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The Journal of Immunology, 2008, 180, 179 -187
Copyright © 2008 by The American Association of Immunologists, Inc.
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Early Effector T Cells Producing Significant IFN-{gamma} Develop into Memory1

J. Jeremiah Bell2, Jason S. Ellis, F. Betul Guloglu, Danielle M. Tartar, Hyun-Hee Lee3, Rohit D. Divekar, Renu Jain, Ping Yu4, Christine M. Hoeman and Habib Zaghouani5

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Currently, transition of T cells from effector to memory is believed to occur as a consequence of exposure to residual suboptimal Ag found in lymphoid tissues at the waning end of the effector phase and microbial clearance. This led to the interpretation that memory arises from slightly activated late effectors producing reduced amounts of IFN-{gamma}. In this study, we show that CD4 T cells from the early stage of the effector phase in which both the Ag and activation are optimal also transit to memory. Moreover, early effector T cells that have undergone four divisions expressed significant IL-7R, produced IFN-{gamma}, and yielded rapid and robust memory responses. Cells that divided three times that had marginal IL-7R expression and no IFN-{gamma} raised base level homeostatic memory, whereas those that have undergone only two divisions and produced IFN-{gamma} yielded conditioned memory despite low IL-7R expression. Thus, highly activated early effectors generated under short exposure to optimal Ag in vivo develop into memory, and such transition is dependent on a significant production of the cell’s signature cytokine, IFN-{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Memory is one of the cardinal features associated with the adaptive immune response and provides the fundamental basis for the development of vaccines (1, 2, 3, 4, 5). Usually, an initial exposure to a microbe triggers a primary response comprising pathogen-specific effector lymphocytes, most of which engage in the clearance of the pathogen (1, 2, 3, 4, 5, 6). However, very few of these effector cells evolve as long-lasting memory lymphocytes (1, 6). These cells then contribute a substantial increase in the frequency of pathogen-specific cells within the lymphocyte repertoire (7, 8) and mount a rapid and robust response highly effective for protection against reinfection (9, 10, 11, 12). To date, very little information is available on the initial events that dictate whether a cell in the primary response should simply be an effector cell programmed to die shortly after microbial clearance or differentiate into a long-lived memory cell (3, 13, 14, 15). This is mostly due to the low frequency of T cells that commit to memory during the effector phase and the lack of specific markers that could trace the effector to memory transition (1, 10, 16, 17). Lately, it has been suggested that memory development occurs as a result of continual exposure 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 raise the question as to whether the immune system can generate memory against acute infections in which the lymphocytes could be exposed to an optimal dose of Ag for a short period early in the effector phase. In other words, do highly activated T cells transit into memory? To address this premise, we designed a model in which naive lymphocytes are exposed to an optimal dose of Ag for a limited time during the initial period of the effector phase and tested for development of memory. Accordingly, OVA-specific TCR transgenic T cells from DO11.10/scid mice (20) were labeled with the intracellular fluorescent dye, CFSE (21), and transferred into compatible BALB/c mice, and the recipients were immunized with a previously defined optimal dose of OVA peptide. The dividing effector LN cells were isolated within 48 h of immunization, sorted on the basis of cell division, which is indicative of their state of activation, and transferred into RAG-2-deficient (RAG-2–/–) BALB/c mice for parking without Ag. The memory response from each sorted division was then quantified over time before and after immunization of the hosts with a suboptimal dose of OVA. The rationale for parking the effector cells in a RAG-2–/– host stems from the following reasoning: the lymphopenic environment should sustain survival of memory precursors that arise at a very low frequency (22, 23, 24), thereby making the readout of memory responses feasible. Second, the effector cells of all divisions are subjected to identical survival mechanisms in the absence of Ag, and any difference in the memory responses will solely reflect how each cell division affects memory development. The results indicated that the effector division D4, which included cells that have undergone four divisions, express IL-7R, and produce IFN-{gamma}, gave rise to a high frequency of memory lymphocytes that sustained a rapid and robust memory response in both the spleen (SP) and LN. D3 effectors, which divided three times, expressed minimal IL-7R, and did not produce IFN-{gamma}, yielded base level homeostatic memory responses in both lymphoid organs. D2 cells that divided only two times expressed low IL-7R, but produced significant IFN-{gamma}, and yielded conditioned memory responses. Thus, early effectors can develop into memory, and such transition is dependent on their ability to produce IFN-{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

DO11.10/scid transgenic mice (H-2d) expressing a TCR specific for OVA peptide were used as donors of T cells (25). BALB/c mice (H-2d) were used 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 used for adoptive transfer of sorted effector DO11.10 cells and analysis of memory T cell responses. All animals were from our breeding colony and were used in accordance with the guidelines of the institutional animal care and use committee.

Antigens

OVA peptide (SQAVHAAHAEINEAGR) encompasses aa residues 323–339 of OVA and is immunogenic in BALB/c mice (25). Influenza virus hemagglutinin (HA) aa residues 110–120 peptide (SFERFEIFPKI), which is 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 T cells (10 x 106 cells/ml) were incubated with 10 µM CFSE at 37°C for 13 min. The labeled cells were then washed with ice-cold DMEM-10% FCS and once with PBS before adoptive transfer into mice.

