of November 20, 2014.This information is current as

Develop into MemoryγIFN-Early Effector T Cells Producing Significant

Yu, Christine M. Hoeman and Habib ZaghouaniPingM. Tartar, Hyun-Hee Lee, Rohit D. Divekar, Renu Jain,

J. Jeremiah Bell, Jason S. Ellis, F. Betul Guloglu, Danielle

http://www.jimmunol.org/content/180/1/179doi: 10.4049/jimmunol.180.1.179

2008; 180:179-187; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/180/1/179.full#ref-list-1

, 20 of which you can access for free at: cites 44 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Early Effector T Cells Producing Significant IFN-� Developinto Memory1

J. Jeremiah Bell,2 Jason S. Ellis, F. Betul Guloglu, Danielle M. Tartar, Hyun-Hee Lee,3

Rohit D. Divekar, Renu Jain, Ping Yu,4 Christine M. Hoeman, and Habib Zaghouani5

Currently, transition of T cells from effector to memory is believed to occur as a consequence of exposure to residual suboptimalAg found in lymphoid tissues at the waning end of the effector phase and microbial clearance. This led to the interpretation thatmemory arises from slightly activated late effectors producing reduced amounts of IFN-�. In this study, we show that CD4 T cellsfrom the early stage of the effector phase in which both the Ag and activation are optimal also transit to memory. Moreover, earlyeffector T cells that have undergone four divisions expressed significant IL-7R, produced IFN-�, and yielded rapid and robustmemory responses. Cells that divided three times that had marginal IL-7R expression and no IFN-� raised base level homeostaticmemory, whereas those that have undergone only two divisions and produced IFN-� yielded conditioned memory despite lowIL-7R expression. Thus, highly activated early effectors generated under short exposure to optimal Ag in vivo develop intomemory, and such transition is dependent on a significant production of the cell’s signature cytokine, IFN-�. The Journal ofImmunology, 2008, 180: 179–187.

M emory is one of the cardinal features associated withthe adaptive immune response and provides the fun-damental basis for the development of vaccines (1–5).

Usually, an initial exposure to a microbe triggers a primary re-sponse comprising pathogen-specific effector lymphocytes, mostof which engage in the clearance of the pathogen (1–6). However,very few of these effector cells evolve as long-lasting memorylymphocytes (1, 6). These cells then contribute a substantial in-crease in the frequency of pathogen-specific cells within the lym-phocyte repertoire (7, 8) and mount a rapid and robust responsehighly effective for protection against reinfection (9–12). To date,very little information is available on the initial events that dictatewhether a cell in the primary response should simply be an effectorcell programmed to die shortly after microbial clearance or differ-entiate into a long-lived memory cell (3, 13–15). This is mostlydue to the low frequency of T cells that commit to memory duringthe effector phase and the lack of specific markers that could tracethe effector to memory transition (1, 10, 16, 17). Lately, it has beensuggested that memory development occurs as a result of continualexposure to low amounts of Ag that persist subsequent to viral

clearance (18). Also, late arrival of T cells to local lymph nodes(LN),6 which subjects the lymphocytes to suboptimal residual Ag,leads to the generation of memory (19). These observations raisethe question as to whether the immune system can generate mem-ory against acute infections in which the lymphocytes could beexposed to an optimal dose of Ag for a short period early in theeffector phase. In other words, do highly activated T cells transitinto memory? To address this premise, we designed a model inwhich naive lymphocytes are exposed to an optimal dose of Ag fora limited time during the initial period of the effector phase andtested for development of memory. Accordingly, OVA-specificTCR transgenic T cells from DO11.10/scid mice (20) were labeledwith the intracellular fluorescent dye, CFSE (21), and transferred intocompatible BALB/c mice, and the recipients were immunizedwith a previously defined optimal dose of OVA peptide. The di-viding effector LN cells were isolated within 48 h of immuniza-tion, sorted on the basis of cell division, which is indicative of theirstate of activation, and transferred into RAG-2-deficient (RAG-2�/�) BALB/c mice for parking without Ag. The memory re-sponse from each sorted division was then quantified over timebefore and after immunization of the hosts with a suboptimal doseof OVA. The rationale for parking the effector cells in a RAG-2�/� host stems from the following reasoning: the lymphopenicenvironment should sustain survival of memory precursors thatarise at a very low frequency (22–24), thereby making the readoutof memory responses feasible. Second, the effector cells of alldivisions are subjected to identical survival mechanisms in theabsence of Ag, and any difference in the memory responses willsolely reflect how each cell division affects memory development.The results indicated that the effector division D4, which includedcells that have undergone four divisions, express IL-7R, and pro-duce IFN-�, gave rise to a high frequency of memory lymphocytesthat sustained a rapid and robust memory response in both the

Department of Molecular Microbiology and Immunology, University of MissouriSchool of Medicine, Columbia, MO 65212

Received for publication April 11, 2007. Accepted for publication October 27, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grants R01 AI48541 and 2R01 NS37406 (to H.Z.)from the National Institutes of Health. J.J.B., J.S.E., and C.M.H. were supported bytraining grants from National Institute of General Medical Sciences. D.M.T. wassupported by Life Sciences fellowship from University of Missouri.2 Current address: Department of Pathology and Laboratory Medicine, University ofPennsylvania School of Medicine, Philadelphia, PA 19104.3 Current address: Division of Immunology, Karp Laboratories, Children’s Hospital,Harvard Medical School, Boston, MA 02115.4 Current address: National Cancer Institute, Metabolism Branch, Bethesda, MD20892.5 Address correspondence and reprint requests to Dr. Habib Zaghouani, Departmentof Molecular Microbiology and Immunology, University of Missouri School of Med-icine, M616 Medical Sciences Building, Columbia, MO 65212. E-mail address:zaghouanih@health.missouri.edu

6 Abbreviations used in this paper: LN, lymph node; HA, hemagglutinin; SP, spleen;TCoM, conditioned memory T cell; THM, homeostatic memory T cell; TRM, rapidmemory T cell.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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spleen (SP) and LN. D3 effectors, which divided three times, ex-pressed minimal IL-7R, and did not produce IFN-�, yielded baselevel homeostatic memory responses in both lymphoid organs. D2cells that divided only two times expressed low IL-7R, but pro-duced significant IFN-�, and yielded conditioned memory re-sponses. Thus, early effectors can develop into memory, and suchtransition is dependent on their ability to produce IFN-�.

