high frequencies of virus-specific cd8 t-cell precursors

JOURNAL OF VIROLOGY, Dec. 2009, p. 12907–12916 Vol. 83, No. 240022-538X/09/$12.00 doi:10.1128/JVI.01722-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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,

Worcester, Massachusetts

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 ofpeptide-major histocompatibility complex-specific CD8� T cells in naïve mice, but precursor frequencies forentire 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 naïveCD8� T-cell precursors of 1 in 1,444 for vaccinia virus (VV) (�13,850 VV-specific CD8� T cells per mouse) and1 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, dramaticallyincreased to 1 in 13 (�1,538,462 VV-specific CD8� T cells per mouse), consistent with estimates of VV-specificmemory 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 everyT cell transferred underwent a full proliferative expansion in response to LCMV infection. This high efficiencywas 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 memoryCD8� T cell precursors for whole viruses can be remarkably high.

The immune response to a viral infection often involves therapid proliferation of CD8� effector T cells that recognizevirus-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-cellreceptors (TCRs) that are generated through largely randomDNA recombination events of the many TCR� and -� genes,encoding polypeptide chains that heterodimerize to form therecognition structure of T cells. The recombination of thesegments also involves addition or deletion of nucleotides dur-ing the joining process, causing even greater diversity, andthese processes allow for a very broad range of T-cell specific-ities, with a calculated theoretical diversity of �1015 TCRs inthe mouse (7). By use of PCR, CDR3 spectratyping, and se-quencing techniques, it was estimated that there are approxi-mately 2 � 106 distinct TCR specificities in a mouse spleen (1,5). This is far below the theoretical level of T-cell diversity, butconsidering estimates of T-cell degeneracy that propose thata single TCR can recognize up to 106 peptide-MHC (pMHC)complexes (17, 36), it is likely that the functional diversity ismuch greater than the number of individual TCRs.

It has been of interest to calculate the number of T cells thatwould either recognize or respond to a pathogen or to a spe-cific pMHC complex. Early estimates of numbers of CD8� Tcells 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 precursorfrequencies to be on the order of 1 in 100,000 in naïve mice andpredicted that these cells needed to undergo about 15 divisionsto reach the higher precursor frequencies found at day 8postinfection (29, 30). The efficiency of such assays, however, isrelatively 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 differentTCRs had been sequenced was reached, exhaustive sequencingwas then used to identify the frequencies of these sequencesin naïve mice. These studies found that there were about 600CD8� T cells specific for that pMHC complex in naïve mice(4). A second strategy used an in vivo competition assay withH-2Db-restricted lymphocytic choriomeningitis virus (LCMV)GP33-specific P14-transgenic T cells to estimate the number ofGP33-specific CD8 T cells in naïve mice and calculated thenumber 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 thathad 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-cellclones 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 thecentral nervous systems of acutely infected mice, with �100 to900 clonotypes calculated to be in chronically infected mice(24). Later studies used a gamma interferon (IFN-�) captureassay instead of tetramer sorting and estimated 1,100 to 1,500H-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: [email protected].

† Present address: Department of Immunology, University of Con-necticut Health Center, Farmington, CT.

� Published ahead of print on 7 October 2009.

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clonotypes of the subdominant H-2Kb-restricted MHV S598peptide-specific T cells in the spleens of acutely infected mice(25). Those studies also estimated that there were 1,000 to1,200 different H-2Db-restricted GP33-specific clonotypes thatcould respond to an LCMV infection.

More-recent studies have taken advantage of magnetic tet-ramer binding enrichment and double tetramer staining ofcells from the spleen and lymph nodes of naïve mice to deter-mine pMHC precursor frequencies, with the assumption thatmost CD8� T cells in a naïve mouse reside in lymphoid organsand will react with tetramers. This technique was first de-scribed by Moon et al. for CD4� T cells, and it detected �190I-Ab 2W1S 52-68-specific T cells, �20 I-Ab Salmonella entericaserovar Typhimurium FLiC 427-441-specific T cells, and �16I-Ab chicken ovalbumin (OVA) 323-339-specific T cells permouse (19). This same technique was then used to determinenumbers of pMHC-specific CD8� T cells for epitopes derivedfrom a variety of viruses and found 15 to 1,070 pMHC-specificCD8� T cells per mouse, depending on the specificity of thepMHC tetramer (10, 15, 23). Determinations of CD8� T-cellprecursor 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-cellresponse has suggested that there are at least 141 differentclonotypes that can grow out in response to an in vitro stimu-lation with peptide, providing a minimum number of T cellsthat can respond to this pMHC complex in humans (22).

