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Chemokine-Guided CD4⁺ T Cell Help Enhances Generation of IL-6R^highIL-7R^high Prememory CD8⁺ T Cells¹

Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD4⁺ T cells promote effective CD8⁺ T cell-mediated immunity, but the timing and mechanistic details of such help remain controversial. Furthermore, the extent to which innate stimuli act independently of help in enhancing CD8⁺ T cell responses is also unresolved. Using a noninfectious vaccine model in immunocompetent mice, we show that even in the presence of innate stimuli, CD4⁺ T cell help early after priming is required for generating an optimal pool of functional memory CD8⁺ T cells. CD4⁺ T cell help increased the size of a previously unreported population of IL-6R^highIL-7R^high prememory CD8⁺ T cells shortly after priming that showed a survival advantage in vivo and contributed to the majority of functional memory CD8⁺ T cells after the contraction phase. In accord with our recent demonstration of chemokine-guided recruitment of naive CD8⁺ T cells to sites of CD4⁺ T cell-dendritic cell interactions, the generation of IL-6R^highIL-7R^high prememory as well as functional memory CD8⁺ T cells depended on the early postvaccination action of the inflammatory chemokines CCL3 and CCL4. Together, these findings support a model of CD8⁺ T cell memory cell differentiation involving the delivery of key signals early in the priming process based on chemokine-guided attraction of naive CD8⁺ T cells to sites of Ag-driven interactions between TLR-activated dendritic cells and CD4⁺ T cells. They also reveal that elevated IL-6R expression by a subset of CD8⁺ T cells represents an early imprint of CD4⁺ T cell helper function that actively contributes to the survival of activated CD8⁺ T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The precursor frequencies of naive T and B lymphocytes specific for any given Ag are extremely low and rapid clonal expansion plays a crucial role in generating the large pool of effector T cells and Ab-forming B cells needed to combat an acute infection. This initial expansion gives rise to a smaller number of residual memory cells that can be activated more rapidly by lower Ag levels and with less dependence on costimulatory molecules than their naive precursors (1, 2, 3, 4). The ability of memory T cells to limit the pathologic consequences of reinfection has made their induction one of the major goals of current vaccine efforts, with particular attention being paid to the CD8⁺ subset (5, 6).

Early studies on the cellular requirements for the generation of cytotoxic CD8⁺ T cell responses, most often involving immunization with sterile cell preparations across minor H barriers (7, 8, 9, 10), suggested a critical role for CD4⁺ T cells, T cell help (TH)³. When subsequent studies using viral challenge showed strong and effective acute CD8⁺ T cell responses in the absence of such CD4⁺ T cell cooperation (11), it was suggested that CD8⁺ T cell responses to noninflammatory Ags were TH dependent, whereas those to infections with live viruses and bacteria were not. This dichotomy was proposed to be due to a requirement for the activation of APCs that could be achieved by infectious agents in a CD4⁺ T cell-independent manner via innate signaling pathways (12) or, in the absence of such signals, by CD4⁺ T cells via CD40-CD40L interaction (13, 14, 15). However, maximal expression of CD40 on dendritic cells (DCs) requires inflammatory signals through receptors like TLRs (16), and dendritic cell activation for high-level production of cytokines such as IL-12 depends on the extent of CD40 stimulation by CD40L (17). Such findings make it unlikely that CD40-dependent enhancement of APC (mainly DC) function is optimal under conditions in which innate stimuli are not available.

Most recently, a new paradigm has emerged proposing that whether or not inflammatory signals are present, CD4⁺ T cells are dispensable for initial CD8⁺ T cell clonal expansion and effector generation but critical for chronic and/or memory CD8⁺ T cell responses (18, 19, 20, 21, 22, 23, 24). Although this concept has gained broad acceptance, two very distinct mechanisms have been proposed for the contribution of CD4⁺ T cell help to postacute CD8⁺ T cell responses. The "programming" model suggests that Ag-activated CD4⁺ T cells function at the time of priming to permit a subpopulation of CD8⁺ T cells to develop into competent memory cells. This hypothesis is based on evidence that the depletion of CD4⁺ T cells 3 days after Ag exposure does not impair secondary CD8⁺ T cell responses, whereas depletion before immunization does so (21, 22, 23). It is also consistent with very recent data showing a role for early paracrine IL-2 exposure in the development of CD8⁺ T cell memory (25), although there is no evidence that CD4⁺ T cells provide the IL-2. The alternative view suggests that Ag-specific CD4⁺ T cell function is not required early in the response but that polyclonal CD4⁺ T cells sustain the viability of memory CD8⁺ T cells in the maintenance phase through late, Ag-unspecific effects (26).

