure 4A is an example of a transfer at limiting
dilution in VV-infected or uninfected mice. Responders versus
nonresponders were scored as described in Materials and
Methods. Figure 4B is an example of an in vivo LDA for VV,
where the numbers of responders per concentration are three
of four at the high concentration, two of four at the interme-
diate concentration, and one of four at the low concentration.
Figure 4C is an example of an in vivo LDA using Ly5.1?
H-2Db-restricted LCMV GP33-specific P14-transgenic T cells
(P14-transgenic T cells) transferred into C57BL/6J (Ly5.2?)
mice subsequently infected with LCMV (Fig. 4C). In this ex-
periment, the numbers of responders per concentration were
three of four at the high concentration, one of four at the
intermediate concentration, and zero of four at the lowest
Determination of donor take. One caveat for determination
of precursor frequencies by use of an adoptive transfer method
involves the donor take. Not all adoptively transferred donor
CD8?T cells survive in the host, and the percentage that does
survive in a host mouse has been referred to as the donor take.
Figure 5 is a graph of data from 91 uninfected mice and plots
the number of Ly5.2?Thy1.1?donor CD8?T cells transferred
into Ly5.1?Thy1.2?hosts against the number of Ly5.2?
Thy1.1?donor CD8?T cells found in the spleen. These data
give us a formula that allows us to determine a splenic donor
take. This take, along with the assumption that approximately
67% of CD8?T cells reside in the spleen, a number calculated
by others (6, 8), allows us to estimate a full mouse CD8?T-cell
take of approximately 3.8% at low cell numbers. This may be
FIG. 4. In vivo LDA. (A) Ly5.2?Thy1.1?donor splenocytes
(1.25 ? 105) were labeled with CFSE and adoptively transferred into
Ly5.1?Thy1.2?host mice, which were subsequently infected with VV.
Six days later, mice were sacrificed and stained for FACS analysis. The
gating scheme is shown for the in vivo LDA. Analysis was done (from
left to right) on singlets by gating on forward scatter area (FSC-A)
versus forward scatter width (FSC-W), on lymphocytes by gating on
FSC-A versus side scatter area (SSC-A), on CD8?T cells by gating on
CD3?CD8?cells, and on donor cells by Thy1.1?and Ly5.2?gates.
Panel ? represents an uninfected mouse at a limiting-dilution dose
(1.25 ? 105splenocytes) of donor CD8?T cells. Panels ?? and ??? are
examples of responders at this same dose, and panels IV and V are
examples of nonresponders. (B) Ly5.2?Thy1.1?donor splenocytes
were labeled with CFSE, two twofold dilutions made, and each dilution
was adoptively transferred into five Ly5.1?Thy1.2?host mice. At each
dilution, one mouse was left uninfected and the other mice were
infected with VV, and FACS analysis was performed on splenocytes as
described for panel A. Responder-versus-nonresponder determina-
tions were as described in Materials and Methods (6, 8). (C) Spleno-
cytes from Ly5.1?H-2Db-restricted LCMV GP33-specific P14-trans-
genic animals were labeled with CFSE, and twofold dilutions were
adoptively transferred into five C57Bl/6J (Ly5.2?Thy1.2?host) mice.
The gating scheme and responder-versus-nonresponder determina-
tions were as described for panel B.
VOL. 83, 2009NUMBERS OF NAI¨VE CD8?T-CELL PRECURSORS12911