Most of the assays estimate the number of T cells specific tosingle peptides in individual mice. These assays, therefore, donot determine the numbers of CD8� T cells that can prolifer-ate in response to an entire virus, especially if the virus isknown to have many epitopes or if epitopes for the virus havenot been described. By examining the average number ofpMHC-specific CD8� T cells in a naïve mouse and comparingthis to the number of pMHC-specific CD8� T cells that are ina mouse at the peak of the T-cell response, it can be calculatedthat CD8� T cells divide approximately 12 to 14 times aftervirus 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 chickenOVA 257-264-specific OT-1-transgenic T cells have confirmedthat the progeny from a single cell can be detected in a mouseafter infection (31), an in vivo LDA was set up to take advan-tage of the extensive division and proliferation of virus-specificCD8� T cells in order to determine virus-specific CD8� T-cellprecursor frequencies.

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 findingthe 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% endpointcalculations (3, 26), we are able to calculate CD8� T-cellprecursor frequencies. We show here that frequencies of naïveCD8� T-cell precursors for whole viruses are quite high andthat our in vivo LDA calculates whole-virus precursor frequen-cies in line with determinations using other methods with naïveand immune mice.

MATERIALS AND METHODS

Mice. B6.SJL (Ly5.1� Thy1.2� host) mice were used between 6 and 20 weeksof age and were either obtained from Taconic Farms (Germantown, NY) or bredin our own mouse-breeding colony. B6.Cg-IgHa Thy-1a GPi-1a/J (Ly5.2�

Thy1.1� donor) mice were used at 6 to 32 weeks of age and bred in our ownmouse-breeding colony. Transgenic TCR-LCMV-P14 mice were used at 6 to 20weeks of age and bred in our own mouse-breeding colony. All experiments weredone in compliance with the Institutional Animal Care and Use Committee ofthe University of Massachusetts Medical School (Worcester, MA).

Viruses and viral infections. LCMV strain Armstrong was propagated inBHK21 baby hamster kidney cells (34, 38). Vaccinia virus (VV) strain WesternReserve was propagated on L929 cells (38). Mice were inoculated intraperito-neally with 5 � 104 PFU of LCMV or 1 � 106 PFU VV in 0.2 ml for acute viralinfections, and some of these mice were tested for memory T-cell responses 3 to6 months later.

CFSE label of mouse splenocytes. CFSE labeling of splenocytes was per-formed as previously described (9, 16). Briefly, a single-cell suspension wasprepared 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 [14175]; Invitrogen, Carlsbad, CA) and resuspended in HBSS for count-ing. Spleen leukocytes were then resuspended in a 2 �M CFSE solution in HBSSat 2 � 107 per ml and labeled for 15 min in a 37°C water bath, with mixing every5 min. After CFSE labeling, cells were again washed twice with cold HBSS andcounted immediately before transfer. An aliquot of splenocytes was used for asurface stain, and the rest of the splenocytes were diluted in HBSS for adoptivetransfer.

FACS staining. Single-cell suspensions of the spleen, lymph nodes, bone mar-row, blood, peritoneal cavity, and lungs (minced finely with a razor blade andfiltered) 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 andresuspended in fluorescence-activated cell sorting (FACS) buffer for staining. Fcreceptors were blocked with antibody to CD16/CD32 (Fc� III/II receptor[553142]; BD Biosciences, San Diego, CA), and cells were then stained in 96-wellplates. For the in vivo LDA, the single-cell suspension from each whole spleenwas divided into 8 to 16 wells of a 96-well plate for staining and later recombinedfor 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, permeabilizedusing 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 chlorophyllprotein (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), CD44PE-Cy7 (25-0441-82; eBioscience, San Diego, CA), Thy1.1 PE (554898; BD), andLy5.2 PerCP-Cy5.5 (552950; BD) were used for the surface stain, and MAb toIFN-� APC (554413; BD) was used for the intracellular stain. Peptides forstimulation 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 CenturyBiochemicals) or purified MAb to CD3ε (1 �g/ml) (553058; BD) for a polyclonalstimulation, with human recombinant interleukin-2 (10 U/ml) and GolgiPlug(555029; BD).

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 log10 of thenumber of donor CD3� CD8� cells transferred by the log10 of the number ofCD3� CD8� donor cells recovered in the spleens of uninfected host mice. Micethat received fewer than 1.25 � 105 splenocytes 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 tocalculate a percent take in the spleen. Then, using the assumption that 67% ofall CD3� CD8� cells in a naïve uninfected mouse reside in the spleen (6, 8), wewere able to calculate a total donor take.

