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The Journal of Immunology, 2006, 177: 869-876.
Copyright © 2006 by The American Association of Immunologists

Divergent Generation of Heterogeneous Memory CD4 T Cells1

Vaishali R. Moulton*,{dagger}, Nicholas D. Bushar*,{dagger}, David B. Leeser*, Deepa S. Patke* and Donna L. Farber2,*

* Department of Surgery, Division of Transplantation, and {dagger} Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mechanisms for the generation of memory CD4 T cells and their delineation into diverse subsets remain largely unknown. In this study, we demonstrate in two Ag systems, divergent generation of heterogeneous memory CD4 T cells from activated precursors in distinct differentiation stages. Specifically, we show that influenza hemagglutinin- and OVA-specific CD4 T cells activated for 1, 2, and 3 days, respectively, exhibit gradations of differentiation by cell surface phenotype, IFN-{gamma} production, and proliferation, yet all serve as direct precursors for functional memory CD4 T cells when transferred in vivo into Ag-free mouse hosts. Using a conversion assay to track the immediate fate of activated precursors in vivo, we show that day 1- to 3-activated cells all rapidly convert from an activated phenotype (CD25highIL-7RlowCD44high) to a resting memory phenotype (IL-7RhighCD25lowCD44high) 1 day after antigenic withdrawal. Paradoxically, stable memory subset delineation from undifferentiated (day 1- to 2-activated) precursors was predominantly an effector memory (CD62Llow) profile, with an increased proportion of central memory (CD62Lhigh) T cells arising from more differentiated (day 3-activated) precursors. Our findings support a divergent model for generation of memory CD4 T cells directly from activated precursors in multiple differentiation states, with subset heterogeneity maximized by increased activation and differentiation during priming.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of memory T cells is essential to the development of long-lived protective immunity; however, basic mechanisms involved in this process remain unknown. Although it is generally accepted that memory T cells develop from activated T cells generated during the initial Ag encounter, the pathway(s) and cellular precursor(s) to memory T cells are not defined. The classic pathway for linear differentiation of memory T cells directly from differentiated effector cells remains the favored model based on evidence demonstrating emergence of memory T cells from effector T cells in vivo (1, 2, 3, 4). However, memory T cell development has also been shown to occur in the absence of an initial effector response (5, 6, 7). Whether memory T cell generation predominantly follows one type of pathway from a specific precursor population or whether multiple pathways and precursors for memory T cell generation coexist, is not known.

The cellular precursors to memory T cells are distinguished by their selective survival within an activated/effector T cell pool that is otherwise susceptible to apoptosis. It was recently shown that expression of the cytokine receptor IL-7R and/or the CD8{alpha}{alpha} homodimer marked precursors for lymphocytic choriomeningitis virus-specific memory CD8 T cell development (8, 9). However, this correlation has not been upheld for memory CD8 T cells in similar systems (10, 11, 12), suggesting that the expression of specific genes may not drive memory T cell generation, but may be a secondary effect of other factors (not yet defined) that directly promote memory T cell generation. The identification of genes or factors directly involved in memory CD4 T cell generation has not yet been accomplished, although IL-7R expression has been implicated in memory CD4 T cell survival (13, 14).

An additional complication in deciphering the cellular origin of memory T cells is the remarkable heterogeneity of memory CD4 and CD8 T cells in phenotype and tissue distribution (15, 16). Two memory T cell subsets have been defined based on expression of the lymph node homing receptor(s) CD62L and/or CCR7, and are designated central memory T cells (TCM3; CD62LhighCCR7+), which primarily reside in lymphoid tissue, and effector memory T cells (TEM; CD62LlowCCR7–), which are the predominant subset in nonlymphoid tissue (17, 18, 19). We and others have also identified additional variations in tissue-resident memory T cells (20, 21), suggesting that multiple memory subsets exist. Mechanisms for the generation of these heterogeneous memory subsets have not been elucidated, including whether distinct type of memory subsets are derived from specific precursors, or via distinct pathways.

Thus, the heterogeneity of memory T cells, the difficulty of associating specific gene expression with memory precursors, and the lack of information on factors affecting the fate of an activated/effector T cell, all present formidable challenges toward elucidating mechanisms for memory T cell generation. It has not been possible to establish a precursor-product relationship between activated/effector cells and the resultant memory T cells with the current in vivo models (largely viral infection (22)) for three reasons. First, the primary effector response in vivo is heterogeneous in terms of extent of activation and differentiation (23), and one cannot determine from which activated/effector population(s) memory T cells develop. Second, the fate of individual activated T cell clones in vivo cannot be tracked, and even T cells derived from TCR-transgenic mice expressing a fixed TCR develop into heterogeneous effector and memory populations (2, 19, 21, 23, 24). Finally, primary responses are typically examined at the peak effector response (after 1 wk) and memory responses months thereafter (8, 25, 26), with no analysis during the intervening period, such that the immediate fate of activated T cells and their potential for memory development are not known.