Materials and MethodsMice

DO11.10/scid transgenic mice (H-2d) expressing a TCR specific for OVApeptide were used as donors of T cells (25). BALB/c mice (H-2d) wereused as hosts for adoptive transfer of naive DO11.10 T cells. RAG-2-deficient BALB/c mice (C.129S6(B6)-RAG-2tm1FwaN12) (H-2d) were usedfor adoptive transfer of sorted effector DO11.10 cells and analysis of mem-ory T cell responses. All animals were from our breeding colony and wereused in accordance with the guidelines of the institutional animal care anduse committee.

Antigens

OVA peptide (SQAVHAAHAEINEAGR) encompasses aa residues 323–339 of OVA and is immunogenic in BALB/c mice (25). Influenza virushemagglutinin (HA) aa residues 110–120 peptide (SFERFEIFPKI), whichis also immunogenic in BALB/c mice, was used as a negative control (25).All peptides were purchased from Metabion.

CFSE labeling

Naive splenic DO11.10 CD4� T cells were isolated using magnetic beads.The cells were then labeled with CFSE, as described (25). Briefly, the Tcells (10 � 106 cells/ml) were incubated with 10 �M CFSE at 37°C for 13min. The labeled cells were then washed with ice-cold DMEM-10% FCSand once with PBS before adoptive transfer into mice.

Analysis of CFSE-labeled T cell division upon Ag stimulationin vitro

CFSE-labeled T cells (2 � 106/well) were incubated in a 6-well plate alongwith bulk splenic APCs (8 � 106/well) and graded amounts of OVA pep-tide in a total volume of 4 ml of culture medium for 72 h. The cells werethen washed in 0.5% BSA buffer, fixed for 20 min in a 2% formaldehydesolution, and analyzed for CFSE dilution, which is indicative of cell divi-sion on a BD FACScan flow cytometer.

Preparation and analysis of effector divisions

Forty DO11.10/scid mice were used at a time to harvest and CFSE-labelCD4 T cells, which yields a number of cells sufficient to generate fourBALB/c effector hosts. This number of mice was chosen because opti-mized sorting takes 7–8 h, and this is a maximum period of time in whichthe sorted cells will remain alive and each division will comprise enoughcells to generate two to five recall or memory hosts.

CFSE-labeled T cells (5 � 106 cells/mouse) were adoptively transferredinto BALB/c mice by i.v. injection into the tail vein. Two days later, thehosts were immunized s.c. with 125 �g of OVA in 200 �l of PBS/CFA(1v/1v). The hosts were sacrificed at a given time point, as indicated, andtheir draining LN were harvested and analyzed.

For analysis of cell division as in Fig. 2B, a group of mice was sacrificedevery 12 h beginning 1 day after immunization, and cell division wasanalyzed by flow cytometry.

For analysis of CD25, CD44, BCL-2, and IL-7R expression on the ef-fector LN cells (Figs. 2 and 3), the mice were sacrificed 2 days after im-munization and assessed for marker expression by surface or intracellularstaining, as described below.

Analysis of proliferative and IFN-� responses of effector cells

Sorted D2, D3, and D4 effector divisions were adoptively transferred (5 �104 cells/mouse) into new BALB/c hosts by i.v. injection into the tail vein.One day later, the mice were immunized s.c. with a defined suboptimaldose (20 �g per mouse) of OVA peptide in 200 �l of PBS/CFA (1v/1v).The proliferative and IFN-� responses from draining LN were analyzed at2–8 days following immunization. Proliferation was measured by [3H]thy-midine incorporation, and IFN-� secretion was detected by ELISA.

For proliferation, the LN cells (0.1, 0.3, and 0.9 � 106/well/100 �l)were incubated with 10 �M OVA peptide for 72 h. Subsequently, 1 �Ci of[3H]thymidine was added per well, and the culture was continued for an

additional 14.5 h. The cells were then harvested, and incorporated [3H]thy-midine was counted, as described (26).

For detection of IFN-� by ELISA, LN cells (0.1, 0.3, and 0.9 � 106/well/100 �l) were incubated with 10 �M OVA peptide for 24 h. Followingincubation, IFN-� was detected in culture supernatants, as described (26).

Analysis of memory T cell responses

D2, D3, and D4 effector divisions were separated on the basis of CFSEdilution by cell sorting, and 5 � 104 cells were adoptively transferred intoRAG-2�/� host mice by i.v. injection. The mice were rested for 4 mo forcell-parking purposes, and the memory responses were subsequentlyanalyzed.

For kinetic analysis of memory responses (Figs. 4 and 5), the mice wereimmunized s.c. with 20 �g of OVA peptide in 200 �l of PBS/CFA (1v/1v),and the splenic and LN IFN-� responses were analyzed over time. The LN(0.1 � 106 cells/well) and the SP (0.1, 0.3, and 0.9 � 106 cells/well) cellswere stimulated with the indicated amounts of OVA or control HA peptidefor 24 h, and IFN-� was detected in culture supernatants by ELISA, asdescribed. Only the highest concentration of HA peptide is shown.