In vivo LDA. Splenocytes were labeled with CFSE as described above anddiluted in HBSS to appropriate concentrations. Pilot experiments gave an indi-

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cation as to the limiting number of T cells that would need to be in host mice torespond to each viral infection. To determine precursor frequencies, twofolddilutions of splenocytes were made in HBSS. Each dilution was transferred intofour to six (usually five) host mice. In each experiment, one mouse at eachdilution was left uninfected to serve as a negative control. An infected mouse wasscored as a responder if donor Thy1.1� Ly5.2� CFSElo cells were detected afterFACS analysis to be above a determined threshold as described below. If therewere Thy1.1� Ly5.2� CFSElo cells in any dilution of any of the uninfectedanimals, this number of cells was multiplied by three (with a correction fornumber 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 miceof 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 averagenumber of CFSElo cells detected in all uninfected mice in all experiments, andit was also the average number of CFSElo cells in uninfected mice that haddetectable CFSElo cells plus 1 standard deviation. After determination of re-sponders versus nonresponders, probit and Reed and Muench 50% endpointanalyses were performed, and the resulting number was multiplied by two todetermine 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 twoanalyses resulted in comparable but not identical precursor numbers. Almost allindividual experiments also included two control host mice that received adop-tive transfers of a large number of donor splenocytes, with one mouse infectedand one uninfected, serving as positive and negative controls.

RESULTS

An in vivo LDA was designed to estimate the CD8� T-cellprecursor frequency for entire viruses. This was achieved byadoptive transfer of donor splenocytes into host mice thatdiffered by using two congenic markers (Ly5 and Thy1) todecrease the fluorescent background when donor CD8� T-cellpopulations were stained for and by increasing the detectionlimit of resultant T-cell progeny by counting only CD8� eventsby FACS. By ignoring non-CD8� events, we increase the totalnumber of CD8� events that we were able to collect, and wecould collect just over 4 � 106 CD8� events, about one-fifth of

the total number of CD8� T cells in an uninfected animal(assuming 2 � 107 CD8 T cells per mouse) and, because ofT-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-tions.

The phenotype of adoptively transferred donor CD8� T cellsis naïve and shows linear take after transfer. To set up the invivo LDA, we verified that the adoptive transfer of donor cellsinto host mice did not alter the phenotype of donor cells andthat adoptive transfer of decreasing numbers of T cells resultedin a linear decrease of donor T cells in host mice. B6.Cg-IgHa

Thy-1a GPi-1a/J (Ly5.2� Thy1.1� donor) splenocytes were la-beled with CFSE and diluted so that each mouse would receive�5 � 107 splenocytes. Twofold dilutions of this stock weremade, and these samples were transferred intravenously intogroups of four B6.SJL (Ly5.1� Thy1.2� host) mice. After 3days, mice were sacrificed and immunophenotyping of thesplenocytes was performed. Donor CD8� T cells had pheno-types that remained largely naïve, with high levels of CD127and CD62L and mostly low levels of CD44, and were similar tothe phenotype of host CD3� CD8� T cells (Fig. 1A). Asdecreasing numbers of T cells were transferred into host mice,the number of donor CD8� T cells detected in the spleenlinearly decreased, with an R2 value of 0.994 (Fig. 1B).

Adoptively transferred CD8� T cells traffic to lymphoidorgans and peripheral sites at similar frequencies. To testwhether adoptively transferred T cells would traffic normallythroughout the body, Ly5.2� Thy1.1� donor splenocytes werelabeled with CFSE, and 1.35 � 107 of these cells was trans-ferred into Ly5.1� Thy1.2� hosts. After 5 days, mice weresacrificed, 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-IgHa Thy-1a GPi-1a/J (Ly5.2� Thy1.1� donor) splenocytes were labeled with CFSE, three twofold dilutions were made (mice at thehighest dilution received 5 � 107 splenocytes), and each dilution was adoptively transferred into four B6.SJL (Ly5.1� Thy1.2� host) mice. On day5, the mice were sacrificed and FACS analysis was performed on splenocytes. Immunophenotyping (CD62L, CD127, and CD44) of donor and hostCD8� 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 cellsdetected in the spleens of host mice.

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was performed. We found that Ly5.2� Thy1.1� donor CD8� Tcells trafficked at similar frequencies to the mediastinal lymphnodes, the axillary lymph nodes, the peribronchial lymphnodes, the spleen, and, to some extent, the bone marrow (Fig.

2A). When total numbers of recovered Ly5.2� Thy1.1� donorCD8� 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 wasa reproducibly detectable number of donor T cells in periph-eral sites such as the peritoneal cavity and lungs, although thetake in such peripheral sites was lower than that in lymphoidsites such as the spleen or mediastinal lymph nodes (Fig. 2B).