In this study, we used an adoptive transfer system where we could follow the fate of activated memory precursors in vivo, to investigate mechanisms for development of heterogeneous memory CD4 T cells. We modeled heterogeneous activation during a primary response by altering the duration of Ag exposure during priming, and hypothesized that these differently activated cells could serve as specific precursors for development of distinct memory T cell subsets. We found that CD4 T cells specific either for influenza hemagglutinin (HA) or chicken OVA activated for 1, 2, or 3 days in vitro exhibited gradations of differentiation by cell surface phenotype, IFN-{gamma} production, and entry into the cell cycle; however, they all developed into memory CD4 T cells in vivo in Ag-free intact mouse hosts. Using a conversion assay to track the immediate fate of differentially activated precursors in vivo, we found that acquisition of memory T cell markers such as IL-7R expression occurs as early as 1 day following Ag withdrawal. Paradoxically, effector memory (CD62Llow) CD4 T cells were the predominant subset arising from short-term activated, undifferentiated CD4 T cells, with increased proportions of central memory CD4 T cells developing from differentiated precursors activated for longer times. Our results support a new divergent model for memory CD4 T cell generation directly from activated precursors independent of differentiation state, with subset heterogeneity maximized by increased activation and differentiation during priming.


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

BALB/c mice (8–16 wk of age) were obtained from Charles River Laboratories. HA-TCR transgenic mice (27) and DO11.10 OVA-TCR mice (28) bred as heterozygotes onto BALB/c (Thy 1.2) hosts, were maintained in the animal facility at the University of Maryland (Baltimore, MD) under specific pathogen-free conditions, and all animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Maryland School of Medicine.

Abs and peptides

The following Abs were purified from bulk culture supernatants and purchased from BioExpress: anti-CD8 (TIB 105), anti-CD4 (GK1.5), anti-I-Ad (212.A1), and anti-Thy-1 (TIB 238). The 6.5 anti-clonotype Ab directed against the HA-TCR (27), was purified and conjugated to biotin (Pierce). The following fluorochrome-conjugated Abs were purchased from BD Pharmingen: PerCP-, FITC-, and allophycocyanin-conjugated anti-CD4 (clones GK1.5, RM4-5), PE-conjugated anti-IFN-{gamma} (clone XMG1.2, rat IgG1), PE-conjugated anti-CD25 (clone PC61, rat IgG1), allophycocyanin- and PE-conjugated anti-CD62L (clone MEL-14, rat IgG2a), FITC-conjugated anti-Thy1.2 (clone 30-H12), FITC-conjugated anti-CD44 (clone IM7, rat IgG2b), and PerCP- and allophycocyanin-conjugated streptavidin. PE-conjugated anti-IL-7R{alpha} (clone A7R34, rat IgG2a) was purchased from eBioscience, and allophycocyanin-conjugated anti-DO11.10-TCR KJ1-26 clonotypic Ab from Caltag Laboratories.

HPLC-purified HA (110–119, SFERFEIFPKE) and OVA (323–339, ISQAVHAAHAEINEAGR) peptides were synthesized by the Biopolymer Lab at the University of Maryland (Baltimore, MD).

Generation of effector and memory CD4 T cells

CD4 T cells were purified from spleens of HA-TCR and OVA-TCR mice as described previously (1) and sorted for CD44 expression by magnetic sorting using AutoMACS (Miltenyi Biotec) resulting in 99–100% CD44low CD4 T cells, and cultured with 5.0 µg/ml HA peptide or 1.0 µg/ml OVA peptide, respectively, and mitomycin C-treated APCs prepared from BALB/c splenocytes as described previously (1) in complete Clicks medium (Irvine Scientific) for 1, 2, or 3 days at 37°C. Differentially activated cells were purified by Ficoll centrifugation (LSM; ICN/Cappel), and sorted for CD25 expression using AutoMACS (Miltenyi Biotec), resulting in 90–95% pure CD25+ CD4 T cells. Activated CD25+ cells or naive CD4 T cells (1.5 x 106/mouse) were injected i.v. into BALB/c (Thy1.1) adoptive hosts as described (24, 29), and persisting memory CD4 T cells were harvested 1–9 mo posttransfer.