Analysis of the frequency of memory precursors

After parking of D2, D3, and D4 effectors for 4 mo in the RAG�/� mice,the splenic cells were harvested without any immunization. Determination ofthe frequency of IFN-�-producing memory T cells was done by ELISPOTusing an Immunospot analyzer, as described (27). Briefly, HA-multiscreenplates were coated with 100 �l/well 1 M NaHCO3 buffer containing 2�g/ml capture Ab. After an overnight incubation at 4°C, the plates werewashed three times with sterile PBS and free sites were saturated withDMEM culture medium containing 10% FCS for 2 h at 37°C. Subse-quently, the blocking medium was removed and 1 � 106 splenic cells wereadded per well along with the indicated amount of peptide. After 24-hincubation at 37°C in a 7% CO2 humidified chamber, the plates werewashed and 100 �l of biotinylated anti-cytokine Ab (1 �g/ml) was added.The Ab pairs used in this study were those described for the ELISA tech-nique. Following overnight incubation at 4°C, the plates were washed and100 �l of avidin-peroxidase (2.5 �g/ml) was added. The plates were thenincubated for 1 h at 37°C. Subsequently, spots were visualized by adding100 �l of substrate (3-amino-9-ethylcarbazole) in 50 mM acetate buffer(pH 5.0) and counted on an Immunospot series 3B analyzer using Immu-nospot version 3.2 software.

Cell surface and intracellular staining

Effector cells. CFSE-labeled cells (1 � 106 cells/ml) were incu-bated with biotin anti-CD25 (7D4), PE Cy5.5 anti-CD44 (IM7),PE-SB/199 anti-IL-7R, allophycocyanin-A7R34 anti-IL-7R, orisotype control Ab for 30 min at 4°C. The cells were then washedand fixed, and the data were collected using a FACScan flow cy-tometer and analyzed with CellQuest software.

BCL-2 expression was analyzed by intracellular staining. Ac-cordingly, CFSE-labeled effector cells (5 � 106 cells/ml) werefixed with 2% formaldehyde, permeabilized with 2% saponin inFACS buffer for 10 min at room temperature, and incubated withPE anti-BCL-2 (3F11) or isotype-matched control for 30 min at4°C. The data were collected and analyzed, as above.

Memory precursors. For analysis of phenotypic markers on IFN-�-producing memory cells (Fig. 7), surface staining was performedfirst, followed by intracellular IFN-� detection. Accordingly,RAG-2�/� BALB/c hosts recipient of effector divisions were sac-rificed after 4-mo parking without any immunization and thesplenic cells were analyzed. For detection of intracellular IFN-�,the memory cells were first subjected to a short 4-h stimulationwith 10 �M OVA peptide to enhance cytokine accumulation andfacilitate intracellular detection, as has been previously defined(28). Cytokine secretion was then blocked by addition of 10 �g/mlbrefeldin A for 2 h. The cells were then washed to remove theexcess of brefeldin A and surface stained with PE-Cy5.5 anti-TCROVA clonotypic mAb, KJ1-26 (mouse IgG2a), and FITC anti-CD44 (IM7), or FITC anti-CD62L (Mel-14), or biotin anti-CCR7(4B12), or FITC anti-VLA-4 (PS/2). Subsequently, the cells werefixed with 2% formaldehyde, permeabilized with 2% saponin in

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FACS buffer for 10 min at room temperature, and incubated withPE anti-mouse IFN-� or isotype-matched controls. All the datawere collected using a FACScan flow cytometer and analyzed withCellQuest software. All Abs were from BD Biosciences.

ResultsCorrelation of the pattern of cell division with the state ofT cell activation early during the effector phase

Upon encounter with Ag, a naive cell undergoes activation, theresult of which is cell division, generation of daughter cells, anddevelopment of effector functions. It is thus logical to contemplatethat more divisions at the first Ag seen would lead to both strongprimary response and accumulation of memory precursors. Thelimitations associated with the low frequency at which memoryprecursors arise within the primary effector response represent amajor setback for investigation of the initial events that drive celltransition from effector to memory. In this study, a T cell transfermodel was developed that could overcome some of these limita-tions and was used to determine whether activation and cell divi-sion early during the effector phase play a role in the developmentof memory. In this model, which is illustrated in Fig. 1, SP areharvested from TCR transgenic DO11.10 mice, and CD4 T cellsare purified, labeled with CFSE, and transferred to BALB/c mice.The host are immunized with OVA, and effector cells that migrateto the LN are harvested within 48 h after immunization. These

effector cells are then analyzed for cell division on the basis ofCFSE dilution, and cells from each division are transferred toRAG-2�/� mice for a 4-mo parking. Subsequently, the RAG-2�/�

hosts are used to analyze memory frequency without rechallengewith OVA or immunized with a suboptimal dose of OVA and usedto analyze memory responses by measuring the volume and speedof the responses. Extensive preliminary experiments were per-formed to define optimal conditions in each step, and these wereused throughout the experiments.

Presumably, two factors, the amount of Ag and the duration ofpresentation, would influence cell activation and division duringthe first Ag seen and, consequently, the transition from effector tomemory. To begin testing this premise, naive DO11.10 T cellswere purified from the SP of DO11.10/SCID mice using anti-CD4microbeads, labeled with CFSE, stimulated with different concen-trations of OVA peptide over the same period of time, and assessedfor CFSE dilution to determine division/proliferation. As can beseen in Fig. 2A, different doses of Ag gave rise to different patternsof cell divisions. Moreover, as the dose of OVA peptide increased,greater numbers of cells with more divisions were observed. This

FIGURE 1. Experimental model for analysis of effector to memorytransition. SP cells were harvested from adult DO11.10/SCID mice, and theCD4 T cells were purified by positive selection on anti-CD4 microbeads.The CD4 T cells were labeled with CFSE, and 5 � 106 were given toBALB/c mice, which were subsequently immunized with 125 �g of OVA.The LN were harvested 48 h later, and the early effectors were sorted intodivisions on the basis of CFSE dilution. The divisions were either analyzedfor expression of CD25, CD44, BCL-2, and IL-7R, or transferred to RAG-2�/� host (50 � 103 cells/mouse) for a 4-mo parking. The hosts weredivided into two groups, one of which was used to harvest the SP withoutany immunization, and the memory cells were analyzed for frequency andphenotypic marker expression. The second group of mice is immunizedwith a suboptimal dose of OVA, and the IFN-� response was analyzed overtime to determine the volume and the speed of the memory response.