In this study we describe experiments in a vaccination model involving immunocompetent hosts aimed at assessing quantitatively in a single system the contributions of innate stimuli and CD4⁺ TH to early and late phases of CD8⁺ T cell responses, as well as probing how and when TH is delivered. We find that while innate signals are sufficient to generate a large number of acute effector CD8⁺ T cells, early Ag-specific CD4⁺ TH is required to produce a substantial pool of functional memory CD8⁺ T cells, even in the presence of a normal repertoire of bystander polyclonal CD4⁺ T cells. We document that activated CD8⁺ T cells marked by dual IL-6R (CD126)IL-7R (CD127) expression represent prememory cells whose survival in the contraction phase of the response shows IL-6 dependence and whose frequency increases when high levels of TH are provided at the time of initial priming. In accord with our recent intravital imaging data showing that the interaction of naive CD8⁺ T cells with Ag-activated CD4⁺ T cell-DC clusters is directed by the chemokines CCL3 and CCL4 (27), we find that the activity of these chemokines is required for the efficient development of both IL-6R^highIL-7R^high prememory CD8⁺ T cells and a large pool of functional memory CD8⁺ T cells. Taken together, these data provide new insights into how inflammation, chemokines, and cellular associations act in concert to optimize the development of useful acute effector and memory CD8⁺ T cell responses.

	Materials and Methods

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Mice

OT-I RAG^–/– mice that possess CD8⁺ T cells expressing transgenes encoding a TCR specific for OVA peptide 257–264 (SIINFEKL) presented by the MHC class I molecule K^b (28), OT-II mice that possess CD4⁺ T cells expressing transgenes encoding a TCR specific for OVA peptide 323–339 presented by the MHC class II molecule I-A^b (29), and CD45.1 congenic mice, all on the C57BL/6 background, were obtained from Taconic Farms. C57BL/6 wild-type and IL-6^–/– mice were purchased from The Jackson Laboratory. All animals were housed and treated in compliance with American Association of Laboratory Animal Care and National Institutes of Health requirements under an approved animal study protocol.

Reagents

CpGs (GCTAGACGTTAGGT and TCAACGTTGA) (30) were admixed at equimolar concentrations and used at 20 µg of total CpG per mouse. CpGs were synthesized at the Center for Biological Evaluation and Research Core Facility; all had <0.1 endotoxin units per milligram. All Abs were purchased from BD Pharmingen with the exception of the anti-human granzyme B (Caltag), anti-mouse IL-15R (Santa Cruz Biotechnology), anti-KLRG1 (Southern Biotech), and neutralizing anti-IL-6, CCL3, and CCL4 or isotype-matched controls (R&D Systems). K^b-SIINFEKL tetramers were obtained from the National Institute of Allergy and Infectious Disease tetramer facility.

Cell preparation, adoptive transfer, and immunization

CD8⁺ OT-1 cells and CD4⁺ OT-II T cells were enriched from single cell suspensions of lymph node (LN) and spleen by negative selection using magnetic beads (Dynal) with Abs to B220 and CD4 or CD8, according to the manufacturer’s directions. After isolation, T cells were labeled with 2.5 µM CFSE (Molecular Probes) and transferred i.v. into congenic recipients (3 x 10⁶ or 3 x 10³ purified T cells of the indicated type per recipient). One day later the mice were immunized s.c. with alum (Pierce) admixed with an endotoxin-free SIINFEKL peptide (1 µg/mouse) in the presence or absence of an endotoxin-free OVA 323–339 peptide (10 µg/mouse) and/or CpGs (20 µg/mouse).

Cell harvest for ex vivo phenotypic analysis

The mice were sacrificed at different time points after immunization, perfused with PBS-heparin, and LNs, spleens, livers, and lungs were collected. The different organs were treated with collagenase and DNase (Calbiochem) before cell isolation and staining for flow cytometry. OT-I cells were identified by staining with specific K^b-SIINFEKL tetramers labeled with allophycocyanin or Abs to CD45.2 and CD8. Gates were set on the matched isotype controls.

Cytokine measurement

For the analysis of IFN-, IL-2, and granzyme B responses, the isolated cells were restimulated with SIINFEKL peptide (10 ng/ml). Supernatants of stimulated cells were analyzed for cytokine content by flow cytometry using the mouse TH1/TH2 cytometric bead array kit (BD Pharmingen). For intracellular staining, cells were restimulated for 6 h in the presence of brefeldin A, stained for surface markers, permeabilized using the Cytofix/Cytoperm Plus kit according to the manufacturer’s directions (BD Pharmingen), and then stained for cytokine content and analyzed by flow cytometry.