Immunodominance hierarchies of host and donor CD8� Tcells are similar after LCMV or VV infection. To ensure thattransferred 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 ofLCMV infection, intracellular IFN-� assays were performed bystimulating spleen cells with LCMV peptides or by polyclonalstimulation with MAb to CD3ε. The percentages of host anddonor CD8� T cells that produced IFN-� when stimulatedwith MAb to CD3ε or with LCMV-specific peptides GP33,NP396, GP276, GP118, and NP205 were similar in donor andhost CD8� T cells (Fig. 3). Comparable experiments wereperformed using VV, and the hierarchies of CD8� T cells thatproduced 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 ofsplenocytes were transferred into host mice at limiting-dilutionnumbers 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 � 106 dose, some donorCD8� T cells proliferated, as shown by a CFSElo cell peak in

FIG. 2. Adoptively transferred donor CD8� T cells traffic to similarfrequencies to lymph organs and to peripheral sites. (A) Ly5.2� Thy1.1�

donor splenocytes were labeled with CFSE, and 1.35 � 107 splenocyteswere adoptively transferred into Ly5.1� Thy1.2� host mice. Five dayslater, FACS analysis was performed on lymphocytes from the bone mar-row, spleen, peribronchial lymph nodes (LN), axillary lymph nodes, andmediastinal lymph nodes to examine donor CD8� T-cell take in lymphnodes of host mice. (B) Ly5.2� Thy1.1� donor splenocytes were labeledwith CFSE and adoptively transferred into Ly5.1� Thy1.2� host mice, andFACS analysis was performed on lymphocytes isolated from the spleen,blood, peritoneal cavity, and lungs to examine donor CD8� T-cell take inperipheral sites of host mice.

FIG. 3. Transferred donor and host CD8� T cells have similarimmunodominance hierarchies for LCMV and VV infections. Ly5.2�

Thy1.1� donor splenocytes (5 � 107) were labeled with CFSE andadoptively transferred into Ly5.1� Thy1.2� host mice, which weresubsequently infected with LCMV. Seven days later, mice were sacri-ficed, splenocytes isolated, and peptide and polyclonal stimulationsperformed, followed by an intracellular-cytokine stain for IFN-�.FACS analysis was then performed. Results are plotted as percentagesof donor or host CD8� T cells that are IFN-� positive (IFNg�).



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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 inresponders and nonresponders, so these dilutions were usedfor in vivo LDA calculations. A small aliquot of the transferredsplenocytes was stained, and FACS analysis was performed todetermine the exact number of CD8� T cells transferred intohost mice. Previous experiments (data not shown) indicatedthat VV-specific CD8� T-cell responses peaked at day 6, so onday 6, mice were sacrificed and their splenocytes analyzed byFACS under two conditions. For each spleen, a small aliquotwas run, analyzing all events to determine the CD3� CD8�

percentage, and then, to allow detection of the small numberof donor CD3� CD8� progeny at limiting dilutions, the rest ofthe spleen was analyzed, with the threshold set to collect onlyCD8� events. Figure 4A is an example of a transfer at limitingdilution in VV-infected or uninfected mice. Responders versusnonresponders were scored as described in Materials andMethods. Figure 4B is an example of an in vivo LDA for VV,where the numbers of responders per concentration are threeof 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 werethree of four at the high concentration, one of four at theintermediate concentration, and zero of four at the lowestconcentration.

Determination of donor take. One caveat for determinationof precursor frequencies by use of an adoptive transfer methodinvolves the donor take. Not all adoptively transferred donorCD8� T cells survive in the host, and the percentage that doessurvive in a host mouse has been referred to as the donor take.Figure 5 is a graph of data from 91 uninfected mice and plotsthe number of Ly5.2� Thy1.1� donor CD8� T cells transferredinto Ly5.1� Thy1.2� hosts against the number of Ly5.2�

Thy1.1� donor CD8� T cells found in the spleen. These datagive us a formula that allows us to determine a splenic donortake. This take, along with the assumption that approximately67% of CD8� T cells reside in the spleen, a number calculatedby others (6, 8), allows us to estimate a full mouse CD8� T-celltake 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 intoLy5.1� Thy1.2� host mice, which were subsequently infected with VV.Six days later, mice were sacrificed and stained for FACS analysis. Thegating scheme is shown for the in vivo LDA. Analysis was done (fromleft to right) on singlets by gating on forward scatter area (FSC-A)versus forward scatter width (FSC-W), on lymphocytes by gating onFSC-A versus side scatter area (SSC-A), on CD8� T cells by gating onCD3� 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 � 105 splenocytes) of donor CD8� T cells. Panels and areexamples of responders at this same dose, and panels IV and V areexamples of nonresponders. (B) Ly5.2� Thy1.1� donor splenocyteswere labeled with CFSE, two twofold dilutions made, and each dilutionwas adoptively transferred into five Ly5.1� Thy1.2� host mice. At eachdilution, one mouse was left uninfected and the other mice wereinfected with VV, and FACS analysis was performed on splenocytes asdescribed 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 wereadoptively 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.