FIGURE 2. Effect of Ag dose and duration of presentation on cell ac-tivation and division. A, In vitro division of CFSE-loaded DO11.10 CD4�

T cells upon stimulation with graded amounts of OVA peptide. NaiveDO11.10 T cells (0.5 � 106 cells/ml) were stimulated with splenic APCs(2 � 106 cells/ml) loaded with the indicated doses of OVA peptide for72 h. CFSE dilution, which is indicative of cell division, was then analyzedby flow cytometry. B, In vivo profile and cellularity of effector divisions ofDO11.10 CD4� T cells within 48 h of immunization with OVA peptide.Forty DO11.10/scid mice were sacrificed, and their splenic CD4 T cellswere purified on anti-CD4 microbeads (Miltenyi Biotec). Forty millionnaive CD4 T cells were then labeled with CFSE and transferred intoBALB/c mice (5 � 106 per mouse), and the animals were immunized with125 �g of OVA peptide in CFA. The LN cells were harvested every 12 hafter transfer, and cell division was determined by flow cytometry analysisof CFSE dilution. At 48 h, a total of five divisions was observed, but onlythree divisions designated D2, D3, and D4, which have undergone two,three, and four divisions, respectively, had sufficient numbers of cells(100–250 � 103 cells) for subsequent transfer and analysis of memorydevelopment. C, Activation status of the dividing effectors. The 48-h di-vided effectors were stained with anti-CD25 and anti-CD44 Abs and wereanalyzed by flow cytometry. Insets, Illustrate staining with isotype-matched Ab instead of anti-CD25 or anti-CD44 Ab.

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indicates that the dose of Ag plays a role in the number of divisionsa cell undergoes while developing into an effector cell. The secondfactor that could shape cell division in vivo is the exposure time toAg, which is usually related to the persistence of Ag in lymphoidtissues. Because the dose of 125 �g was previously defined asoptimal for in vivo immunization and induction of effector re-sponse (25), it was used to assess for the effect duration of Agexposure might have on division of effector cells in vivo. Accord-ingly, naive DO11.10 T cells were purified from the SP ofDO11.10/SCID mice using anti-CD4 microbeads, labeled withCFSE, and transferred into BALB/c mice. The hosts were thenimmunized with 125 �g of OVA, the LN were harvested, and theproliferative (dividing) effector cells were analyzed for CFSE di-lution, an indicator of cell division. The results indicated that at24-h exposure very little division has occurred, but by 36 h celldivision began, continued through 60 h, and by 3 days (72 h)complete CFSE dilution is achieved, indicating advanced cell di-vision (data not shown). This kinetics study also indicated that 48 his the optimal time point in which a number of cell divisions haveoccurred at this early stage of the effector phase and had significantcellularity (Fig. 2B). Three of these divisions designated D2, D3,and D4, which have undergone two, three, and four divisions invivo, respectively, yielded between 100 and 250 � 103 effectorcells, corresponding to 0.2–0.7% of the total LN lymphocyte pop-ulation. Analysis of the expression of activation markers on thesedivisions indicated that both CD25 (IL-2R�) and CD44 are highlyexpressed on the cell divisions relative to isotype controls (Fig.2C), indicating a significant state of activation at this early timepoint of the effector phase. Overall, a pattern of cell division hasbeen identified within the early stage of the effector phase thatyielded sufficient numbers of activated effectors useful for evalu-ation of the role cell division and state of activation might play inthe transition to memory.

D2, D3, and D4 effectors display different patterns of IL-7Rexpression and IFN-� production

To define the phenotype of the effector cells in each division,which most likely reflects a potential for transition to memory, weanalyzed expression of markers usually associated with memorydevelopment as well as the ability of the cells to produce IFN-�,the signature cytokine for these cells. Accordingly, BALB/c micewere given CFSE-labeled naive DO11.10 T cells and the hostswere immunized with OVA peptide in CFA. Forty-eight hourslater, the LN were harvested, the effector cells were stained forsurface IL-7R, and intracellular BCL-2 and expression was ana-lyzed in the context of cell division. As indicated in Fig. 3, BCL-2expression had little discrepancy among the effector divisions (Fig.3A), but IL-7R showed a significant increase in the most advancedD4 division, as assessed by two different anti-IL-7R Abs (Fig. 3B),possibly indicating differentiation of effectors with potential formemory transition (29).