Proliferation assays

Naive HA- or OVA-specific CD4 T cells were labeled with 2.5 mM CFSE (Molecular Probes), and 1 x 106 T cells were cultured with 3 x 106 splenic APCs plus HA peptide (5 µg/ml) or OVA peptide (1 µg/ml) in 24-well plates at 37°C, harvested at time points described in the text, and analyzed by flow cytometry. Analysis of cell division was conducted as described previously (30), with the number of undivided cohorts obtained by dividing the number of events (x) at each cell division n, by 2n. The percentage of dividing cells was obtained by the equation: percent dividing cells = (total no. of cohorts undergoing 1–9 divisions/total no. of cohorts undergoing 0–9 divisions) x 100.

Assays for cytokine production

Cytokine production from memory CD4 T cells was assessed by 18-h intracellular cytokine staining (ICS) analysis, as previously described (21, 31). Briefly, HA- or OVA-specific CD4 T cells (1 x 106) were cultured with syngeneic APC (3 x 106) with or without HA peptide (5 µg/ml) or OVA peptide (1 µg/ml) for 18 h. Monensin (Golgistop; BD Pharmingen) was added to cultures and cells were harvested after 6 h, surface stained with fluorescent mAbs to CD4, transgenic TCR and Thy1.2, fixed (Cytofix buffer; BD Pharmingen), permeabilized, and stained intracellularly with anti-IFN-{gamma} Ab or its isotype control as described previously (21, 31), and analyzed using the FACSCalibur and CellQuest software (BD Biosciences). Cytokine production from day 1-, 2-, and 3-activated cells was assessed similarly by adding monensin in the last 6 h of each culture.

In vivo conversion assay

CD4 T cells isolated from naive HA-TCR mice were labeled with 2.5 µM CFSE (Molecular Probes) as described previously (32), and 1 x 106 T cells were cultured with 3 x 106 BALB/c splenic APC plus HA peptide (5 µg/ml) in 24-well plates at 37°C, harvested at 1, 2, or 3 days, sorted for CD25 expression as above, and adoptively transferred into intact BALB/c hosts. Each successive day, spleens and/or peripheral blood were harvested from individual mice, and CD4 T cells were isolated, stained for cell surface markers, and analyzed by flow cytometry. For analysis of IFN-{gamma} production, splenocytes were isolated on successive days from recipients of CD25+ day 1- to 3-activated cells, directly cultured for 4 h in vitro with monensin with or without PMA/ionomycin as described previously (21), and harvested for analysis of IFN-{gamma} production by ICS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
We previously found that priming of influenza HA-specific CD4 T cells from HA-TCR transgenic mice with HA peptide and APC for 3–5 days in vitro provided the necessary signals for generation of a long-lived HA-specific memory CD4 T cell population when transferred into Ag-free BALB/c and RAG2–/– hosts, with all of the phenotypic and functional qualities of memory T cells—including rapid effector function, heterogeneity in CD62L expression, and diverse distribution in lymphoid and nonlymphoid tissues (21, 24, 29, 31, 33). To test our hypothesis that differentially activated T cells give rise to distinct subsets of memory T cells, we used a variation of this in vitro priming/adoptive transfer system to isolate sufficient quantities of activated cells for subsequent tracking and analysis of memory development in vivo (see Fig. 1). We generated differentially activated T cells by stimulating purified naive TCR-transgenic (CD44low) CD4 T cells with peptide Ag and APC in vitro for 1, 2, or 3 days, respectively. The resultant activated cells (Thy1.2+) were sorted based on expression of the activation marker, CD25, to isolate primed cells and remove residual APC, transferred in equal numbers into syngeneic BALB/c (Thy1.1) mouse hosts (with control transfers of naive CD4 T cells) and assayed for development of memory CD4 T cells after 1–9 mo in vivo (Fig. 1). To verify that our findings were not specific to one antigenic system, we used purified CD4 T cells from both HA-TCR and DO11.10 (OVA-specific) transgenic mice.


Figure 1
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  		FIGURE 1. Schematic for memory CD4 T cell generation from differentially activated precursors. CD4 T cells purified from Thy1.2+ HA-TCR or OVA-TCR transgenic mice were sorted to obtain CD44low naive cells that were activated with HA/APC or OVA/APC in vitro for 1, 2, or 3 days, and at each time point, activated CD25+ T cells were sorted and transferred in equal numbers (1.5 x 106) into syngeneic Thy1.1+ BALB/c hosts. Persisting Thy1.2+ OVA-specific (KJ1-26+) or HA-specific (6.5+) CD4 T cells were analyzed 1–9 mo posttransfer. As controls, sorted CD44low OVA- or HA-specific naive CD4 T cells were transferred in parallel.