Analysis of CFSE-labeled T cell division upon Ag stimulation in vitro

CFSE-labeled T cells (2 x 106/well) were incubated in a 6-well plate along with bulk splenic APCs (8 x 106/well) and graded amounts of OVA peptide in a total volume of 4 ml of culture medium for 72 h. The cells were then washed in 0.5% BSA buffer, fixed for 20 min in a 2% formaldehyde solution, and analyzed for CFSE dilution, which is indicative of cell division 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-label CD4 T cells, which yields a number of cells sufficient to generate four BALB/c effector hosts. This number of mice was chosen because optimized sorting takes 7–8 h, and this is a maximum period of time in which the sorted cells will remain alive and each division will comprise enough cells to generate two to five recall or memory hosts.

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

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

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

Analysis of proliferative and IFN-{gamma} responses of effector cells

Sorted D2, D3, and D4 effector divisions were adoptively transferred (5 x 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 suboptimal dose (20 µg per mouse) of OVA peptide in 200 µl of PBS/CFA (1v/1v). The proliferative and IFN-{gamma} responses from draining LN were analyzed at 2–8 days following immunization. Proliferation was measured by [3H]thymidine incorporation, and IFN-{gamma} secretion was detected by ELISA.

For proliferation, the LN cells (0.1, 0.3, and 0.9 x 106/well/100 µl) were incubated with 10 µM OVA peptide for 72 h. Subsequently, 1 µCi 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]thymidine was counted, as described (26).

For detection of IFN-{gamma} by ELISA, LN cells (0.1, 0.3, and 0.9 x 106/well/100 µl) were incubated with 10 µM OVA peptide for 24 h. Following incubation, IFN-{gamma} 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 CFSE dilution by cell sorting, and 5 x 104 cells were adoptively transferred into RAG-2–/– host mice by i.v. injection. The mice were rested for 4 mo for cell-parking purposes, and the memory responses were subsequently analyzed.

For kinetic analysis of memory responses (Figs. 4 and 5), the mice were immunized s.c. with 20 µg of OVA peptide in 200 µl of PBS/CFA (1v/1v), and the splenic and LN IFN-{gamma} responses were analyzed over time. The LN (0.1 x 106 cells/well) and the SP (0.1, 0.3, and 0.9 x 106 cells/well) cells were stimulated with the indicated amounts of OVA or control HA peptide for 24 h, and IFN-{gamma} was detected in culture supernatants by ELISA, as described. 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 of the frequency of IFN-{gamma}-producing memory T cells was done by ELISPOT using an Immunospot analyzer, as described (27). Briefly, HA-multiscreen plates were coated with 100 µl/well 1 M NaHCO3 buffer containing 2 µg/ml capture Ab. After an overnight incubation at 4°C, the plates were washed three times with sterile PBS and free sites were saturated with DMEM culture medium containing 10% FCS for 2 h at 37°C. Subsequently, the blocking medium was removed and 1 x 106 splenic cells were added per well along with the indicated amount of peptide. After 24-h incubation at 37°C in a 7% CO2 humidified chamber, the plates were washed and 100 µl of biotinylated anti-cytokine Ab (1 µg/ml) was added. The Ab pairs used in this study were those described for the ELISA technique. Following overnight incubation at 4°C, the plates were washed and 100 µl of avidin-peroxidase (2.5 µg/ml) was added. The plates were then incubated for 1 h at 37°C. Subsequently, spots were visualized by adding 100 µl of substrate (3-amino-9-ethylcarbazole) in 50 mM acetate buffer (pH 5.0) and counted on an Immunospot series 3B analyzer using Immunospot version 3.2 software.

Cell surface and intracellular staining

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

BCL-2 expression was analyzed by intracellular staining. Accordingly, CFSE-labeled effector cells (5 x 106 cells/ml) were fixed with 2% formaldehyde, permeabilized with 2% saponin in FACS buffer for 10 min at room temperature, and incubated with PE anti-BCL-2 (3F11) or isotype-matched control for 30 min at 4°C. The data were collected and analyzed, as above.

Memory precursors. For analysis of phenotypic markers on IFN-{gamma}-producing memory cells (Fig. 7), surface staining was performed first, followed by intracellular IFN-{gamma} detection. Accordingly, RAG-2–/– BALB/c hosts recipient of effector divisions were sacrificed after 4-mo parking without any immunization and the splenic cells were analyzed. For detection of intracellular IFN-{gamma}, the memory cells were first subjected to a short 4-h stimulation with 10 µM OVA peptide to enhance cytokine accumulation and facilitate intracellular detection, as has been previously defined (28). Cytokine secretion was then blocked by addition of 10 µg/ml brefeldin A for 2 h. The cells were then washed to remove the excess of brefeldin A and surface stained with PE-Cy5.5 anti-TCR OVA 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 were fixed with 2% formaldehyde, permeabilized with 2% saponin in FACS buffer for 10 min at room temperature, and incubated with PE anti-mouse IFN-{gamma} or isotype-matched controls. All the data were collected using a FACScan flow cytometer and analyzed with CellQuest software. All Abs were from BD Biosciences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Correlation of the pattern of cell division with the state of T cell activation early during the effector phase