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a slight underestimate in comparison to our own results sug-gesting that less than 65% of donor CD3� CD8� cells reside inthe spleen (Fig. 2).

Precursor frequency determination. Using multiple in vivoLDAs, our take value, and probit analysis for 50% endpointtimes 2 (3), we determined T-cell precursor frequencies innaïve and immune mice as shown in Table 1. There were about1 in 2,958 392 CD8� T cells in naïve mice that proliferatedin response to LCMV, while there were almost twice as manyCD8� T cells, 1 in 1,444 171, that proliferated in response toVV (P � 0.0001 in comparison to LCMV precursors). Thenumber of CD8� T cells in VV-immune mice that proliferatedin response to VV was, as expected, greatly increased, with 1 in13 2 CD8� T cells able to proliferate in response to thishomologous infection (P � 0.0001 in comparison to the naïveimmune state). As expected, the LCMV-specific CD8� T-cellprecursor frequency was not elevated in VV-immune mice; infact, it was slightly, although significantly, decreased, withabout 1 in 4,425 1,705 CD8� T cells proliferating (P � 0.05in comparison to the naïve immune state).

We calculated the number of P14-transgenic T cells thatresponded to an LCMV infection by our in vivo LDA anddetermined a frequency by probit analysis of 1 in 0.93 0.04.This suggests a virtually 100% efficiency in the outgrowth ofthe transgenic T cells and reinforces the calculations that wehave made concerning T-cell take after transfer.

We also employed the commonly used Reed and Muench50% endpoint analysis to determine precursor frequencies (26)and found that these calculations resulted in precursor fre-quencies comparable but not identical to those obtained withthe probit method. By Reed and Muench analysis, naïve micehad 1 in 3,121 291 CD8� T cells specific to LCMV and 1 in1,615 409 CD8� T cells specific to VV, while in VV-immunemice, 1 in 3,956 787 were specific to LCMV, and 1 in 13 1 were specific to VV in these immune mice. Using the Reedand Muench method, we calculated that 1 in 1.22 0.11P14-transgenic T cells responded to an LCMV infection, againa figure close to 100%.

A summary of C57BL/6J mouse precursor frequency deter-

minations by different methods (2, 10, 12, 15, 19, 23–25, 27, 35)is given in Table 2 and is discussed further below.

DISCUSSION

The broad possible pMHC reactivity generated by randomgene rearrangements of � and � TCR chains on T cells wouldseem to ensure reactivity against a diverse array of pathogens,but this broad diversity then raises the question of how many Tcells in a host would respond to a specific pMHC complex oragainst an entire pathogen. Many determinations of CD8�

T-cell precursor frequencies have now been used to calculatethe number of pMHC-specific CD8� T cells within a mouse(Table 2), but the determination of the total number of CD8�

T cells that could respond to a viral infection would be possiblewith these methods only if all of the epitopes of the virus wereknown. However, the numbers of epitopes found to stimulateT-cell responses are now becoming quite large, as means fordetecting them have become more sensitive. A virus like VV isnow reported to encode close to 50 H-2Kb- and H-2Db-re-stricted epitopes (20), a number that would make it very dif-ficult, if not prohibitive, if the precursor frequency for theentire virus was to be determined with the usual methods.Herein, we have described an in vivo LDA that allows an

FIG. 5. Determination of donor take. The number of donor CD8�

T cells transferred was plotted against the number of donor CD8� Tcells detected in host spleens after transfer. The graph is based on 91uninfected mice used in 26 different experiments. Donor take wascalculated by using the equation generated and the assumption thatsplenic CD8� T cells account for 67% of CD8� T cells.