 
Generation of differentially activated Ag-specific CD4 T cells

We initially evaluated the differentiation state of naive/day 0 (CD44low) Ag-specific CD4 T cells activated in vitro for 1 (day 1), 2 (day 2), and 3 days (day 3) with APC plus antigenic peptide, by assessing their phenotype, proliferation, and capacity for effector cytokine production (Fig. 2). Phenotypically, there was a progressive up-regulation of the activation markers CD44 and CD25 (Fig. 2A, second and third rows), and a progressive increase in cell size (first row) with increased Ag exposure from 1 to 3 days of activation that was comparable in both HA- (Fig. 2A) and OVA- (data not shown) specific CD4 T cells. By contrast, IL-7R was extensively down-regulated on day 1- to 3-activated Ag-specific CD4 T cells, with only a small proportion (2–3%) of IL-7R+ T cells remaining at each time point (Fig. 2A, fourth row). For proliferation, there was minimal division of the day 1-activated CD4 T cells, a low proportion of dividing cells by day 2, yet a substantial proportion of cells undergoing multiple divisions by day 3 for both HA- (Fig. 2B) and OVA-specific (data not shown) CD4 T cells. Functionally, day 1-activated CD4 T cells produced very low levels of IFN-{gamma}, with increased levels of IFN-{gamma} from day 2- and day 3-activated CD4 T cells (Fig. 2C). For HA-specific CD4 T cells, peak IFN-{gamma} production was observed from day 2 cells (Fig. 2C, left), whereas for OVA-specific CD4 T cells, peak IFN-{gamma} production occurred from day 3 cells (right). These results indicate that altering the duration of Ag exposure affects the differentiation state of Ag-specific CD4 T cells with respect to phenotype, effector function, and proliferation; day 1-activated cells are undifferentiated by all parameters, day 3-activated cells are maximally differentiated by all criteria, and day 2-activated cells are intermediate-differentiated in effector cytokine production (particularly for HA-specific T cells), but not yet proliferating. Our results are likewise consistent with earlier studies that found limiting Ag exposure did not enable effector CD4 T cell differentiation (34, 35).


Figure 2
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  		FIGURE 2. Phenotype, proliferation, and function of differentially activated Ag-specific CD4 T cells. A, Naive (day 0) and in vitro day 1- to 3-activated HA-specific CD4 T cells were analyzed for size (first row) and expression of CD44, CD25, and IL-7R{alpha} (rows 2–4, filled histograms), compared with isotype controls (open histograms) gated on clonotypic 6.5+CD4+ T cells. Numbers indicate percentages, with markers drawn based on isotype controls at each time point. Numbers in parentheses (row 4) indicate MFI. Results are representative of >10 experiments. B, Proliferation of day 0 and day 1- to 3-activated HA-specific CD4 T cells. Numbers represent percentage of dividing cells after stimulation for 1–3 days with Ag, calculated as described in Materials and Methods, and are representative of three experiments. C, IFN-{gamma} production from day 1-, 2-, and 3-activated HA-specific (left panel) and OVA-specific (right panel) CD4 T cells assessed by ICS indicated by percentage of 6.5+ or KJ1-26+IFN-{gamma}+ CD4 T cells, with results shown as mean ± SD from three experiments for each cell type. D, MFIs for CD44, and CD25 (left graph), IL7R{alpha} and forward scatter (right graph) of day 0 and day 1–3 CD25+ HA-specific CD4 T cells.

 
We sorted day 1- to 3-activated cells for CD25 up-regulation before adoptive transfer. Fig. 2D shows mean fluorescence intensities (MFIs) of CD44 and CD25 (left), IL-7R{alpha} and forward scatter (right) of the naive (day 0) and sorted CD25+ day 1-, day 2-, and day 3-activated HA-specific CD4 T cells used for adoptive transfer. The progressive alterations in these phenotypic parameters are similar to those observed on unfractionated day 1- to 3-activated cells (Fig. 2A and data not shown). To ensure that the adoptively transferred cells were not being further stimulated in vivo, we cultured the day 1, day 2 and day 3 CD25+ cells with freshly isolated APCs, overnight. We found that CD25 expression remained unchanged or was slightly decreased after culture, and no IFN-{gamma} production could be detected from these cultured cells (data not shown), suggesting that the level of Ag carryover was minute and not sufficient to further stimulate these cells in vivo.