Upon encounter with Ag, a naive cell undergoes activation, the result of which is cell division, generation of daughter cells, and development of effector functions. It is thus logical to contemplate that more divisions at the first Ag seen would lead to both strong primary response and accumulation of memory precursors. The limitations associated with the low frequency at which memory precursors arise within the primary effector response represent a major setback for investigation of the initial events that drive cell transition from effector to memory. In this study, a T cell transfer model was developed that could overcome some of these limitations and was used to determine whether activation and cell division early during the effector phase play a role in the development of memory. In this model, which is illustrated in Fig. 1, SP are harvested from TCR transgenic DO11.10 mice, and CD4 T cells are purified, labeled with CFSE, and transferred to BALB/c mice. The host are immunized with OVA, and effector cells that migrate to the LN are harvested within 48 h after immunization. These effector cells are then analyzed for cell division on the basis of CFSE dilution, and cells from each division are transferred to RAG-2–/– mice for a 4-mo parking. Subsequently, the RAG-2–/– hosts are used to analyze memory frequency without rechallenge with OVA or immunized with a suboptimal dose of OVA and used to analyze memory responses by measuring the volume and speed of the responses. Extensive preliminary experiments were performed to define optimal conditions in each step, and these were used throughout the experiments.


Figure 1
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  		FIGURE 1. Experimental model for analysis of effector to memory transition. SP cells were harvested from adult DO11.10/SCID mice, and the CD4 T cells were purified by positive selection on anti-CD4 microbeads. The CD4 T cells were labeled with CFSE, and 5 x 106 were given to BALB/c mice, which were subsequently immunized with 125 µg of OVA. The LN were harvested 48 h later, and the early effectors were sorted into divisions on the basis of CFSE dilution. The divisions were either analyzed for expression of CD25, CD44, BCL-2, and IL-7R, or transferred to RAG-2–/– host (50 x 103 cells/mouse) for a 4-mo parking. The hosts were divided into two groups, one of which was used to harvest the SP without any immunization, and the memory cells were analyzed for frequency and phenotypic marker expression. The second group of mice is immunized with a suboptimal dose of OVA, and the IFN-{gamma} response was analyzed over time to determine the volume and the speed of the memory response.

 
Presumably, two factors, the amount of Ag and the duration of presentation, would influence cell activation and division during the first Ag seen and, consequently, the transition from effector to memory. To begin testing this premise, naive DO11.10 T cells were purified from the SP of DO11.10/SCID mice using anti-CD4 microbeads, labeled with CFSE, stimulated with different concentrations of OVA peptide over the same period of time, and assessed for CFSE dilution to determine division/proliferation. As can be seen in Fig. 2A, different doses of Ag gave rise to different patterns of cell divisions. Moreover, as the dose of OVA peptide increased, greater numbers of cells with more divisions were observed. This indicates that the dose of Ag plays a role in the number of divisions a cell undergoes while developing into an effector cell. The second factor that could shape cell division in vivo is the exposure time to Ag, which is usually related to the persistence of Ag in lymphoid tissues. Because the dose of 125 µg was previously defined as optimal for in vivo immunization and induction of effector response (25), it was used to assess for the effect duration of Ag exposure might have on division of effector cells in vivo. Accordingly, naive DO11.10 T cells were purified from the SP of DO11.10/SCID mice using anti-CD4 microbeads, labeled with CFSE, and transferred into BALB/c mice. The hosts were then immunized with 125 µg of OVA, the LN were harvested, and the proliferative (dividing) effector cells were analyzed for CFSE dilution, an indicator of cell division. The results indicated that at 24-h exposure very little division has occurred, but by 36 h cell division began, continued through 60 h, and by 3 days (72 h) complete CFSE dilution is achieved, indicating advanced cell division (data not shown). This kinetics study also indicated that 48 h is the optimal time point in which a number of cell divisions have occurred at this early stage of the effector phase and had significant cellularity (Fig. 2B). Three of these divisions designated D2, D3, and D4, which have undergone two, three, and four divisions in vivo, respectively, yielded between 100 and 250 x 103 effector cells, corresponding to 0.2–0.7% of the total LN lymphocyte population. Analysis of the expression of activation markers on these divisions indicated that both CD25 (IL-2R{alpha}) and CD44 are highly expressed on the cell divisions relative to isotype controls (Fig. 2C), indicating a significant state of activation at this early time point of the effector phase. Overall, a pattern of cell division has been identified within the early stage of the effector phase that yielded sufficient numbers of activated effectors useful for evaluation of the role cell division and state of activation might play in the transition to memory.