In vivo challenge

Thirty days after priming, mice were challenged i.v. with a mixture of equal numbers (10 x 10⁶) of pulsed and unpulsed B cells. B cells were purified from C57BL/6 mice by negative selection and either pulsed with SIINFEKL (10 ng/ml) before labeling with a high concentration of CFSE (2.5 µM CFSE) or left unpulsed before labeling with a low concentration of CFSE (0.12 µM CFSE). For the analysis of in vivo cytotoxicity, the mice were sacrificed 48 h after challenge and the ratio between the two populations of transferred B cells was analyzed in the spleen by flow cytometry. For the analysis of the frequency of OT-I T cells producing IFN-, splenocytes were analyzed by intracellular staining 48 h after challenge without additional in vitro restimulation.

Sorting

Seven days after immunization, single cell suspensions from spleen were prepared and the cells stained for CD45.2 and CD8 or for CD45.2, CD8, IL-6R, and IL-7. OT-I IL-7R^high cells were sorted into OT-I IL-6R^low (the dimmest 10% of IL-6R-expressing cells) or OT-I IL-6R^high (the brightest 10% of IL-6R-expressing cells) subsets and transferred into naive recipients at the indicated numbers.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Contribution of TH and innate signals to early and late postvaccination CD8⁺ T cell numbers and effector capacity

Our aim was to explore how and when innate signals and TH influence the early and late phases of an in vivo CD8⁺ T cell response. A vaccine approach consisting of nonreplicating bland Ag with or without added adjuvant was chosen to separate the effects of Ag-dependent TH from those of innate stimuli such as TLR ligands, which is difficult to do in models using an infectious challenge. To this end, we adoptively transferred both OT-I (CD8⁺) (28) and OT-II (CD4⁺) (29) TCR transgenic T cells of known Ag specificity into congenic immunocompetent recipients. These mice were then immunized s.c with alum admixed with specific peptide Ags. Alum was chosen because it is the principal approved vehicle for human vaccination. All mice were vaccinated with alum plus the MHC class I-binding peptide OVA 257–264 (SIINFEKL). When TH was also desired at priming, the MHC class II-binding peptide OVA 323–339 was added into the immunization mixture. This approach permitted the assessment of Ag-specific TH function without otherwise disrupting the state of the immune system and in the presence of a polyclonal CD4⁺ T cell repertoire. As a stimulus for innate activation, we used unmethylated CpG-containing oligonucleotides, which are being examined for their use as adjuvants both in animal models and in humans (30).

For all immunization conditions tested, analyses of CFSE dilution revealed that the majority of OT-I T cells found in the draining LNs had divided similarly (6–8 times) during the first 72 h following vaccination and that the expansion of OT-I T cells was maximal between days 6 and 8 (data not shown). Given these findings and for ease of comparison to published reports, day 7 results are reported as the peak of the response. In agreement with a previous report (31), CpGs induced a 4- to 6-fold increase in the acute expansion of OT-I CD8⁺ T cells and their accumulation in both lymphoid organs and peripheral tissues, as well as a 2-fold increase in the frequency of cells showing IFN- or granzyme B synthesis upon restimulation (Fig. 1a and Table I). This increased proportion of functional effectors, combined with the more robust expansion of the Ag-specific CD8⁺ T cell population, resulted in the generation of a 10-fold greater total number of effector cells at the peak of the response, when CpGs were administered. Conversely, TH had a limited effect on the acute expansion of CD8⁺ T cells and their accumulation in peripheral tissues as well as the acquisition of effector functions; such a TH effect was more pronounced when provided in association with innate signals (Fig. 1a and Table I).

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Influence of innate stimuli and TH on the expansion and long-term survival of Ag-stimulated CD8+ T cells. Naive OT-I (3 x 106) and OT-II (3 x 106) T cells were adoptively transferred i.v. into naive CD45 congenic recipients. One day later the mice were immunized s.c. with the indicated vaccine constituents. At different time points after vaccination the mice were sacrificed and perfused and the accumulation of OT-I CD8+ T cells was analyzed in pooled cells recovered from LNs, spleens, livers, and lungs based on staining with Abs to CD45.2 and CD8. a, Mice were immunized with alum plus SIINFEKL (A+S) (gray box), alum plus SIINFEKL plus OVA peptide 323–339 (A+S+TH) (gray circle), alum plus SIINFEKL plus CpGs (A+S+CpG) (black box), or alum plus SIINFEKL plus CpGs plus OVA peptide 323–339 (A+S+CpG+TH) (black circle). b, Mice were immunized as in a and the total recovered OT-I T cells was determined 60 days after immunization. In a one representative experiment of six similar experiments is shown. In b the averages and the SE values were computed from six independent experiments.