Memory T cell generation from differentially activated precursors

We transferred sorted, CD25+ day 1- to 3-activated OVA and HA-specific CD4 T cells into BALB/c (Thy1.1) hosts, and found that they all gave rise to functional Ag-specific memory CD4 T cells based on three criteria: long-term persistence, a memory-specific phenotype, and rapid recall function. For OVA-specific CD4 T cells, we found significant numbers of persisting OVA-specific memory CD4 T cells from day 1- to 3-activated precursors in the spleen, lungs, and mesenteric lymph nodes of BALB/c hosts (Fig. 3A). By contrast, we observed complete attrition of OVA-specific naive CD4 T cells, which is known to occur in adoptive transfers into intact mice (36), and further establishes the persisting CD4 T cells in our system as memory. Although substantial proportions of memory CD4 T cells were generated from day 1- to 2-activated precursors, the frequency of persisting memory CD4 T cells from day 3 precursors averaged 5-fold more than that of day 1–2 in the spleen and lung and 10-fold more in the lymph node (Fig. 3A and data not shown). Spleen-derived OVA-specific memory T cells from day 1–3 precursors were small in size and exhibited a memory phenotype (CD25lowCD44highIL-7Rhigh) (Fig. 3B), which was likewise observed in lymph node and lung-derived memory cells (data not shown). Functionally, day 1- to 3-derived memory T cells from spleen and lung all exhibited rapid IFN-{gamma} production following in vitro recall with OVA peptide and APC, with an increased proportion of IFN-{gamma} producers from day 3-derived compared with day 1- to 2-derived memory CD4 T cells (Fig. 3C). These results show that memory CD4 T cells can be generated from CD4 T cells exposed to Ag for only 1 day; however, memory T cell frequency and rapid recall capacity were greatest when derived from CD4 T cells activated with Ag for longer times.


Figure 3
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  		FIGURE 3. Generation of OVA-specific memory T cells from day 1- to 3-activated precursors. A, Recovery of persisting OVA-specific CD4 T cells from day 1- to 3-activated cells 8–10 wk posttransfer. CD4 T cells were isolated from the spleen, lung, and mesenteric lymph nodes of BALB/c (Thy1.1) recipients of naive or day 1- to 3-activated OVA-specific CD4 T cells. Plots show Thy1.2 vs TCR clonotype KJ1-26 expression in individual mice, with percentage of Thy1.2+KJ1-26+ cells indicated in the upper right quadrant. Average absolute yields for memory recovery varied between experiments with an average ± SD for day 1-derived memory, 74,100 ± 50,982.4; day 2-derived memory, 106,325 ± 44,594.18; and day 3-derived memory, 1,076,400 ± 537,684.9. B, Phenotype of persisting spleen-derived OVA-specific CD4 T cells in spleen gated on KJ1-26+CD4+ T cells: first row, size (forward scatter); second through fourth rows, expression of CD25, CD44, and IL-7R{alpha} (filled histograms) compared with isotype-matched controls (open histograms). C, Recall function of persisting OVA-specific memory CD4 T cells from day 1- to 3-activated precursors. CD4 T cells purified from recipients of day 1- to 3-activated cells were stimulated with OVA peptide and APC for 18 h, and IFN-{gamma} production was measured by ICS, with percentage of CD4+IFN-{gamma}+ indicated in the upper right quadrant gated on KJ1-26+ T cells. Quadrants were drawn based on isotype-matched controls, and control cultures with APC alone and no peptide showed no IFN-{gamma} production (data not shown). Results are representative of three experiments.

 
We performed similar adoptive transfers of day 1- to 3-activated cells using HA-specific CD4 T cells into BALB/c (Thy1.1) hosts. Although overall yields were lower compared with OVA-specific CD4 T cells, we also observed memory generation from HA-specific day 1- to 3-activated precursors, with no persisting cells from transfers of naive HA-specific T cells (Fig. 4A, top row). We obtained the lowest frequency of HA-specific memory CD4 T cells from day 1 precursors, and comparable frequencies from day 2- and day 3-activated precursors (Fig. 4A and data not shown). Persisting day 1- to 3-derived HA-specific memory CD4 T cells in intact hosts likewise bore a memory phenotype (CD25lowCD44highIL-7Rhigh) (data not shown), and exhibited rapid IFN-{gamma} and IL-2 production upon restimulation with HA peptide in vitro, with a lower overall recall capacity from day 1-derived compared with day 2- and day 3-derived memory cells (Fig. 4B), similar to the OVA system. However, with HA-specific memory CD4 T cells, we typically find a lower proportion of IFN-{gamma} producers after Ag stimulation compared with OVA-specific memory CD4 T cells (data not shown), due to the lower overall avidity of the 6.5 TCR compared with the KJ1-26 TCR (M. Ndejembi and D. L. Farber, unpublished results). Furthermore, we obtained similar results for memory generation from HA-specific day 1- to 3-activated cells that were not sorted for CD25 up-regulation before transfer. These results establish that memory CD4 T cells can be generated from Ag-specific CD4 T cells at multiple stages of differentiation-from undifferentiated (day 1), to highly differentiated (day 3)—that is, generalizable to two diverse antigenic systems.