Figure 2
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  		FIGURE 2. Effect of Ag dose and duration of presentation on cell activation and division. A, In vitro division of CFSE-loaded DO11.10 CD4+ T cells upon stimulation with graded amounts of OVA peptide. Naive DO11.10 T cells (0.5 x 106 cells/ml) were stimulated with splenic APCs (2 x 106 cells/ml) loaded with the indicated doses of OVA peptide for 72 h. CFSE dilution, which is indicative of cell division, was then analyzed by flow cytometry. B, In vivo profile and cellularity of effector divisions of DO11.10 CD4+ T cells within 48 h of immunization with OVA peptide. Forty DO11.10/scid mice were sacrificed, and their splenic CD4 T cells were purified on anti-CD4 microbeads (Miltenyi Biotec). Forty million naive CD4 T cells were then labeled with CFSE and transferred into BALB/c mice (5 x 106 per mouse), and the animals were immunized with 125 µg of OVA peptide in CFA. The LN cells were harvested every 12 h after transfer, and cell division was determined by flow cytometry analysis of CFSE dilution. At 48 h, a total of five divisions was observed, but only three divisions designated D2, D3, and D4, which have undergone two, three, and four divisions, respectively, had sufficient numbers of cells (100–250 x 103 cells) for subsequent transfer and analysis of memory development. C, Activation status of the dividing effectors. The 48-h divided effectors were stained with anti-CD25 and anti-CD44 Abs and were analyzed by flow cytometry. Insets, Illustrate staining with isotype-matched Ab instead of anti-CD25 or anti-CD44 Ab.

 
D2, D3, and D4 effectors display different patterns of IL-7R expression and IFN-{gamma} production

To define the phenotype of the effector cells in each division, which most likely reflects a potential for transition to memory, we analyzed expression of markers usually associated with memory development as well as the ability of the cells to produce IFN-{gamma}, the signature cytokine for these cells. Accordingly, BALB/c mice were given CFSE-labeled naive DO11.10 T cells and the hosts were immunized with OVA peptide in CFA. Forty-eight hours later, the LN were harvested, the effector cells were stained for surface IL-7R, and intracellular BCL-2 and expression was analyzed in the context of cell division. As indicated in Fig. 3, BCL-2 expression had little discrepancy among the effector divisions (Fig. 3A), but IL-7R showed a significant increase in the most advanced D4 division, as assessed by two different anti-IL-7R Abs (Fig. 3B), possibly indicating differentiation of effectors with potential for memory transition (29).


Figure 3
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  		FIGURE 3. Highly divided effectors produce significant IFN-{gamma} and express elevated IL-7R. BALB/c mice recipient of CFSE-loaded naive DO11.10 CD4 T cells (5 x 106 cells/mouse) were immunized with 125 µg of OVA peptide in CFA, and their LN cells were harvested 48 h after imunization 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) was performed to ensure reproducibility. The plots were obtained by analysis of CFSE intensity and expression of the indicated markers on gated live cells. The percentage of CFSE-positive cells expressing the indicated marker was obtained by overlaying the isotype control plot (inset in the left-hand side) over the sample plot in the right-hand corner. The boxed cells represent those positive for marker expression. C, D4 and D2, but not D3 effectors produce significant amounts of IFN-{gamma}. BALB/c mice recipient of 5 x 106 CFSE-loaded naive DO11.10 CD4 T cells were immunized with 125 µg of OVA peptide in CFA. Forty-eight hours later, the LN cells were harvested and cell division was assessed by CFSE dilution. The D2, D3, and D4 divisions were sorted and retransferred (5 x 104 sorted cells per mouse) into new naive BALB/c hosts. To measure production of IFN-{gamma} responses, these new recipients were immunized 1 day later with the suboptimal dose of 20 µg of OVA peptide in CFA. The LN effector proliferative (upper panel) and IFN-{gamma} (lower panel) responses were analyzed by in vitro stimulation with 10 µM OVA peptide at the indicated time points after immunization. A group recipient of naive unsorted DO11.10 T cells was included for control purposes. HA peptide was used as a negative control for the in vitro stimulation. Day 2 of D2 division was not done because there was no sufficient cellularity.

 
To test the divisions for production of IFN-{gamma}, in vitro measurement is not feasible because cytokine production requires long stimulation in vitro, which could trigger further cell division and interfere with the readout. We then decided to sort each division after the 48-h Ag exposure in vivo, transfer the divisions into BALB/c hosts, immunize with a suboptimal dose of OVA peptide (20 µg/mouse) that does not trigger an immune response by endogenous BALB/c cells, and then harvest the LN and measure IFN-{gamma} production. The 48-h pattern of cell division illustrated in Fig. 2B was obtained from the LN of four BALB/c hosts recipient of 5 x 106 CFSE-labeled DO11.10 T cells each. Given the low frequency of effectors (0.2–0.7%) among the total LN cells, 7-h sorting was required to obtain 100–250 x 103 effector cells per division (only two divisions can be sorted at a time). Because the cells might not survive longer in the sorting buffer, the following procedure was adopted for the generation and sorting of effector divisions. Forty DO11.10/scid mice are used as donors of T cells for CFSE labeling and adoptive transfer to four BALB/c hosts. The recipients were sacrificed 48 h after immunization, their LN are harvested, and the effector cells are sorted based on CFSE dilution. Because preliminary studies indicated that 50 x 103 effectors will be required to generate one RAG-2–/– host for memory parking (experimental producedure illustrated in Fig. 1), each sort usually generates two to five mice per division for two divisions at a time.