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Effector function of CD8+ T cells primed in the presence of different combinations of innate stimuli and TH. Naive OT-I (3 x 106) and OT-II (3 x 106) T cells were adoptively transferred i.v. into naive CD45 congenic recipients. One day later the mice were immunized s.c. with the indicated vaccine constituents. Fifty days after priming, cells recovered from the indicated organs were restimulated for 6 h with SIINFEKL (10 ng/ml) and the responses of the OT-I population were analyzed by intracellular staining for IFN-. Equal numbers of recovered OT-I were analyzed. The number represents the cytokine positive OT-I T cells as a percentage of the total OT-I T cells recovered from the organ; the number in parentheses represents the mean fluorescent intensity of the cytokine positive population. Gates are set on the staining obtained with isotype controls. One of five experiments with comparable results is shown. A + S + CpG, alum plus SIINFEKL plus CpGs; A + S + CpG + TH, alum plus SIINFEKL plus CpGs plus OVA peptide 323–339.

Recent reports have suggested that at the peak of the response to the lymphocytic choriomeningitis virus it is possible to identify prememory CD8⁺ T cells by either of two unique cell surface phenotypes, namely higher expression of IL-7R and/or CD8 (33, 34, 35). In one report, the IL-7R^high CD8⁺ T cells represented 5–12% of lymphocytic choriomeningitis virus-specific CD8⁺ T cells 8 days after infection but comprised the bulk of surviving, functional memory T cells several weeks later (33). However, in other studies IL-7R^high CD8⁺ T cells represented 30 or 70% of Ag-specific CD8⁺ T cells at the peak of the response (35, 36), corresponding to absolute numbers of Ag-reactive T cells that greatly exceeded the final memory CD8⁺ T cell pool size. Furthermore, IL-7R expression did not correlate with the availability of TH at priming (26).

We therefore examined whether either of the reported markers identified the prememory CD8⁺ T cells generated under the present vaccination conditions, which do not involve a replicating Ag source, and whether these phenotypes were influenced by the availability of TH. For these experiments, all of the mice were immunized with CpGs to eliminate innate signals as a variable. To test for potential effects of different precursor frequency among the cells in the CD8⁺ T cell responder pool (37), we compared the behavior of transgenic T cells in mice adoptively transferred with high (3 x 10⁶) and low (3 x 10³) numbers of naive OT-I cells. In addition to IL-7R, we also analyzed the surface expression of additional cytokine receptors (IL-6R and IL-15R) that have antiapoptotic functions in hemopoietic cells and that have been reported to contribute to CD8⁺ T cell responses (38).

We first monitored the kinetics of surface expression of the above cytokine receptors following Ag stimulation. In agreement with previous reports, at both frequencies of transferred T cells, IL-6R and IL-7R were down modulated shortly after T cell activation in vivo (39, 40) (Fig. 3, a and b). At the peak of the acute response, an average of 70% of OT-I T cells re-expressed IL-7R at levels similar to that on naive CD8⁺ T cells (Fig. 3c), in agreement with a recent report (36). This corresponds to a substantially greater number of cells than the number of memory CD8⁺ T cells present after the contraction phase of the response (Fig. 1 and Ref. 36), suggesting that high surface expression of IL-7R alone does not reliably identify prememory CD8⁺ T cells. Likewise, IL-15R expression was maintained on all activated CD8⁺ T cells (Fig. 3b and data not shown), indicating that this cytokine receptor cannot be used for identifying committed prememory cells. Animals primed in the presence or absence of TH had a similar frequency of cells with CD8 expression, even though the number of memory cells generated in the presence of TH was 3- to 5-fold higher (data not shown). Other potentially relevant cell surface molecules including CD27, the common cytokine receptor -chain (_c), the IL-2R-chain (CD122), killer cell lectin-like receptor G1 (KLRG1), Bcl-2, and CD62L showed only small differences at day 7 between OT-I derived from mice primed in the presence of innate signals and TH or without TH (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Phenotype of Ag-activated CD8+ T cells during the acute and late phases of the response. Analysis of the surface expression of IL-6R and IL-7R (a, c, and d) and IL-15R (b) on OT-I T cells is depicted. Mice received 3 x 106 OT-I and 3 x 106 OT-II (upper panels) or 3 x 103 OT-I and 3 x 106 OT-II (lower panel) cells and were then immunized with either PBS (No vaccination), alum plus SIINFEKL plus CpGs (A + S + CpG), or alum plus SIINFEKL plus CpGs plus OVA peptide 323–339 (A + S + CpG + TH). a and b, The phenotype of OT-I from the draining LNs of mice immunized three days earlier. c and d, The phenotype of OT-I from the spleens of mice immunized 7 days (c) and 30 days earlier (d). The number in certain quadrants represents the frequency of OT-I T cells with the indicated phenotype expressed as a percentage of the total OT-I T cells recovered from the organ. One of 10 independent experiments for 3 x 106 OT-I cells and one of three independent experiments with 3 x 103 OT-I cells with comparable results are shown.