To test the divisions for production of IFN-�, in vitro measure-ment is not feasible because cytokine production requires longstimulation in vitro, which could trigger further cell division andinterfere with the readout. We then decided to sort each divisionafter the 48-h Ag exposure in vivo, transfer the divisions intoBALB/c hosts, immunize with a suboptimal dose of OVA peptide(20 �g/mouse) that does not trigger an immune response by en-dogenous BALB/c cells, and then harvest the LN and measureIFN-� production. The 48-h pattern of cell division illustrated inFig. 2B was obtained from the LN of four BALB/c hosts recipientof 5 � 106 CFSE-labeled DO11.10 T cells each. Given the lowfrequency of effectors (0.2–0.7%) among the total LN cells, 7-hsorting was required to obtain 100–250 � 103 effector cells per

FIGURE 3. Highly divided effectors produce significant IFN-� andexpress elevated IL-7R. BALB/c mice recipient of CFSE-loaded naiveDO11.10 CD4 T cells (5 � 106 cells/mouse) were immunized with 125�g of OVA peptide in CFA, and their LN cells were harvested 48 h afterimunization and stained for intracellular BCL-2 (A) and surface IL-7R(B). B, Staining with both PE anti-IL-7R Ab clone SB/199 (top panel)and allophycocyanin anti-IL-7R Ab clone A7R34 (bottom panel) wasperformed to ensure reproducibility. The plots were obtained by anal-ysis of CFSE intensity and expression of the indicated markers on gatedlive cells. The percentage of CFSE-positive cells expressing the indi-cated marker was obtained by overlaying the isotype control plot (insetin the left-hand side) over the sample plot in the right-hand corner. Theboxed cells represent those positive for marker expression. C, D4 andD2, but not D3 effectors produce significant amounts of IFN-�. BALB/cmice recipient of 5 � 106 CFSE-loaded naive DO11.10 CD4 T cellswere immunized with 125 �g of OVA peptide in CFA. Forty-eighthours later, the LN cells were harvested and cell division was assessedby CFSE dilution. The D2, D3, and D4 divisions were sorted and re-transferred (5 � 104 sorted cells per mouse) into new naive BALB/chosts. To measure production of IFN-� responses, these new recipientswere immunized 1 day later with the suboptimal dose of 20 �g of OVApeptide in CFA. The LN effector proliferative (upper panel) and IFN-�(lower panel) responses were analyzed by in vitro stimulation with 10�M OVA peptide at the indicated time points after immunization. Agroup recipient of naive unsorted DO11.10 T cells was included forcontrol purposes. HA peptide was used as a negative control for the invitro stimulation. Day 2 of D2 division was not done because there wasno sufficient cellularity.

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division (only two divisions can be sorted at a time). Because thecells might not survive longer in the sorting buffer, the followingprocedure was adopted for the generation and sorting of effectordivisions. Forty DO11.10/scid mice are used as donors of T cellsfor CFSE labeling and adoptive transfer to four BALB/c hosts. Therecipients were sacrificed 48 h after immunization, their LN areharvested, and the effector cells are sorted based on CFSE dilution.Because preliminary studies indicated that 50 � 103 effectors willbe required to generate one RAG-2�/� host for memory parking(experimental producedure illustrated in Fig. 1), each sort usuallygenerates two to five mice per division for two divisions at a time.

Accordingly, cells from each division were separated by sortingand transferred into new BALB/c hosts, and 1 day later the animalswere immunized with 20 �g of OVA peptide, a dose that does notinduce a measurable immune response in normal BALB/c mice(data not shown). The proliferative and IFN-� LN responses werethen analyzed over time. As indicated in Fig. 3C, all divisionsyielded Ag-specific proliferative responses slightly higher thanmice recipient of naive DO11.10 T cells, indicating that cell sort-ing and transfer to these new hosts did not severely affect theviability of the effector cells. Moreover, each division displayed aunique proliferation pattern over time, suggesting that cell divisionat the initial encouter with Ag plays a role in the subsequent re-sponse. IFN-� cytokine analysis also showed that each divisionyielded an IFN-� response with a different pattern. Indeed, divisionD4 developed a strong IFN-� response that reached a plateau byday 6, whereas D3 did not produce significant IFN-� at any pointdespite the fact that the cells were proliferative. Division D2 gen-erated a strong response as early as day 4, but declined quickly.Some IFN-� was observed with the control HA, possibly reflectingan ongoing ex vivo T cell activation. Thus, D4 effectors that ex-pressed significant IL-7R produced strong IFN-� (IL-7Rhigh/IFN-��), D3 had low IL-7R and insignificant IFN-� (IL-7Rlow/IFN-��), whereas D2 that manisfested low IL-7R expression hadsignificant IFN-� production (IL-7Rlow/IFN-��). Overall, early ef-fector cells that have undergone different divisions display differ-ent patterns of IL-7R expression and IFN-� production.

IL-7Rhigh/IFN-�� D4 effectors gave rise to rapid splenicmemory responses

Memory responses are always stronger (higher volume) andquicker to come about (more rapid) when compared with primaryresponses. These two parameters were then used to evaluate mem-ory response of the effector cells of each division. Accordingly,DO11.10 effectors were generated in BALB/c host, and LN cellsfrom each division were sorted on the basis of CFSE dilution andtransferred into RAG-2�/� mice for parking (see experimentalprocedure in Fig. 1). Four months later, the RAG-2�/� recipients,which we designate memory hosts, were immunized with the 20�g suboptimal dose of OVA peptide and the splenic IFN-� re-sponses were assessed over time. Again, RAG-2�/� mice werechosen as memory hosts because the lymphopenic environmentprovides a significant source of IL-7 that sustains survival of thetransferred low frequency potential memory cells (22–24) and op-timizes the readout of memory responses. As can be seen in Fig. 4,the specific memory IFN-� splenic response emanating from D4effectors developed as early as day 2 after immunization andreached a plateau by day 4 postchallenge. The response is specificbecause stimulation of the cells with the negative control HA pep-tide did not yield any measurable IFN-� response. The kinetics ofthe D4 memory response parallels with the response of mice thatreceived unseparated effector cells, which include the sum of po-tential memory cells of all effector divisions (labeled memory).The D3 and D2 divisions, however, yielded delayed memory re-

sponses similar to mice recipient of naive DO11.10 T cells. Indeed,IFN-� production was not significant until day 4 after immuniza-tion. These responses were also specific because stimulation withHA peptide did not yield any significant IFN-� response over the4-day evaluation period. Overall, IL-7Rhigh/IFN-�� D4 effectorsyielded strong (volume) and rapid (speed) splenic memory re-sponses, whereas D3 (IL-7Rlow/IFN-��) and D2 (IL-7Rlow/IFN-��) effector produced delayed memory responses.