Accordingly, cells from each division were separated by sorting and transferred into new BALB/c hosts, and 1 day later the animals were immunized with 20 µg of OVA peptide, a dose that does not induce a measurable immune response in normal BALB/c mice (data not shown). The proliferative and IFN-{gamma} LN responses were then analyzed over time. As indicated in Fig. 3C, all divisions yielded Ag-specific proliferative responses slightly higher than mice recipient of naive DO11.10 T cells, indicating that cell sorting and transfer to these new hosts did not severely affect the viability of the effector cells. Moreover, each division displayed a unique proliferation pattern over time, suggesting that cell division at the initial encouter with Ag plays a role in the subsequent response. IFN-{gamma} cytokine analysis also showed that each division yielded an IFN-{gamma} response with a different pattern. Indeed, division D4 developed a strong IFN-{gamma} response that reached a plateau by day 6, whereas D3 did not produce significant IFN-{gamma} at any point despite the fact that the cells were proliferative. Division D2 generated a strong response as early as day 4, but declined quickly. Some IFN-{gamma} was observed with the control HA, possibly reflecting an ongoing ex vivo T cell activation. Thus, D4 effectors that expressed significant IL-7R produced strong IFN-{gamma} (IL-7Rhigh/IFN-{gamma}+), D3 had low IL-7R and insignificant IFN-{gamma} (IL-7Rlow/IFN-{gamma}–), whereas D2 that manisfested low IL-7R expression had significant IFN-{gamma} production (IL-7Rlow/IFN-{gamma}+). Overall, early effector cells that have undergone different divisions display different patterns of IL-7R expression and IFN-{gamma} production.

IL-7Rhigh/IFN-{gamma}+ D4 effectors gave rise to rapid splenic memory responses

Memory responses are always stronger (higher volume) and quicker to come about (more rapid) when compared with primary responses. These two parameters were then used to evaluate memory response of the effector cells of each division. Accordingly, DO11.10 effectors were generated in BALB/c host, and LN cells from each division were sorted on the basis of CFSE dilution and transferred into RAG-2–/– mice for parking (see experimental procedure in Fig. 1). Four months later, the RAG-2–/– recipients, which we designate memory hosts, were immunized with the 20 µg suboptimal dose of OVA peptide and the splenic IFN-{gamma} responses were assessed over time. Again, RAG-2–/– mice were chosen as memory hosts because the lymphopenic environment provides a significant source of IL-7 that sustains survival of the transferred low frequency potential memory cells (22, 23, 24) and optimizes the readout of memory responses. As can be seen in Fig. 4, the specific memory IFN-{gamma} splenic response emanating from D4 effectors developed as early as day 2 after immunization and reached a plateau by day 4 postchallenge. The response is specific because stimulation of the cells with the negative control HA peptide did not yield any measurable IFN-{gamma} response. The kinetics of the D4 memory response parallels with the response of mice that received unseparated effector cells, which include the sum of potential memory cells of all effector divisions (labeled memory). The D3 and D2 divisions, however, yielded delayed memory responses similar to mice recipient of naive DO11.10 T cells. Indeed, IFN-{gamma} production was not significant until day 4 after immunization. These responses were also specific because stimulation with HA peptide did not yield any significant IFN-{gamma} response over the 4-day evaluation period. Overall, IL-7Rhigh/IFN-{gamma}+ D4 effectors yielded strong (volume) and rapid (speed) splenic memory responses, whereas D3 (IL-7Rlow/IFN-{gamma}–) and D2 (IL-7Rlow/IFN-{gamma}+) effector produced delayed memory responses.


Figure 4
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  		FIGURE 4. Kinetics of splenic memory responses generated from distinct effector cell divisions. Sorted effector D2, D3, and D4 cells were transferred into RAG-2–/– mice (5 x 104 cells/mouse) and parked for 4 mo. To analyze the memory response generated from each of the divisions, the recipient mice were immunized with the suboptimal dose of 20 µg of OVA peptide in CFA, and their splenic memory IFN-{gamma} responses were measured by ELISA at the indicated time points after immunization. The column labeled "Memory" represents responses of RAG-2–/– mice recipient of 5 x 104 unlabeled naive DO11.10 CD4 T cells that were immunized with 125 µg of OVA peptide, rested for 4 mo, and reimmunized with 20 µg of OVA peptide. The column labeled "Naive" represents the responses of RAG-2–/– mice recipient of 5 x 104 naive DO11.10 T cells that were not given the primary 125 µg OVA peptide immunization, but did receive a 20 µg challenge regimen 4 mo after cell transfer. The splenic cells (100, 300, and 900 x 103 cells/well) were stimulated with graded concentrations of OVA peptide for 24 h. IFN-{gamma} production was measured in cell culture supernatant by ELISA. Only the highest concentration was used with the negative control HA peptide. Each point represents the mean of triplicate wells ± SD. The results shown are those obtained with 900 x 103 cells/well.