By days 20–40, at both frequencies of transferred T cells the majority of OT-I T cells expressed high levels of both IL-6R and IL-7R (Fig. 3d), with the difference between priming in the presence and absence of TH being the absolute number of surviving T cells. Strikingly, the total number of OT-I T cells with the IL-6R^highIL-7R^high phenotype remained relatively constant between day 6 and day 40 after priming, unlike the IL-6R^lowIL-7R^high population that rapidly declined during the contraction phase. This was seen in each condition of vaccination and with both high (Fig. 4, a and b) and low numbers of transferred T cells (Fig. 4c). This strongly suggested that the subpopulation of activated CD8⁺ T cells expressing high surface levels of both IL-6R and IL-7R had a survival and/or proliferation advantage in vivo and preferentially contributed to the memory pool.

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Selective persistence of IL-6RhighIL-7Rhigh Ag-activated CD8+ T cells. IL-6R and IL-7R expression was analyzed on OT-I T cells from mice that received an adoptive transfer of naive OT-I (3 x 106) and OT-II (3 x 106) T cells (a and b) or naive OT-I (3 x 103) and OT-II (3 x 106) T cells (c). One day later mice were immunized with alum plus SIINFEKL plus CpGs (a) or alum plus SIINFEKL plus CpGs plus OVA peptide 323–339 (b and c). Cells were analyzed on day 7 (open bars), day 21 (gray bars), and day 40 (filled bars) after priming. The total number (x104) of OT-I expressing the indicated cytokine receptors in cells recovered from LNs, spleens, livers, and lungs is shown. Data are from one of three experiments with similar results. OT-I T cells were identified based on staining with Abs to CD45.2 and CD8.