Figure 4
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  		FIGURE 4. Generation and function of HA-specific memory CD4 T cells from day 1 to 3 precursors. A, Recovery of persisting splenic HA-specific memory CD4 T cells from BALB/c recipients of HA-specific naive or day 1- to 3-activated CD4 T cells, 6–10 wk posttransfer. Plots show expression of Thy1.2 vs TCR clonotype 6.5 on a live cell gate with percentage Thy1.2+6.5+ indicated. B, Recall function of HA-specific memory CD4 T cells from day 1- to 3-activated precursors activated with HA/APC for 18 h; IFN-{gamma} (top) and IL-2 (bottom) production shown gated on Thy1.2+6.5+ T cells. Unstimulated controls showed <3.0% background and isotype-matched controls <0.5% background (data not shown).

 
Direct conversion of differentially activated precursor cells into Ag-specific memory CD4 T cells

Our finding that even short-term activated yet undifferentiated Ag-specific CD4 T cells (day 1-activated) gave rise to long-lived memory T cells contrasted with the prevailing view that memory T cells derive from differentiated effector cells. We therefore asked whether memory generation from day 1- to 3-activated precursors in our system occurred via direct "resting down" to a memory phenotype, or whether differentiation to effector cells continued in vivo due to an initial activation program, as suggested for CD8 T cells (37). To distinguish these possibilities, we developed a conversion assay to track the acquisition of memory characteristics from day 1- to 3-activated CD4 T cells on successive days following their transfer into adoptive hosts in vivo. For this assay, we transferred CFSE-labeled day 1–3 CD25+ activated HA-specific CD4 T cells into BALB/c mice, and recovered T cells from peripheral blood and spleen each successive day up to 4 days posttransfer. From this analysis, we found that the CD25highIL-7RlowCD44+ phenotype of activated T cells rapidly converted to a resting memory (CD25lowIL-7RhighCD44high) phenotype after 1 day in vivo (Fig. 5 and data not shown). After only 24 h in vivo, CD25 expression was significantly down-regulated (Fig. 5A, first row), and IL-7R expression was substantially up-regulated (Fig. 5B, first row) from day 1- to 3-activated cells, and these phenotypic changes continued after 2–3 additional days in vivo (Fig. 5, A and B, second and third rows). Fig. 5C depicts the corresponding MFIs for CD25 (left panel) and IL-7R{alpha} (right panel) expression of the cells in vitro (black symbols) and in vivo (red symbols). CD44 expression was also further up-regulated after an additional 1–3 days in vivo on day 1- to 3-activated CD4 T cells (data not shown).


Figure 5
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  		FIGURE 5. Rapid conversion of day 1- to 3-activated precursors to a memory phenotype. CFSE-labeled HA-TCR CD4 T cells activated for 1–3 days were sorted for CD25 expression and transferred into BALB/c hosts, recovered on successive days posttransfer, and analyzed phenotypically and functionally. Plots show CD25 expression (A) and IL-7R expression (B), of day 1- to 3-activated input cells ("in vitro") and cells recovered from spleen on successive days in vivo (outlined by red boxes), gated on CD4+6.5+ T cells. Numbers in A and B indicate percentage of cells expressing the phenotypic marker (filled histograms) compared with isotype-matched controls (open histograms). C, MFIs for CD25 and IL-7R{alpha} are shown with black symbols denoting in vitro profiles and red symbols denoting in vivo profiles and how they change over time. D, Proliferation of day 1- to 3-activated cells recovered on successive days posttransfer in vivo. Numbers in plots indicate percentage of dividing cells (see Materials and Methods). Similar results were obtained with peripheral blood T cells (data not shown).

 
To address whether the day 1- to 3-activated cells were undergoing further turnover and/or functional differentiation, we likewise monitored proliferation and IFN-{gamma} production at successive days posttransfer into Ag-free hosts. As shown in Fig. 5D, day 1- to 3-activated precursors all continued to divide in vivo, with a similar extent of proliferation observed at day 4 for all precursors. By contrast, analysis of de novo IFN-{gamma} production from day 1- to 3-activated cells recovered ex vivo did not reveal significant IFN-{gamma} production (Table I; columns marked "in vivo"), indicating that these cells were not producing IFN-{gamma} de novo, although they could be further stimulated in vitro with PMA/ionomycin to produce IFN-{gamma} (Table I). These results indicate that day 1-activated cells acquire memory T cell characteristics (i.e., CD25lowIL-7RhighCD44high phenotype and ability to produce IFN-{gamma} upon restimulation) without eliciting substantial de novo effector function during priming. When taken together, our results demonstrate a rapid conversion of CD25+ day 1- to 3-activated precursors into resting memory CD4 T cells in vivo upon removal of the antigenic stimulus, in the absence of further differentiation, yet accompanied by proliferative turnover.