 
IL-7Rhigh/IFN-{gamma}+ D4 and IL-7Rlow/IFN-{gamma}+ D2, but not IL-7Rlow/ IFN-{gamma}– D3 effectors yielded rapid LN memory responses

Memory development from each division was also analyzed in the LN of the memory mice. As indicated in Fig. 5, the IL-7Rhigh/IFN-{gamma}+ D4 effectors yielded a memory IFN-{gamma} response that was at plateau level by day 2 after immunization and parallels with the response of unseparated effector cells (memory). These responses are specific to OVA because stimulation with HA peptide produced no significant responses. Because D4 effectors produced similar kinetics in both the SP and LN and IFN-{gamma} response was rapid in both organs, we designate this response as rapid memory T cell (TRM) response. IL-7Rlow/IFN-{gamma}– D3 effectors again generated a delayed memory response that was not evident until day 4 after immunization. This delay occurred despite the fact that the LN were available on day 2 and had sufficient cellularity, whereas the mice recipient of naive DO11.10 cells had similar delay, but the LN did not form until day 4 after immunization (Fig. 5). We designate these responses as homeostatic memory T cell (THM) because they were delayed in both the SP and LN, have similar kinetics as the naive cells, and proliferated without significant IFN-{gamma} production at the effector stage (30, 31, 32) (Fig. 3C). Intriguingly, the IL-7Rlow/IFN-{gamma}+ D2 division, which generated a delayed memory response in the SP, showed significant IFN-{gamma} production in the LN as early as day 2 after immunization. These responses were designated conditioned memory T cell (TCoM) because they were rapid in the LN, but tardy in the SP. As we shall see below, expression of molecules involved in T cell trafficking may have been responsible for these discrepancies.


Figure 5
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  		FIGURE 5. Kinetics of LN memory responses generated from distinct effector cell divisions. The LN IFN-{gamma} memory responses of RAG-2–/– mice recipient of 5 x 104 D2, D3, or D4 sorted cells described in Fig. 3 were also measured by ELISA at different time points after immunization, as indicated. The same memory and naive groups described in Fig. 3 were included for comparison. Analysis of the response of mice transferred with naive cells was not feasible on days 2 and 3 due to the fact that the LN from these animals had little cellularity and did not form until day 4. Only the highest concentration was used with the negative control HA peptide. Each point represents the mean of triplicate wells ± SD. The results shown are those obtained with 100 x 103 cells/well.

 
Rapid responses correlate with higher frequency of IFN-{gamma}-producing memory precursors

The different patterns of memory responses among the effector divisions could be due to a difference in the frequency of responding memory cells, the amount of IFN-{gamma} these cells produce, or to the ability to traffic through the organs. To test these premises, splenic cells from 4-mo-parking RAG-2–/– mice were harvested before any immunization with OVA and the frequency of IFN-{gamma}-producing memory cells as well as total IFN-{gamma} production were evaluated. ELISPOT analysis, which measures the frequency of IFN-{gamma}-producing memory responders, shows that the D4 division had the highest frequency, followed by D2, but D3 had very few detectable IFN-{gamma}-producing cells (Fig. 6A). No detectable IFN-{gamma}-producing cells were observed with the negative control HA peptide. Evaluation of the total amount of IFN-{gamma} by ELISA indicated that splenic cells from mice recipient of D4 effectors produced significant amounts of IFN-{gamma} upon in vitro stimulation with OVA peptide (Fig. 6B). Splenic cells from mice recipient of D3 effectors did not produce significant IFN-{gamma}, whereas those cells from animals harboring D2 effectors did secrete IFN-{gamma} at significant levels when the stimulation used 10 µM OVA peptide. Again, IFN-{gamma} production is specific because HA peptide did not induce any measurable IFN-{gamma} production. The frequency of IFN-{gamma}-producing memory precursors parallels with total amount of cytokine production in that more IFN-{gamma} is secreted when the frequency of memory precursors is higher (Fig. 6, compare A and B). Thus, it is likely that the frequency of memory precursors able to produce IFN-{gamma} is responsible for the kinetics of memory responses observed after challenge with OVA peptide (Figs. 4 and 5).


Figure 6
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  		FIGURE 6. Frequency of memory precursors in RAG-2–/– mice transferred with D4, D3, or D2 sorted cells. Sorted D2, D3, and D4 divisions were transferred (5 x 104 cells/mouse) and parked in RAG-2–/– mice. Four months later, the mice were sacrificed without any immunization, and the frequency of IFN-{gamma}-producing memory splenic precursors was determined by ELISPOT (A) upon in vitro stimulation with OVA peptide. The total amount of IFN-{gamma} produced in the culture was also determined by ELISA (B) upon in vitro stimulation with OVA peptide. Each point represents the mean of triplicate wells ± SD.