To examine directly whether the subpopulation of activated OT-I T cells expressing high levels of IL-6R had a survival advantage in vivo after the peak of the response, we immunized mice in the presence of both innate signals and TH, sorted OT-I T cells that had undergone more than seven rounds of divisions into IL-6R^highIL-7R^high and IL-6R^lowIL-7R^high cells, and transferred each population into distinct naive congenic recipients. Two weeks later the mice were sacrificed and the number of OT-I cells present in a pool of diverse tissues was determined. Two to three times as many OT-I T cells were recovered from the animals that received IL-6R^highIL-7R^high compared with the IL-6R^lowIL-7R^high cells (Fig. 5a). At this time point all of the surviving cells, irrespective of their phenotype at the time of transfer, expressed similar high levels of IL-6R (Fig. 5a, inset) in accordance with the memory phenotype described above (Fig. 3d), Upon in vivo challenge, the animals that had received the IL-6R^highIL-7R^high OT-I T cells showed greater Ag-specific T cell expansion (Fig. 5b), a higher ability to kill Ag-pulsed targets in vivo (Fig. 5c), and an increased frequency and absolute number of IFN- secreting cells (Fig. 5d) as compared with recipients of IL-6R^lowIL-7R^high cells. Due to the increased survival of transferred OT-I cells in the recipients of IL-6R^highIL-7R^high cells, however, the memory pool derived from both sorted populations was, on a per cell basis, functionally equivalent.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Preferential survival and memory effector function of IL-6RhighIL-7Rhigh primed CD8+ T cells. Congenic mice received an adoptive transfer of naive OT-I (3 x 106) and OT-II (3 x 106) T cells and were immunized with alum plus SIINFEKL plus CpGs plus OVA peptide 323–339. Seven days later, spleen cells were sorted into OT-I IL-6RlowIL-7Rhigh (gray bar in a) and OT-I IL-6RhighIL-7Rhigh (filled bar in a) subsets. Equal numbers of sorted cells (2.5 x 105) were transferred i.v. into naive congenic recipients. a, Two weeks later, the recipients were sacrificed and the recovery of OT-I T cells was analyzed in LNs, spleens, livers, and lungs; the inset shows the surface expression of IL-6R on OT-I recovered from the spleens of mice transferred 2 wk earlier with IL-6RlowIL-7Rhigh (shaded histogram) and OT-I IL-6RhighIL-7Rhigh (black line) subsets. b–d, Two weeks after the transfer of sorted OT-I IL-6RlowIL-7Rhigh (left) and OT-I IL-6RhighIL-7Rhigh (right), the recipients were challenged with equal numbers of differentially CFSE-labeled peptide-pulsed and unpulsed B cells i.v. b, Two days after challenge the recipients were sacrificed and the percentage of OT-I cells was analyzed; the number represents the frequency of OT-I T cells as a percentage of total spleen cells. c, Two days after in vivo challenge the number of residual CFSE-labeled targets of each type was analyzed in the spleen. The value in the plot represents the fraction of peptide-pulsed cells (high CFSE) recovered relative to the number of peptide-unpulsed cells (low CFSE) recovered. d, Two days after rechallenge, IFN- production by OT-I cells in the spleen was analyzed without additional in vitro restimulation. In a, the data are expressed as the average of recovered OT-I T cells in four independent experiments. In b–d, one experiment of three with similar results is shown in each case.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. IL-6 contributes to the in vivo survival of activated CD8+ T cells. a, Total OT-I T cells were sorted from the spleens of mice that had received an adoptive transfer of naive OT-I and OT-II T cells and that had been immunized 7 days earlier with alum plus SIINFEKL plus CpGs plus OVA peptide 323–339. Equal numbers of sorted cells (1 x 106) were transferred i.v. into naive C57BL/6 (wild type; gray bar) or IL-6 KO recipients (filled bar). Two weeks later the recipients were sacrificed and the number of OT-I T cells recovered from the spleen was determined. OT-I T cells were identified using specific Kb-SIINFEKL tetramers and staining for CD44. b, Congenic mice received an adoptive transfer of naive OT-I and OT-II T cells and were immunized with alum plus SIINFEKL plus CpGs plus OVA peptide 323–339. Eight days later one group of animals was injected i.v. with an IL-6-neutralizing Ab (filled bar) and the other with an isotype-matched control (gray bar). Survival of the OT-I T cells was analyzed 3 wk later. The averages and SE values are calculated from the data of three independent experiments.

The data reported above agree with other evidence indicating that TH acts early after immunization to enhance the pool of prememory CD8⁺ T cells ("programming") (21, 22, 23). To further study the relationship between IL-6R^highIL-7R^high prememory CD8⁺ T cell generation and the delivery of TH as well as probe the nature of the cell interactions involved in the generation of this cohort of prememory T cells, we turned to our recent observation that naive CD8⁺ T cells are actively recruited by the inflammatory chemokines CCL3 and CCL4 to Ag-dependent clusters of DC and CD4⁺ T cells and that the action of these chemokines contributes to optimal delivery of TH (27). To this end, mice were immunized with vaccines containing CpGs in the presence or absence of TH and in the presence or absence of neutralizing Abs to CCL3, CCL4, or isotype-matched controls. Interfering with CCL3/CCL4 function at the time of priming had a small effect on the acute expansion of OT-I T cells (Fig. 7a) but abolished the enhancing effect of TH on the generation of IL-6R^highIL-7R^high prememory OT-I T cells (Fig. 7b) without any detectable effects on the expansion of OT-II T cells and their functionality at this time point (Fig. 7c and data not shown). In accord with our previous report (27) and in agreement with the reduction in the pool of IL-6R^highIL-7R^high cells, neutralization of CCL3 and CCL4 at the time of priming eliminated the enhancing effect of TH on both the number (Figs. 7d and 8a) and the functionality (IFN- production) of memory cells present several weeks later (Fig. 8b). In addition, if priming occurred in the presence of both help and blocking Abs to CCL3 and CCL4, the surviving memory cells showed a reduced in vivo ability to mount a CTL response (Fig. 8c) and to expand upon boosting (Fig. 8d), the latter being a characteristic feature of CD8⁺ T cells primed in the absence of TH (21, 41). Similar results were obtained following the transfer of lower numbers of naive OT-I T cells (data not shown). These results further link the generation of IL-6R^highIL-7R^high prememory CD8⁺ T cells to optimal delivery of TH, which in turn depends on the local action of inflammatory chemokines that optimize cell-cell interactions in LNs during the early priming period (27).