IL-7Rhigh/IFN-�� D4 and IL-7Rlow/IFN-�� D2, but not IL-7Rlow/IFN-�� D3 effectors yielded rapid LN memory responses

Memory development from each division was also analyzed in theLN of the memory mice. As indicated in Fig. 5, the IL-7Rhigh/IFN-�� D4 effectors yielded a memory IFN-� response that was atplateau level by day 2 after immunization and parallels with theresponse of unseparated effector cells (memory). These responsesare specific to OVA because stimulation with HA peptide pro-duced no significant responses. Because D4 effectors producedsimilar kinetics in both the SP and LN and IFN-� response wasrapid in both organs, we designate this response as rapid memoryT cell (TRM) response. IL-7Rlow/IFN-�� D3 effectors again gen-erated a delayed memory response that was not evident until day4 after immunization. This delay occurred despite the fact that theLN were available on day 2 and had sufficient cellularity, whereasthe mice recipient of naive DO11.10 cells had similar delay, butthe LN did not form until day 4 after immunization (Fig. 5). Wedesignate these responses as homeostatic memory T cell (THM)because they were delayed in both the SP and LN, have similarkinetics as the naive cells, and proliferated without significant

FIGURE 4. Kinetics of splenic memory responses generated from dis-tinct effector cell divisions. Sorted effector D2, D3, and D4 cells weretransferred into RAG-2�/� mice (5 � 104 cells/mouse) and parked for 4mo. To analyze the memory response generated from each of the divisions,the recipient mice were immunized with the suboptimal dose of 20 �g ofOVA peptide in CFA, and their splenic memory IFN-� responses weremeasured by ELISA at the indicated time points after immunization. Thecolumn labeled “Memory” represents responses of RAG-2�/� mice recip-ient of 5 � 104 unlabeled naive DO11.10 CD4 T cells that were immunizedwith 125 �g of OVA peptide, rested for 4 mo, and reimmunized with 20�g of OVA peptide. The column labeled “Naive” represents the responsesof RAG-2�/� mice recipient of 5 � 104 naive DO11.10 T cells that werenot given the primary 125 �g OVA peptide immunization, but did receivea 20 �g challenge regimen 4 mo after cell transfer. The splenic cells (100,300, and 900 � 103 cells/well) were stimulated with graded concentrationsof OVA peptide for 24 h. IFN-� production was measured in cell culturesupernatant by ELISA. Only the highest concentration was used with thenegative control HA peptide. Each point represents the mean of triplicatewells � SD. The results shown are those obtained with 900 � 103

cells/well.

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IFN-� production at the effector stage (30–32) (Fig. 3C). Intrigu-ingly, the IL-7Rlow/IFN-�� D2 division, which generated a de-layed memory response in the SP, showed significant IFN-� pro-duction in the LN as early as day 2 after immunization. Theseresponses were designated conditioned memory T cell (TCoM) be-cause they were rapid in the LN, but tardy in the SP. As we shallsee below, expression of molecules involved in T cell traffickingmay have been responsible for these discrepancies.

Rapid responses correlate with higher frequency ofIFN-�-producing memory precursors

The different patterns of memory responses among the effectordivisions could be due to a difference in the frequency of respond-ing memory cells, the amount of IFN-� these cells produce, or tothe ability to traffic through the organs. To test these premises,splenic cells from 4-mo-parking RAG-2�/� mice were harvestedbefore any immunization with OVA and the frequency of IFN-�-producing memory cells as well as total IFN-� production wereevaluated. ELISPOT analysis, which measures the frequency of

IFN-�-producing memory responders, shows that the D4 divisionhad the highest frequency, followed by D2, but D3 had very fewdetectable IFN-�-producing cells (Fig. 6A). No detectable IFN-�-producing cells were observed with the negative control HA pep-tide. Evaluation of the total amount of IFN-� by ELISA indicatedthat splenic cells from mice recipient of D4 effectors producedsignificant amounts of IFN-� upon in vitro stimulation withOVA peptide (Fig. 6B). Splenic cells from mice recipient of D3effectors did not produce significant IFN-�, whereas those cellsfrom animals harboring D2 effectors did secrete IFN-� at sig-nificant levels when the stimulation used 10 �M OVA peptide.Again, IFN-� production is specific because HA peptide did notinduce any measurable IFN-� production. The frequency ofIFN-�-producing memory precursors parallels with totalamount of cytokine production in that more IFN-� is secretedwhen the frequency of memory precursors is higher (Fig. 6,compare A and B). Thus, it is likely that the frequency of mem-ory precursors able to produce IFN-� is responsible for thekinetics of memory responses observed after challenge withOVA peptide (Figs. 4 and 5).

TRM, THM, and TCoM express markers signature of memory

As indicated above, the different patterns of memory responsesamong the effector divisions could be related to differences in theability of IFN-�-producing precursors to traffic through lymphoidorgans and tissues. CD44 and VLA-4 are molecules expressed onmemory cells and may be involved in regulation of memory cellmigration (33). To test these premises, splenic cells from 4-mo-parking RAG-2�/� mice were harvested before any immunizationwith OVA, and the expression of CD44 and VLA-4 on IFN-�-producing memory precursors was determined ex vivo. As can be

FIGURE 5. Kinetics of LN memory responses generated from distincteffector cell divisions. The LN IFN-� memory responses of RAG-2�/�

mice recipient of 5 � 104 D2, D3, or D4 sorted cells described in Fig. 3were also measured by ELISA at different time points after immunization,as indicated. The same memory and naive groups described in Fig. 3 wereincluded for comparison. Analysis of the response of mice transferred withnaive cells was not feasible on days 2 and 3 due to the fact that the LN fromthese animals had little cellularity and did not form until day 4. Only thehighest concentration was used with the negative control HA peptide. Eachpoint represents the mean of triplicate wells � SD. The results shown arethose obtained with 100 � 103 cells/well.