TABLE 1. Precursor frequencies for naıve and immune states asdetermined by in vivo LDA

Immune state Virus

Precursor frequency calculated byindicated method

Reed and Muench Probit

Naıve LCMV 1/2,880 1/3,1431/3,092 1/2,8321/2,899 1/3,4271/3,461 1/2,8521/3,601 1/3,2041/2,965 1/3,0471/2,946 1/2,202

1/3,121 291 1/2,958 392

Naıve VV 1/1,081 1/1,1421/2,010 1/1,5341/1,280 1/1,4921/1,811 1/1,4921/1,892 1/1,559

1/1,615 409 1/1,444 171

VV-immune LCMV 1/3,708 1/6,7211/3,917 1/6,4561/5,519 1/4,9971/3,606 1/2,9181/3,622 1/2,9851/3,362 1/3,294

1/3,956 787 1/4,562 1,824

VV-immune VV 1/11 1/151/14 1/141/12 1/131/14 1/101/13 1 1/13 2

P14-transgenic LCMV 1/1.34 1/0.921/1.19 1/0.981/1.12 1/0.89

1/1.22 0.11 1/0.93 0.04



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unbiased determination of precursor frequencies for entireviruses without any knowledge of the specificity or number ofepitopes.

It is interesting to note the differences calculated for T-cellprecursor frequencies depending on the method used (Table2). Extrapolation of V� clonotype number per spleen by cal-culation of a measure of diversity by examination of the CDR3sequences of pMHC-specific populations as done previously(12, 24, 25) gives precursor frequencies on the high ends ofmost estimates, with the highest number of H-2Db LCMVGP33-specific CD8� T cells calculated among all methods.This is interesting in that this method is assumed to be anunderestimate, because it does not include TCR� diversity,and it also does not account well for redundancy in T-cellpopulations, i.e., there may be more than one cell of a singleclone in a naïve mouse. However, there is some uncertainty inthese numbers because they rely on extrapolations of the num-

bers and diversities of sequenced clones to estimate T-cellprecursor frequencies. Compared to our results, those ob-tained with this extrapolation method would suggest our cal-culations to be slight underestimates. If there are 1,100 to 1,200GP33-specific CD8� T cells per naïve mouse spleen, and theGP33-specific CD8� T-cell response is approximately 10% ofthe total LCMV-specific response, we would expect to findabout twice as many LCMV-specific precursors. Instead of the6,760 per mouse as we calculated, we would expect 11,000 to12,000 LCMV-specific CD8� T cells.

If precursor frequencies are instead calculated by trans-genic-T-cell competition (2), where transferred monoclonaltransgenic T cells compete against heterogeneous endogenousT-cell populations to determine precursor frequencies, ourresults look to be overestimates. Assuming a 10% take and 2 �107 CD8� T cells per mouse, this method estimates �100H-2Db LCMV GP33-specific CD8� T cells per mouse (2). This

TABLE 2. Precursor frequencies in C57BL/6J mice

Immune state Specificity region Total no. ofprecursors/mouse

Referenceor source Technique

Naıve H2Db LCMV GP33-41 100–200 2 T-cell transgenic competitionH-2Db LCMV GP33-41 1,000–1,200b 25 Diversity estimate by CDR3

sequencingH-2Db LCMV GP33-41 �287 23 Tetramer pulldownH-2Db LCMV GP33-41 �449 15 Tetramer pulldownH-2Db LCMV NP396-404 �151 23 Tetramer pulldownH-2Db LCMV NP396-404 �117 15 Tetramer pulldownH-2Kb LCMV L2062-2069 �90 15 Tetramer pulldownH-2Kb LCMV NP205-212 �57 15 Tetramer pulldownH-2Kb LCMV GP118-125 �43 15 Tetramer pulldownH-2Kb LCMV L156-163 �24 15 Tetramer pulldownH-2Db LCMV L338-346 �15 15 Tetramer pulldownI-Ab LCMV GP61-80 �100 35 T-cell transgenic competition

Naıve LCMV �6,760 Our results In vivo LDAVV-immune LCMV �4,384 Our results In vivo LDA

Naıve H-2Db MHV-JHM S510-518 300–500a 24 Diversity estimate by CDR3sequencing

H-2Db MHV-JHM S510-518 1,100–1,500b 25 Diversity estimate by CDR3sequencing

H-2Kb MHV-JHM S598-605 600–900b 25 Diversity estimate by CDR3sequencing

H-2Db IAV NP366-374 50–300b 12 Diversity estimate by CDR3sequencing

H-2Db IAV PA224-233 350–600b 12 Diversity estimate by CDR3sequencing

H-2Db IAV PA224-233 �120 23 Tetramer pulldownH-2Kb VV B8R20-27 �1,070 10 Tetramer pulldown

Naıve VV �13,850 Our results In vivo LDAVV-immune VV �1,538,500 Our results In vivo LDA

Naıve I-Ab chicken OVA 323-339 �16 19 Tetramer pulldownH-2Kb chicken OVA 257-264 �130 23 Tetramer pulldownH-2Kb chicken OVA 257-264 �170 10 Tetramer pulldownI-Ab S. enterica serovar Typhimurium

FLiC 427-441�20 19 Tetramer pulldown

H-2Db MCMV M45 985-993 �603 23 Tetramer pulldownH-2Kb vesicular stomatitis virus N52-59 �166 23 Tetramer pulldownH-2Kb HSV-1 gB498-505 �489 10 Tetramer pulldownH-2Db Mgp100 25-33 �20 27 T-cell transgenic competitionI-Ab 2W1S 52-68 �190 19 Tetramer pulldown

a Number of clonotypes per infected central nervous system.b Number of clonotypes per infected spleen.