View larger version (24K): [in this window] [in a new window]   FIGURE 7. Role of CCL3 and CCL4 in TH-dependent prememory CD8+ T cell generation. Congenic mice received an adoptive transfer of naive OT-I (3 x 106) and OT-II (3 x 106) T cells and were immunized with alum plus SIINFEKL plus CpGs or alum plus SIINFEKL plus CpGs plus OVA peptide 323–339 in the absence or presence of neutralizing Abs (50 µg of each) to CCL3 and CCL4 or isotype-matched control Abs. a, The total number of OT-I T cells recovered from LNs, spleens, livers, and lung on day 7 after priming. b, The absolute numbers of OT-I T cells with an IL-6RhighIL-7Rhigh phenotype on day 7 after priming. c, The absolute numbers of OT-II T cells recovered in the draining LN on day 3 after priming. d, The total number of OT-I T cells recovered from LNs, spleens, livers, and lungs on day 30 after priming. The averages and SE values are calculated from the data of three independent experiments.

View larger version (51K): [in this window] [in a new window]   FIGURE 8. Role of CCL3 and CCL4 in memory CD8+ T cell differentiation. Congenic mice received an adoptive transfer of naive OT-I (3 x 106) and OT-II (3 x 106) T cells and were immunized with alum plus SIINFEKL plus CpGs (A + S + CpG) or alum plus SIINFEKL plus CpGs plus OVA peptide 323–339 (A + S + CpG + TH) in the absence or presence of neutralizing Abs to CCL3 and CCL4 or isotype-matched control Abs (50 µg of each). a, OT-I T cells recovered from spleen on day 30 after priming. b, Intracellular staining for IFN- 30 days after priming. OT-I T cells were restimulated in vitro for 6 h before intracellular staining. The number represents the cytokine positive OT-I T cells as a percentage of the total OT-I T cells recovered from the spleen. Gates are set on the staining obtained with isotype controls. c and d, Thirty days after priming mice, were challenged with differentially CFSE-labeled peptide-pulsed and unpulsed B cells i.v. c, Two days after challenge the number of residual CFSE-labeled cells of each type was analyzed in the spleen. The value in the plot represents fraction of peptide-pulsed cells (high CFSE) recovered relative to the number of peptide-unpulsed cells (low CFSE) recovered. d, Two days after challenge the expansion of OT-I was analyzed in the spleen. The number represents the recovered number of OT-I expressed as a percentage of total spleen cells.

	Discussion

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In this study, CD4⁺ TH made a minor contribution to the CD8⁺ T cell primary response whereas innate stimuli (represented by CpGs) were essential for robust initial expansion of the CD8⁺ T cell population, development of effector capacity measured as IFN- or granzyme B production, and accumulation of effector cells in peripheral tissues that would typically be the site of active infection These data agree with the previously reported lack of a requirement for CD4⁺ T cells in the CD8⁺ T cell-mediated clearance of acute infections (11, 18, 19) and with studies showing that CpGs are more effective than CD4⁺ T cell help in promoting CD8⁺ T cell-mediated destruction of pre-existing human papillomavirus-induced tumors (31). Yet, despite the high burst size and early development of effector responses, innate stimuli were not sufficient to generate a maximal pool of functional memory CD8⁺ T cells. Both innate stimuli and TH were required at priming for the generation of an optimal functional population of memory CD8⁺ T cells. Under these conditions, the memory pool consisted of 3- to 5-fold more CD8⁺ T cells than that seen with innate stimuli alone, and these cells showed a greater frequency of cells capable of high IFN- production and of proliferation upon Ag re-exposure. The net result was that TH contributed to a 10- to 20-fold enhancement in the number of memory CD8⁺ T cells capable of rapid effector function upon Ag exposure, a result with obvious implications for the effectiveness of this population in defense against reinfection.

The availability of TH at priming also resulted in a substantial increase at the peak of the response in the number of CD8⁺ T cells marked by the re-expression of both IL-6R and IL-7R. Unlike the IL-6R^lowIL-7R^high cells, whose number rapidly declined during the contraction phase of the response, the number of IL-6R^highIL-7R^high CD8⁺ T cells remained relatively constant between day 7 and day 40. Together with transfer studies showing that these CD8⁺ T cells preferentially survived in vivo, these data strongly suggest that this population makes the major contribution to the functional memory pool. When distinct populations of activated CD8⁺ T cells were sorted and transferred into naive recipients, all of the surviving cells found weeks later were IL-6R^highIL-7R^high regardless of the initial phenotype of the transferred cells. This latter finding implies that our posttransfer comparisons probably underestimate the differential survival capacity of IL-6R^highIL-7R^high vs IL-6R^lowIL-7R^high CD8⁺ T cells. This is because at the time of isolation the IL-6R^low/IL-7R^high population includes a significant subpopulation of cells still in the process of re-expressing IL-6R (F. Castellino, unpublished observations). Both transfer and neutralization studies showed that IL-6 has a direct role in the survival of IL-6R^high CD8⁺ T cells during the contraction phase of the immune response, even in the presence of cytokines such as IL-7 and IL-15 that have previously been shown to be necessary for CD8⁺ memory T cell maintenance (40). In addition to this enhanced survival, CFSE labeling studies indicate that the isolated prememory cells continue to divide after the peak of the response (F. Castellino, unpublished observations), adding to their representation in the final memory pool.