FIGURE 6. Frequency of memory precursors in RAG-2�/� mice trans-ferred with D4, D3, or D2 sorted cells. Sorted D2, D3, and D4 divisionswere transferred (5 � 104 cells/mouse) and parked in RAG-2�/� mice.Four months later, the mice were sacrificed without any immunization, andthe frequency of IFN-�-producing memory splenic precursors was deter-mined by ELISPOT (A) upon in vitro stimulation with OVA peptide. Thetotal amount of IFN-� produced in the culture was also determined byELISA (B) upon in vitro stimulation with OVA peptide. Each point rep-resents the mean of triplicate wells � SD.

FIGURE 7. TRM, THM, and TCoM precursors express markers signatureof memory. Sorted D2, D3, and D4 effectors were transferred into RAG-2�/� mice (5 � 104 cells/mouse) and parked for 4 mo. The mice were thensacrificed (without any immunization), and IFN-�-producing KJ1-26 cellswere tested ex vivo for cell surface expression of CD44 (A), VLA-4 (B),CD62L (C), and CCR7 (D) by flow cytometry. In this case, the cells weresubject to a short 4-h stimulation with 10 �M OVA peptide, stained withAbs to the indicated marker, and for identification purpose costained forsurface KJ1-26 and intracellular IFN-�. Live cells were gated on IFN-�,and the plots show KJ1-26 and sample marker expression of IFN-�-posi-tive cells. The numbers of IFN-��/KJ1-26� expressing the test marker areindicated in the upper right corner of the plot.

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seen in Fig. 7, IFN-�/KJ1-26 double-positive splenic cells frommice recipient of D4, D3, or D2 effectors had high expression ofCD44 (Fig. 7A). Note the presence of IFN-�-producing cells thatare negative for KJ1-26 in all divisions, possibly indicating TCRdown-regulation by these cells. Thus, the discrepancies in thememory responses among the three divisions observed in Figs. 4and 5 are likely to be related to the number of IFN-�-producingmemory precursors rather than expression of CD44. As for ex-pression of VLA-4, about one-half of the IFN-�/KJ1-26 double-positive splenic memory precursors from D4 and D3 effectors hadsignificant VLA-4, whereas most (84%) of the precursors emanat-ing from D2 effectors had significant surface VLA-4 expression(Fig. 7B). This biased VLA-4 expression may have contributed toretention of cells in the LN, leading to accumulation of precursorsand rapid memory responses in this organ, but delayed memory inthe SP, hence the TCoM for the precursors giving rise to this mem-ory response.

Recently, L-selectin, or CD62L, and the chemokine receptorCCR7 have been defined as markers whose expression can distin-guish effector and central memory T cells (34, 35). It is now wellestablished that central memory cells express significant CD62Land CCR7 (CD62Lhigh, CCR7high), whereas effector memory cellsdisplay minimal CD62L and CCR7 (CD62Llow, CCR7low) expres-sion (5). To determine whether the newly defined memory cellsbelong to the effector or central memory subset, the IFN-�/KJ1-26splenic memory precursors from mice recipient of D2, D3, and D4effectors were analyzed for CD62L and CCR7 expression. Accord-ingly, 4-mo-parking RAG-2�/� mice recipient of D2, D3, or D4effectors were sacrificed without any immunization and theirsplenic cells were stained with KJ1-26, and anti-IFN-� and anti-CD62L, or anti-CCR7 Ab. The results illustrated in Fig. 7 indicatethat all three populations are comprised of cells with either phe-notype with more prevalence of effector memory. Indeed, expres-sion of CD62L on KJ1-26/IFN-� double-positive memory precur-sors ranged between 18 and 25% among all divisions (Fig. 7C),and CCR7 was observed on 16–30% of the cells (Fig. 7D). Thus,the double-positive (central memory) cells range from 16 to 22%and the double-negative (effector memory) from 78 to 84% of thememory pool. These findings indicate a heterogeneity within TRM,THM, and TCoM, which bodes well with the recently defined flex-ibility associated with effector and central memory phenotypes(36, 37).

DiscussionDue to the low frequency at which memory cells arise within theeffector phase and the lack of memory markers, investigation ofthe transition of effector cells to memory was difficult and themechansims underlying such transition remain largely undefined(1, 3, 10, 13–15, 19, 29, 38). Most of the findings available to dateindicate that memory cells arise from slightly activated effectorsproducing little IFN-� (39), and that the memory precursors em-anate from cells that were exposed to suboptimal Ag as a conse-quence of late arrival to lymphoid tissues (19) or due to encounterof residual Ag that persists after microbial clearance (18). This hasbeen interpreted to suggest that memory arises from cells that seethe Ag at the end of the effector phase with a rather moderate stateof activation (18, 19). In this study, an in vivo approach was de-vised that enabled us to determine whether cells exposed to opti-mal Ag within 48 h of the effector phase can transit to memory(Fig. 1). Also, we were able to investigate the events that controlmemory transition at this stage. The findings indicate that highlyactivated early effectors do indeed transit to memory. Moreover,significant production of IFN-� seems to aid in an efficient tran-sition. Indeed, naive DO11.10 T cells transferred into BALB/c