 
TRM, THM, and TCoM express markers signature of memory

As indicated above, the different patterns of memory responses among the effector divisions could be related to differences in the ability of IFN-{gamma}-producing precursors to traffic through lymphoid organs and tissues. CD44 and VLA-4 are molecules expressed on memory cells and may be involved in regulation of memory cell migration (33). To test these premises, splenic cells from 4-mo-parking RAG-2–/– mice were harvested before any immunization with OVA, and the expression of CD44 and VLA-4 on IFN-{gamma}-producing memory precursors was determined ex vivo. As can be seen in Fig. 7, IFN-{gamma}/KJ1-26 double-positive splenic cells from mice recipient of D4, D3, or D2 effectors had high expression of CD44 (Fig. 7A). Note the presence of IFN-{gamma}-producing cells that are negative for KJ1-26 in all divisions, possibly indicating TCR down-regulation by these cells. Thus, the discrepancies in the memory responses among the three divisions observed in Figs. 4 and 5 are likely to be related to the number of IFN-{gamma}-producing memory precursors rather than expression of CD44. As for expression of VLA-4, about one-half of the IFN-{gamma}/KJ1-26 double-positive splenic memory precursors from D4 and D3 effectors had significant VLA-4, whereas most (84%) of the precursors emanating from D2 effectors had significant surface VLA-4 expression (Fig. 7B). This biased VLA-4 expression may have contributed to retention of cells in the LN, leading to accumulation of precursors and rapid memory responses in this organ, but delayed memory in the SP, hence the TCoM for the precursors giving rise to this memory response.


Figure 7
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  		FIGURE 7. TRM, THM, and TCoM precursors express markers signature of memory. Sorted D2, D3, and D4 effectors were transferred into RAG-2–/– mice (5 x 104 cells/mouse) and parked for 4 mo. The mice were then sacrificed (without any immunization), and IFN-{gamma}-producing KJ1-26 cells were 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 were subject to a short 4-h stimulation with 10 µM OVA peptide, stained with Abs to the indicated marker, and for identification purpose costained for surface KJ1-26 and intracellular IFN-{gamma}. Live cells were gated on IFN-{gamma}, and the plots show KJ1-26 and sample marker expression of IFN-{gamma}-positive cells. The numbers of IFN-{gamma}+/KJ1-26+ expressing the test marker are indicated in the upper right corner of the plot.

 
Recently, L-selectin, or CD62L, and the chemokine receptor CCR7 have been defined as markers whose expression can distinguish effector and central memory T cells (34, 35). It is now well established that central memory cells express significant CD62L and CCR7 (CD62Lhigh, CCR7high), whereas effector memory cells display minimal CD62L and CCR7 (CD62Llow, CCR7low) expression (5). To determine whether the newly defined memory cells belong to the effector or central memory subset, the IFN-{gamma}/KJ1-26 splenic memory precursors from mice recipient of D2, D3, and D4 effectors were analyzed for CD62L and CCR7 expression. Accordingly, 4-mo-parking RAG-2–/– mice recipient of D2, D3, or D4 effectors were sacrificed without any immunization and their splenic cells were stained with KJ1-26, and anti-IFN-{gamma} and anti-CD62L, or anti-CCR7 Ab. The results illustrated in Fig. 7 indicate that all three populations are comprised of cells with either phenotype with more prevalence of effector memory. Indeed, expression of CD62L on KJ1-26/IFN-{gamma} double-positive memory precursors 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 the memory pool. These findings indicate a heterogeneity within TRM, THM, and TCoM, which bodes well with the recently defined flexibility associated with effector and central memory phenotypes (36, 37).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Due to the low frequency at which memory cells arise within the effector phase and the lack of memory markers, investigation of the transition of effector cells to memory was difficult and the mechansims underlying such transition remain largely undefined (1, 3, 10, 13, 14, 15, 19, 29, 38). Most of the findings available to date indicate that memory cells arise from slightly activated effectors producing little IFN-{gamma} (39), and that the memory precursors emanate from cells that were exposed to suboptimal Ag as a consequence of late arrival to lymphoid tissues (19) or due to encounter of residual Ag that persists after microbial clearance (18). This has been interpreted to suggest that memory arises from cells that see the Ag at the end of the effector phase with a rather moderate state of activation (18, 19). In this study, an in vivo approach was devised that enabled us to determine whether cells exposed to optimal Ag within 48 h of the effector phase can transit to memory (Fig. 1). Also, we were able to investigate the events that control memory transition at this stage. The findings indicate that highly activated early effectors do indeed transit to memory. Moreover, significant production of IFN-{gamma} seems to aid in an efficient transition. Indeed, naive DO11.10 T cells transferred into BALB/c mice gave rise to a number of LN effector divisions in vivo 48 h after immunization with OVA peptide (Fig. 2). Because these effector divisions occurred 2 days after immunization and expressed significant levels of activation marker such as CD25 and CD44, we refer to them as highly activated early effector divisions. Three of these, D2, D3, and D4, which have undergone two, three, and four divisions, respectively, had sufficient cellularity that enabled isolation of the divisions and retransfer into naive RAG-2–/– host. When the hosts were challenged with OVA peptide 4 mo after transfer, the recipients of effectors from the most advanced D4 division 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 level memory responses in both lymphoid organs that were comparable to naive cells and most likely represent homeostatic memory (30, 31, 32). We refer to these cells as THM. The mice transferred with effectors from division D2 produced homeostatic-like responses in the SP, but rapid responses in the LN; hence, we designated these cells as TCoM for conditioned memory cells. A schematic representation of these findings is illustrated in Fig. 8, providing the type of memory response each division gave rise to as defined by this in vivo study. The latest models of memory development suggest that advanced activation/proliferation leads to reduced numbers of responding memory cells (18, 39). However, recent reports postulated that advanced proliferation would most likely result in more CD4 T cell memory (15). Our findings show that within the early effector phase, the cells express activation markers to the same extent (Fig. 2), and both advanced division (D4) as well as cells that have undergone fewer division (D2) yielded higher frequency of memory precursors (Fig. 6) that enabled rapid memory responses (TRM and TCoM) upon challenge with Ag. Cells with intermediate division (D3) had minimal memory precursor frequency (Fig. 6) and sustained only homeostatic memory responses both in the SP and LN (THM). Although this is intriguing, because one cell division more or less influences memory development, the findings raise interesting observations. Indeed, whereas D4 effectors expressed the activation markers CD25 and CD44 to a similar extent as D3 and D2, they had significant IL-7R expression and produced significant IFN-{gamma} (Figs. 2 and 3). These highly activated IL-7Rhigh/IFN-{gamma}+ effectors gave rise to high frequency of memory precursors (Fig. 6) that yielded rapid TRM responses (Figs. 4 and 5). D3 effectors, which were highly activated, but had low IL-7R expression and no IFN-{gamma} production (IL-7Rlow/IFN-{gamma}–), generated minimal memory precursor frequency (Fig. 6) and only base level homeostatic memory responses (Figs. 4 and 5). D2 effectors, which expressed CD25 and CD44 activation markers, had low IL-7R expression and produced significant IFN-{gamma}. These highly activated IL-7Rlow/IFN-{gamma}+ effectors gave rise to significant memory precursors that yielded significant memory response that were localized in the LN, but not the SP, hence the designation (TCoM) responses (Figs. 4, 5, and 8). Thus, it seems that production of significant amounts of IFN-{gamma} is a requirement at this early stage of the effector phase for transition to memory. IL-7R expression may influence survival of memory precursors leading to robust memory responses in the SP and LN. VLA-4, a molecule that is involved in regulating T cell trafficking (33, 40, 41), may have played a role in the preferential responses of D2 memory cells in the LN, because more VLA-4 was found on the memory precursors emanating from D2 effector, giving rise to TCoM memory responses (Figs. 7 and 8).