The specific mechanism by which IL-6 synergizes with other signals in promoting memory cell formation is not known; in particular, it is unknown whether the accumulation of IL-6R^highIL-7R^highIL-15R^high CD8⁺ T cells is the consequence solely of a survival advantage of this subpopulation of T cells, of a greater ability of these cells to proliferate in vivo, or both. IL-6 has been reported to promote the survival of naive T and B cells via activation of STAT-1, STAT-3, and PI3K-AKT (41, 42, 43, 44, 45). IL-6 can also interfere with IFN- signaling by activating suppressor of cytokine signaling 1 (SOCS-1), which might help limit T cell death during the inflammatory phase of the immune response (4, 46). IL-6 has been also reported to be required by CD8⁺ T cells for proliferation and synthesis of IL-2 in response to weak TCR stimulation (47, 48), although this does not provide an explanation for the better survival of the IL-6R^high cells during the contraction phase of the response. Recently, IL-6 was suggested to prevent activated T cells from being suppressed by CD25⁺ regulatory T cells (49) and to inhibit the development of CD4⁺ regulatory T cells in the presence of TGF (50, 51), but how this might contribute to augmented survival of memory CD8⁺ cells is unclear.

The finding that in immunocompetent animals the pool of early-arising, prememory IL-6R^highIL-7R^high CD8⁺ T cells is enhanced by CD4⁺ T cell activation provides additional support for a recently proposed model of CD4-CD8 cooperation, postulating that CD8⁺ T cells are "programmed" for memory cell development early during priming by the action of Ag-stimulated CD4⁺ T cells (21, 22, 23). In addition, we found that the augmenting effect of TH on both the generation of IL-6R^highIL-7R^high prememory cells and the long-term CD8⁺ functional memory pool was eliminated when priming occurred in the presence of neutralizing Abs to CCL3 and CCL4. This is likely to be a reflection of the role of these chemokines in directing naive CD8⁺ T cells to optimally activated DC- and/or Ag-dependent CD4-DC clusters as seen in our imaging studies (27) and perhaps also of the reported T cell costimulatory capacity of these mediators (52). It will be of interest to determine whether the prememory phenotype we describe is seen under a variety of immunization conditions and can provide an early way of screening for optimal vaccine formulations, as well as whether the CCL3/CCL4-CCR5 pathway participates in promoting CD8⁺ T cell memory in these other circumstances.

Finally, what do these data tell us about the mechanism that underlies CD4⁺ augmentation of CD8⁺ memory cell development? We suggest that our present findings and many other results in the literature may best be explained by postulating that CD4⁺ T cell-DC interaction does not result in the production of a single unique factor necessary and sufficient to drive the differentiation of such cells. Rather, we propose that innate stimuli and CD4⁺ TH act in a coordinated fashion to increase the frequency of optimally signaled CD8⁺ T cells through chemokine guidance of naive CD8⁺ T cells to the locale of Ag-responsive CD4⁺ T cells and fully activated DCs. In this milieu, the CD8⁺ T cells can efficiently receive signals from both the TH and the DCs, whose production of many of the same mediators initially induced through innate receptor signaling has been amplified or sustained by the activated CD4⁺ T cells.

	Acknowledgments

 
We thank all the members of the Lymphocyte Biology Section for technical help and insightful discussions. Special thanks to Alex Huang, Hai Qi, Gregoire Altan-Bonnet, Jackson Egen, and Rosalind Polley for their helpful comments on the manuscript and to Alex Huang for his assistance with animal manipulations in a number of experiments.

	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 the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

² Address correspondence and reprint requests to Dr. Ronald N. Germain, Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11N-311, 10 Center Drive, Bethesda, MD 20892-1892. E-mail address: rgermain{at}nih.gov

³ Abbreviations used in this paper: TH, T cell help; DC, dendritic cell; KO, knockout; LN, lymph node.

Received for publication August 31, 2006. Accepted for publication November 6, 2006.

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