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  		Table I. Analysis of IFN-{gamma} production in the conversion assay

 
Heterogeneity of differentially generated Ag-specific memory CD4 T cells

Our results above show that Ag-activated CD4 T cells in distinct stages of differentiation can directly give rise to functional memory CD4 T cells. Although we observed heterogeneous tissue distribution in lymphoid and nonlymphoid compartments from day 1–3 precursors (see Fig. 3), we also asked whether the stable distribution of memory TCM and TEM subsets varied according to the extent of priming by examining CD62L expression in long-term memory T cells derived from day 1- to 3-activated precursors. We found that HA- and OVA-specific memory CD4 T cells derived from day 1 precursors were primarily CD62Llow, consistent with a TEM phenotype, with an increased proportion of CD62Lhigh TCM cells in day 2- and day 3-derived memory T cells (Fig. 6A). Expression of CD62L likewise cosegregated with CD45RBhigh expression (data not shown), which we previously found to also delineate mouse TCM (21). The proportion of HA- and OVA-specific TCM (CD62Lhigh) memory CD4 T cells in individual BALB/c mice from multiple experiments is depicted in Fig. 6B, and reveals two consistent findings: first, there was a preponderance of CD62Llow TEM memory CD4 T cells derived from day 1 and day 2 precursors in all experiments, and second, the proportion of CD62Lhigh TCM cells from day 3 precursors was significantly higher than that of day 1 and day 2 precursors (p < 0.001) for both HA- and OVA-specific memory CD4 T cells (Fig. 6B). This trend of increased CD62Lhigh memory T cells from day 3 vs day 1 and 2 precursors was also evident in lymph nodes from recipient mice, and was independent of whether activated precursors were sorted for CD25 before transfer, sorted for pure CD44low naive T cells before activation, or transferred into BALB/c or RAG2–/– hosts (Fig. 6B and data not shown). These results demonstrate that delineation of CD62L subsets of memory CD4 T cells can be affected by the differentiation state of the activated precursor.


Figure 6
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  		FIGURE 6. CD62L heterogeneity of HA-specific memory CD4 T cells derived from differentially activated precursors. A, Phenotypic heterogeneity of HA- and OVA-specific memory CD4 T cells isolated from spleens of BALB/c hosts 2–3 mo posttransfer. CD62L expression (filled histograms) compared with isotype controls (open histograms) gated on CD4+6.5+ T cells (top row) and CD4+KJ1-26+ T cells (bottom row). Numbers indicate percentage of CD62Lhigh cells. Results are representative of more than three experiments for each host with two to three mice per group. B, Graph showing the percentage of splenic TCM (CD62Lhigh) HA-specific (black symbols) and OVA-specific (red symbols) memory CD4 T cells in individual BALB/c hosts (n = 12 for each precursor). Average percentages of CD62Lhigh cells for each time point are 17 ± 11% for day 1- and day 2-derived cells, and 30 ± 9% for day 3-derived cells. Average for day 3-derived cells is significantly different from that of day 1- and day 2-derived memory (p < 0.001).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Mechanisms for the generation of memory CD4 T cells and delineation into subsets have proven difficult to establish. We reveal here that influenza HA- and OVA-specific CD4 T cells activated for 1, 2, and 3 days, respectively, exist in distinct differentiation states, yet all serve as direct precursors for functional memory CD4 T cells upon withdrawal from the antigenic stimulus. Paradoxically, CD62Llow TEM cells represent the predominant subset generated from undifferentiated (day 1-activated) precursors, with more differentiated precursors giving rise to CD62Lhigh TCM cells. Our results support a new model for generation of memory T cells from multiple precursors via branched diversions on the pathway of T cell activation, effector differentiation and death (Fig. 7). In this model, the potential for memory generation from activated cells occurs immediately upon withdrawal of Ag generating a "pre-memory" T cell with up-regulated IL-7R expression and down-regulated CD25 expression (Fig. 7). The less differentiated T cells skew toward development of TEM cells, whereas more differentiated precursors give rise to increased development of TCM cells and increased subset heterogeneity.


Figure 7
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  		FIGURE 7. Divergent model for heterogeneous memory generation. In this model, memory CD4 T cells are generated from multiple activated precursors upon Ag withdrawal, with the extent of heterogeneity proportional to the precursor differentiation state.