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result would put our LCMV-specific CD8� T-cell precursordetermination on the high end.

The tetramer-based enrichment assay, which makes use ofpMHC tetramers, magnetic-bead enrichment and double-tet-ramer FACS staining of spleens and lymph nodes to identifypMHC-specific CD8� T cells (assuming that most naïve T cellsreside in lymph organs), seems to yield numbers that are in linewith results determined by our in vivo LDA. Depending on theindividual determination, there are �287 (23) or �449 (15)H-2Db LCMV GP33-specific CD8� T cells in all lymph organsof a naïve mouse, and this result would be on the low end yetstill compatible with what our results might predict for a fre-quency of GP33-specific naïve CD8� T cell precursors. ForVV, the in vivo LDA calculates about 13,850 responsive CD8�

T cells per C57BL/6J mouse. The tetramer-based enrichmentassay estimated 1,070 CD8� T cells specific for the H-2Kb-restricted VV B8R epitope in the spleen, lymph nodes, andovaries (10), and those results would seem consistent with theresults that we have described, considering that the B8R pep-tide response may represent about 10% of the VV-inducedresponse.

About twice as many T cells were responsive to VV than toLCMV (P � 0.0001). This might in part reflect the observa-tions that the T-cell response to VV peaks earlier than that ofLCMV. Having more CD8� T cells that are specific to VV mayincrease the likelihood that VV-specific CD8� T cells interactwith stimulating antigen-presenting cells earlier, allowing peakT-cell proliferation to occur earlier. VV also encodes moreproteins than LCMV, with almost twice as many VV epitopesdescribed to occur in the C57BL/6J mouse, consistent with theresult showing almost twice as many VV-specific precursor Tcells than LCMV-specific CD8� T cells (14, 20).

Our results also estimate that 8% of CD8� T cells in VV-immune mice are VV responsive, and these data are supportedby results obtained from VV-immune mice by using peptidestimulations and intracellular cytokine stains that estimate thatanywhere from 2 to 11% of CD8� T cells are specific to theVV-encoded immunodominant B8R epitope in D21 or D40VV-immune mice (28, 32), and our own results from intracel-lular cytokine assays estimate that 0.5 to 2% of CD8� T cellsin VV-immune mice are specific to the VV B8R epitope at 3 to8 months postinfection (data not shown). This increase inCD8� T-cell precursor frequency for VV in VV-immune ani-mals by more than 2 orders of magnitude (P � 0.0001) dem-onstrates the expected considerable increase of VV-specificmemory CD8� T cells after VV infection. As expected, therewas no increase in the number of CD8� T cells that respond toLCMV in VV-immune mice, and this helps to validate thespecificity of our assay. The small but significant decrease inLCMV CD8� T-cell precursor frequency in VV-immune miceis interesting and may suggest that memory cells may displacesome naïve cells in the immune response. We have not system-atically addressed changes in VV-specific precursors in LCMV-immune mice because there is a high degree of heterologousimmunity in this virus sequence, and the immunity, due toprivate specificities in the immune repertoire, has such highvariability that our in vivo LDA would likely suffer from re-producibility issues (13).

It is possible to make an approximation of the number ofdivisions a CD8� T cell undergoes after stimulation by exam-

ining the burst size or recovered cell number at the limitingdilution. By determining the frequency of CFSElo donor cellsamong all CD8� events collected, multiplying that frequencyby the total number of CD8� T cells found in the spleen, andthen multiplying that number in accordance with the assump-tion that 67% of all CD8� T cells are present in the spleenduring infection, we are able to calculate the approximatenumber of divisions a CD8� T cell undergoes after virus in-fection. The numbers of divisions that a VV-specific precursorundergoes by day 6 (�11 divisions) and that an LCMV-specificprecursor undergoes by day 7 (�12 or 13 divisions) fall withinpredicted ranges. However, we approximate that a P14-trans-genic T cell undergoes �14 divisions by day 7 of an LCMVinfection, and this is significantly different (P � 0.023) from thenumber (�12 or 13 divisions) that a naïve CD8� T cell from aheterogeneous population of T cells undergoes. This may re-flect differences in avidity between the transgenic T-cell pop-ulation and the expected large range of avidities of T cells in aheterogeneous population as a whole or may instead be relatedto the examination of a monoclonal T-cell population thatresponds to a highly expressed immunodominant epitope ver-sus a heterogeneous population of CD8� T cells that containsT cells responding to immunodominant and subdominantepitopes.