Figure 8
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  		FIGURE 8. Production of IFN-{gamma} by early effectors plays a major role in the transit to memory. Effector divisions develop distinct memory responses depending on the pattern of IFN-{gamma} production and IL-7R expression during the first exposure to Ag.

 
Even with a limited technical feasibility that enabled studies with only three of five early effector divisions, three distinct types of memory cells, TRM, THM, and TCoM, were defined on the basis of the kinetics of the responses (Fig. 8). The reference to kinetics (volume and speed) of the response has always been the method of choice for evaluation of memory development. Because the effector cells have been removed from the Ag and parked in the RAG-2–/– lymphopenic environment, equal survival factors will be available to all effectors. Thus, whereas this environment serves to amplify memory and ease the readout of the responses, the different kinetics of the responses observed among the divisions most likely reflect the effect of Ag-induced cell division rather than RAG-2–/–-driven homeostatic proliferation. The findings in this study reinforce prior suggestions postulating that differentiation of a naive lymphocyte into a memory cell most likely occurs as a result of events emanating from the encounter with Ag rather than an intrinsic predetermination (42). Moreover, recent studies in this area elegantly demonstrated that exposure to limited amount of residual Ag due to late arrival of circulating T cells to regional LN triggers late cell division that specifically guided transition to central memory (19). Our findings add a novel observation demonstrating that early effectors also transit to memory and a variance by a single division leads to different kinetics of the memory response (Fig. 8). Also, significant production of the cell’s signature cytokine (i.e., IFN-{gamma}) is required for such transition, possibly reflecting an epigenetic phenomenon (43). Finally, the three populations, TRM, THM, and TCoM, defined in this study, had a mixture of effector and central memory cells because both CD62Lhigh CCR7high VLA4– and CD62Llow CCR7low VLA4+ cells were components of these responses, and this bodes well with the recently defined flexibility among the central/effector subsets (36, 37).

Overall, these studies show that highly activated early effector CD4 T cells transit to memory, and production of significant IFN-{gamma} is required for such transition. Moreover, a single cell division affects the transition to memory at this stage. During an initial infection or tumor development, microbes or cancer cells may influence division and/or IFN-{gamma} production of effector lymphocytes either by loading variable amounts of Ag or by interference with Ag presentation. This could affect the development of memory and the effectiveness of vaccines (44) especially given that one cell division produces a significant difference on the development, rapidity, and localization of the memory response.


   Acknowledgment
 
We thank Louise Barnett for help with cell sorting.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 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 by training grants from National Institute of General Medical Sciences. D.M.T. was supported by Life Sciences fellowship from University of Missouri. Back

2 Current address: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. Back

3 Current address: Division of Immunology, Karp Laboratories, Children’s Hospital, Harvard Medical School, Boston, MA 02115. Back

4 Current address: National Cancer Institute, Metabolism Branch, Bethesda, MD 20892. Back

5 Address correspondence and reprint requests to Dr. Habib Zaghouani, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, M616 Medical Sciences Building, Columbia, MO 65212. E-mail address: zaghouanih{at}health.missouri.edu Back

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

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


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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