 
Our model for memory generation contrasts the prevailing linear model for memory generation from differentiated effector cells, which was supported by earlier adoptive transfer studies demonstrating memory T cell generation from populations of activated T cells producing effector cytokines (3, 4). In analogous transfer studies, activated T cells that did not produce effector cytokines were also shown to persist in vivo as memory T cells (6, 38). However, the immediate fate of the activated and differentiated T cells transferred in the above studies was not examined, so it is not clear whether true linear or divergent pathways were followed. Here, we demonstrate direct emergence of resting memory T cells from activated T cells exhibiting gradations of activation/differentiation in terms of cell size, expression of activation markers, and effector cytokine capacity (Fig. 5). In humans, functional memory T cells exhibiting heterogeneous expression of chemokine receptors representing phenotypes intermediate between naive and classic memory T cells have recently been identified (39), providing indirect evidence for memory differentiation from activated cells in diverse differentiation states. These results and our findings suggest that multiple precursors for memory T cells exist and exhibit gradations of differentiation (day 1–3 activated; Fig. 7). However, it is also possible that the memory precursors within day 1- and 3-activated T cell pools may have been comparably activated/differentiated, yet on the population level, more memory precursors were present within the day 1 vs day 3 pool, due to accumulation of activated T cells over time. This type of differentiation "switch" leading to apparent gradations of differentiation has been shown to occur in other eukaryotic systems (40). The critical switches for memory generation remain to be determined, and are a challenge for future studies.

Our finding that undifferentiated precursors (day 1 activated) predominantly gave rise to TEM cells was surprising given the lymphoid homing and less differentiated CD62LhighCD45RBhigh phenotype attributed to TCM cells (16, 21). In infection models for generation of memory CD8 T cells, limiting the extent of pathogen exposure by antibiotic administration led to increased development of CD62Lhigh TCM memory CD8 T cells in spleen (26, 41), although it was not possible in those studies to identify the direct precursors to TCM cells. However, Ahmed and colleagues (19) have demonstrated a direct conversion of CD62Llow TEM to TCM in vivo in adoptive transfers, suggesting that TCM cells may represent a more differentiated memory T cell. Moreover, it was recently shown that a low precursor frequency favored development of the TEM rather than the TCM subset (26), and day 1-activated precursors likewise gave a lower frequency of memory T cells and a higher proportion of TEM cells. It is possible that multiple aspects of the activated precursor, including quantitative influences, differentiation state, and proliferative potential may dictate the resultant memory subset generated; in our case, proliferation of these precursors was not limited (Fig. 5D).

Both memory CD8 and CD4 T cells require IL-7 for optimal survival (14, 42, 43). Although it was shown that the IL-7R expression during the peak effector stage marks the cellular precursors to virus-specific memory CD8 T cells (8), this correlation has not been upheld for peptide-primed CD8 T cells (10), and its role in memory CD4 T cell generation is not defined. Our results showing that Ag withdrawal triggers up-regulation of IL-7R on the majority of activated T cells within 1 day in vivo suggest that acquisition of qualities that promote survival of memory T cells is controlled by extrinsic factors, rather than being an inherent property of the activated T cell. We propose that Ag withdrawal is a primary trigger for memory development during activation, although other host factors (e.g., cytokines) may likewise influence memory conversion and subset delineation. We previously found that CD62L expression on effector cells was not maintained in the resultant memory T cell population (21), and we found here that CD62L expression was not altered on day 1–3 precursors up to 1 wk in vivo (data not shown), contrasting the rapid phenotypic conversion of CD25 and IL-7R. These results suggest that stable delineation into CD62L subsets occurs after the initial priming stage, after an extended period in the host in vivo.

In conclusion, identifying pathways for memory T cell differentiation is critical for understanding long-term immunity, and has broad implications for the manipulation of memory heterogeneity in vaccines and autoimmune disease. We reveal here that memory CD4 T cells can derive from multiple cellular precursors upon Ag withdrawal, suggesting a novel divergent model of memory T cell development.


   Acknowledgments
 
We thank Wendy Lai and Elizabeth Kadavil for mouse colony maintenance and Drs. Matthias von Herrath and Mark Shlomchik for helpful discussions.


   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 National Institutes of Health Grants AI50632 and AI42092 (to D.L.F.). Back

2 Address correspondence and reprint requests to Dr. Donna L. Farber, Department of Surgery, University of Maryland School of Medicine, Medical School Teaching Facility, Room 400, 685 West Baltimore Street, Baltimore, MD 21201. E-mail address: dfarber{at}smail.umaryland.edu Back

3 Abbreviations used in this paper: TCM, central memory T cell; TEM, effector memory T cell; HA, hemagglutinin; ICS, intracellular cytokine staining; MFI, mean fluorescence intensity. Back

Received for publication January 5, 2006. Accepted for publication May 4, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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