The immunological environment produced by a specific vi-rus infection can have a profound impact on the burst sizes ofepitope-specific T cells, as has been demonstrated by experi-ments examining the T-cell response to recombinant virusesengineered to express the same T-cell epitope (21). One ex-planation for this would be the expression of insufficient anti-gen to engage all of the T-cell precursors, as shown previously(11, 18). However, we used doses of virus that maximized theburst of the T-cell response, as in Ref (11), and we feel thatvirtually all the precursors should have been engaged. Finally,T cells of differing affinities may undergo different numbers ofdivisions and expand to different peak sizes, as recently dem-onstrated (39). Since the in vivo LDA requires extensive pro-liferation, it is possible that we have missed lower-affinityclones in our assay that would divide fewer than seven or eighttimes, and this would make our calculated precursor frequen-cies underestimates. However, analyses of T-cell responses toseveral epitopes suggest at least 12 to 14 divisions per T cell(23), which would be detected by our assays. Further, our invivo LDA seems to be able to detect the bulk of memory T-cellprecursors detected by intracellular IFN-� assays.

Our calculated take of CD8� T cells is not the normallyquoted 10% figure. If we instead use a 10% value for take, theprecursor frequencies would be decreased with a naïve mousehaving about 1 in 7,805 1,034 CD8� T cells specific forLCMV and 1 in 3,809 452 CD8� T cells specific for VV anda VV-immune mouse having about 1 in 34 6 and 1 in 12,036 4,812 CD8 T� cells specific for VV and LCMV, respectively.Our own rough estimate of total cell numbers (Fig. 2) wouldsuggest that slightly less than 67% of CD8� T cells reside in thespleen, as had been suggested (8), but given that our attemptto count T cells throughout the body was not exhaustive andthat the take would probably only change by at most a 20 to30% value, we remain confident that our calculations arewithin a reasonable range of total virus-specific CD8� T-cellprecursor frequencies. Further, our experiments using P14-



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transgenic T cells also strongly support our estimation of takesince our calculations of numbers of precursors equals thenumber of transgenic T cells obtained using that take value.The efficiencies of T-cell take in our experiments may seem tobe in conflict with studies by others using OT-1-transgenic Tcells, where 25% of the mice injected with a single OT-1-transgenic T cell had detectable responding donor T cells (31).However, these single cells were injected intraperitoneally, fol-lowed by an immediate intraperitoneal infection. We, instead,chose to allow for a total-body distribution of T cells by way ofan intravenous transfer and challenged mice with virus 2 to 5days later. We therefore remain confident of our take valueunder the conditions of our system. Further, this in vivo LDAcalculated at a 3.8% take gives frequencies with high concor-dance with the anticipated number of VV-specific memorycells, which can be measured directly by intracellular-cytokineassays. Considering the large amount of data we have in gen-erating the 3.8% figure (Fig. 5), we believe that our estimate oftake is reasonably accurate in these experiments.

The in vivo LDA could be used to examine virus-specificT-cell precursor frequencies in mice of different ages or phys-iological states or in mice with histories of different infections.For example, a decline in IAV-specific repertoire diversityleading to epitope-specific holes in the repertoire in aged micewas recently reported (37). Future experiments may be able touse the in vivo LDA described herein to determine whetherwhole-virus T-cell precursor frequencies in naïve or immuneaged mice are also changed.

Whereas tetramer-based enrichment assays measure thenumber of cells that are reactive to a particular pMHC com-plex, the in vivo LDA requires CD8� T-cell division and pro-liferation, measuring instead the number of CD8� T cells thatdo proliferate in response to a viral infection. It might seemlikely that not all virus-specific T cells would react with tet-ramer and that not all tetramer-specific T cells would be ca-pable of proliferating in response to their cognate antigen.Remarkably, though, while the in vivo LDA described heretells us something different than the tetramer-based enrich-ment assays described recently, it gives reasonable concor-dance with those techniques.

ACKNOWLEDGMENTS

This work was supported by U.S. National Institutes of Healthresearch grants U19-AI-057330, RO1-AR-35506, and R37-AI-17672and training grant T32 AI07349.

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high frequencies of virus-specific cd8 t-cell precursors

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