mice gave rise to a number of LN effector divisions in vivo 48 hafter immunization with OVA peptide (Fig. 2). Because these ef-fector divisions occurred 2 days after immunization and expressedsignificant levels of activation marker such as CD25 and CD44, werefer to them as highly activated early effector divisions. Three ofthese, D2, D3, and D4, which have undergone two, three, and fourdivisions, respectively, had sufficient cellularity that enabled iso-lation of the divisions and retransfer into naive RAG-2�/� host.When the hosts were challenged with OVA peptide 4 mo aftertransfer, the recipients of effectors from the most advanced D4division yielded rapid memory responses in both the SP and LN,and these were designated TRM for rapid memory (Figs. 4 and 5).Those recipients of cells from division D3 generated base levelmemory responses in both lymphoid organs that were comparableto naive cells and most likely represent homeostatic memory (30–32). We refer to these cells as THM. The mice transferred witheffectors from division D2 produced homeostatic-like responses inthe SP, but rapid responses in the LN; hence, we designated thesecells as TCoM for conditioned memory cells. A schematic repre-sentation of these findings is illustrated in Fig. 8, providing thetype of memory response each division gave rise to as defined bythis in vivo study. The latest models of memory development sug-gest that advanced activation/proliferation leads to reduced num-bers of responding memory cells (18, 39). However, recent reportspostulated that advanced proliferation would most likely result inmore CD4 T cell memory (15). Our findings show that within theearly effector phase, the cells express activation markers to thesame extent (Fig. 2), and both advanced division (D4) as well ascells that have undergone fewer division (D2) yielded higher fre-quency of memory precursors (Fig. 6) that enabled rapid memoryresponses (TRM and TCoM) upon challenge with Ag. Cells with

FIGURE 8. Production of IFN-� by early effectors plays a major role inthe transit to memory. Effector divisions develop distinct memory re-sponses depending on the pattern of IFN-� production and IL-7R expres-sion during the first exposure to Ag.

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intermediate division (D3) had minimal memory precursor fre-quency (Fig. 6) and sustained only homeostatic memory responsesboth in the SP and LN (THM). Although this is intriguing, becauseone cell division more or less influences memory development, thefindings raise interesting observations. Indeed, whereas D4 effec-tors expressed the activation markers CD25 and CD44 to a similarextent as D3 and D2, they had significant IL-7R expression andproduced significant IFN-� (Figs. 2 and 3). These highly activatedIL-7Rhigh/IFN-�� effectors gave rise to high frequency of memoryprecursors (Fig. 6) that yielded rapid TRM responses (Figs. 4 and5). D3 effectors, which were highly activated, but had low IL-7Rexpression and no IFN-� production (IL-7Rlow/IFN-��), gener-ated minimal memory precursor frequency (Fig. 6) and only baselevel homeostatic memory responses (Figs. 4 and 5). D2 effectors,which expressed CD25 and CD44 activation markers, had lowIL-7R expression and produced significant IFN-�. These highlyactivated IL-7Rlow/IFN-�� effectors gave rise to significant mem-ory precursors that yielded significant memory response that werelocalized in the LN, but not the SP, hence the designation (TCoM)responses (Figs. 4, 5, and 8). Thus, it seems that production ofsignificant amounts of IFN-� is a requirement at this early stage ofthe effector phase for transition to memory. IL-7R expression mayinfluence survival of memory precursors leading to robust memoryresponses in the SP and LN. VLA-4, a molecule that is involved inregulating T cell trafficking (33, 40, 41), may have played a role inthe preferential responses of D2 memory cells in the LN, becausemore VLA-4 was found on the memory precursors emanating fromD2 effector, giving rise to TCoM memory responses (Figs. 7 and 8).

Even with a limited technical feasibility that enabled studieswith only three of five early effector divisions, three distincttypes of memory cells, TRM, THM, and TCoM, were defined on thebasis of the kinetics of the responses (Fig. 8). The reference tokinetics (volume and speed) of the response has always been themethod of choice for evaluation of memory development. Be-cause the effector cells have been removed from the Ag andparked in the RAG-2�/� lymphopenic environment, equal sur-vival factors will be available to all effectors. Thus, whereasthis environment serves to amplify memory and ease the read-out of the responses, the different kinetics of the responses ob-served among the divisions most likely reflect the effect of Ag-induced cell division rather than RAG-2�/�-driven homeostaticproliferation. The findings in this study reinforce prior sugges-tions postulating that differentiation of a naive lymphocyte intoa memory cell most likely occurs as a result of events emanat-ing from the encounter with Ag rather than an intrinsic prede-termination (42). Moreover, recent studies in this area elegantlydemonstrated that exposure to limited amount of residual Agdue to late arrival of circulating T cells to regional LN triggerslate cell division that specifically guided transition to centralmemory (19). Our findings add a novel observation demonstrat-ing that early effectors also transit to memory and a varianceby a single division leads to different kinetics of the memoryresponse (Fig. 8). Also, significant production of the cell’s sig-nature cytokine (i.e., IFN-�) is required for such transition, pos-sibly reflecting an epigenetic phenomenon (43). Finally, thethree populations, TRM, THM, and TCoM, defined in this study,had a mixture of effector and central memory cells because bothCD62Lhigh CCR7high VLA4� and CD62Llow CCR7low VLA4�

cells were components of these responses, and this bodes wellwith the recently defined flexibility among the central/effectorsubsets (36, 37).

Overall, these studies show that highly activated early effectorCD4 T cells transit to memory, and production of significant IFN-�is required for such transition. Moreover, a single cell division

affects the transition to memory at this stage. During an initialinfection or tumor development, microbes or cancer cells may in-fluence division and/or IFN-� production of effector lymphocyteseither by loading variable amounts of Ag or by interference withAg presentation. This could affect the development of memory andthe effectiveness of vaccines (44) especially given that one celldivision produces a significant difference on the development, ra-pidity, and localization of the memory response.

AcknowledgmentWe thank Louise Barnett for help with cell sorting.

DisclosuresThe authors have no financial conflict of interest.

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