Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues

Abstract

Expression of the lymph node homing and CC-chemokine receptor 7 (CCR7), with L-selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T-cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62L^high CD44^low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7^high CD62L^high CD44^high (central memory) and CCR7^low CD62L^low CD44^high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short-term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7^high CD4 T cells produced predominantly interleukin (IL)-2, whereas CCR7^low CD4 T cells produced both IL-2 and interferon-γ (IFN-γ). However, in contrast to previously published reports, the CCR7^high CD8 T-cell subpopulation produced both IFN-γ and IL-2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down-regulated CCR7 only after multiple cell divisions, and this coincided with the down-regulation of CD62L and production of IL-4 and IFN-γ. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL-2- and IFN-γ-producing cells are CCR7^low, while few cytokine-expressing CCR7^high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T-cell subpopulations, but indicate additional complexity in that CCR7^high CD8 T cells also may produce IFN-γ.

Introduction

The engagement of T-cell receptors (TCRs) on CD4⁺ or CD8⁺ T cells by antigen peptide–major histocompatibility complex (MHC) complexes that are expressed on the surface of antigen-presenting cells (APCs), leads to proliferation and differentiation into effector T cells.¹ Effector T cells either produce cytokines to co-ordinate the adaptive immune response or acquire cytolytic capability to lyse virally infected cells, normally leading to elimination of the pathogen. The effector phase is followed by a period of death during which most of the effector T cells are believed to undergo apoptosis.^2–4 However, a small subset of antigen-experienced memory cells remain for long-term protection. The induction of these memory T cells provides the potential for a more efficient T-cell response upon re-exposure to the same antigen. The qualitative and quantitative differences between naïve and memory T-cell immune responses have been suggested to be the result of a combination of factors, such as a higher frequency of antigen (Ag)-specific T cells, reduced requirement for costimulation, and faster kinetics of cytokine secretion.^5–7

It is known that subpopulations of memory T cells, displaying distinct phenotypes, exist.⁸ Interestingly, during the past few years a model has been proposed in which human memory T cells can be subdivided into central memory (T_CM)² and effector memory (T_EM) cells on the basis of expression of L-selectin (CD62L) and CC-chemokine receptor 7 (CCR7).⁹ CCR7 and CD62L are known to be key players in the mechanism by which naïve T cells enter lymph nodes and Peyer's patches through high endothelial venules via interaction with peripheral-node addressin (PNAd) and CC-chemokine ligand 19 (CCL19) and/or CC-chemokine ligand 21 (CCL21), respectively.^10,11 Furthermore, analysis of CCR7-deficient mice has demonstrated that CCR7 is an important organizer of the primary immune response. Mice deficient in CCR7 show impaired antibody responses and a lack of delayed-type hypersensitivity reactions. They also exhibit morphological abnormalities in secondary lymphoid organs because of impaired immigration of lymphocytes and dendritic cells, and fail to mount adequate primary T- and B-cell responses.^11,12 Indeed, it has been shown that CCR7^high CD62L^high T_CM efficiently home to peripheral lymph nodes, whereas CCR7^low CD62L^low T_EM can be found in non-peripheral tissue.^13,14 When functional characterization of the two subpopulations was performed, further marked differences were revealed. Following in vitro stimulation of human CCR7^high CD62L^high CD4⁺ T_CM cells_, interleukin (IL)-2 production, but no, or only very low levels of, interferon-γ (IFN-γ), IL-4 or IL-5 were detected. In contrast, CCR7^low CD62L^low CD4⁺ T_EM cells produced substantial amounts of IFN-γ, IL-4 or IL-5, but less IL-2. Moreover, within the CD8⁺ T cells, only CCR7^low cells were identified to contain perforin-containing granules.⁹

Because of the apparent lack of an antibody directed against the murine CCR7 protein, most studies of the expression and function of CCR7 on T cells have been performed on human T cells, although the CCL19 [macrophage inflammatory protein (MIP)-3b/ELC]–immunoglobulin chimera has been used to detect CCR7 on murine T cells.¹⁴ The biological significance of CCR7, and the need for an antibody that can detect this molecule in order to characterize effector and memory T-cell subpopulations in murine in vivo models, led us to generate an antibody against murine CCR7. In the present study, we used this antibody to characterize the expression of CCR7 on murine naïve, effector and memory T-cell subpopulations in different lymphoid organs. Our data support the hypothesis that the expression of CCR7 can separate functionally different memory T-cell subsets in mice, but also suggest additional complexity in the system in that both CCR7^low and CCR7^high memory CD8⁺ T cells are able to produce IFN-γ. Moreover, we show that naïve T cells must undergo multiple cell divisions before they can down-regulate CCR7 (and CD62L), but that the number of cell divisions per se is not sufficient to induce this down-regulation. Finally, analysis of effector T cells during secondary immune responses in vivo indicates that the vast majority of both IL-2 and IFN-γ producers are CCR7^low.

Materials and methods

Mice

Female C57Bl/6 and BALB/c mice were purchased from Charles River (Margate, UK). D0.11.10 mice (BALB/c background) were originally obtained from Dr K. Murphy (Washington University School of Medicine, Howard Hughes Medical Institute, St Louis, MO) and a colony was maintained at GlaxoSmithKline (Bury Green Farm, UK). Recombination-activating gene-2 (RAG-2)^−/− mice were a gift from Dr Terence Barrett (North-western University Medical School, Chicago, IL) and were used to generate D0.11.10 TCR RAG-2^−/− mice by crossbreeding with the D0.11.10 mice. The D0.11.10 TCR RAG-2^−/− mice are characterized by expressing the transgene D0.11.10, which is specific for peptide(323–339) derived from hen egg ovalbumin (OVA), but are deficient in RAG-2, preventing the formation of T cells with endogenous TCRs.¹⁵ The mice were housed under conditions of constant temperature and humidity, with regular 12-hr cycles of light and darkness, sterile bedding, food and water, and were used at 6–10 weeks of age. All experiments were carried out under UK ethical guidelines.

Generation and purification of rabbit anti-mouse polyclonal CCR7 antibody

The antibody used in this study was raised against a polypeptide sequence representing the extreme N-terminus of the murine CCR7 protein (QDEVTDDYIGENTTVDYTLYESVC). This was chosen because the termini of proteins are often located at the surface and available to extrinsic probes, such as antibodies, and therefore represent a suitable choice of immunogen for the generation of such reagents. Furthermore, extensive basic local alignment search tool (blast) analysis revealed that the sequence had no close matches to any other known mouse proteins. The 24-mer peptide was synthesized using standard solid-phase techniques (Severn Biotech, Ltd, Kidderminster, UK) and coupled to purified protein derivative of tuberculin (PPD) using sulpho-succinimidyl-4-[N-maleidomethyl]-cyclohexane-1-carboxy-[6-amidocarproate] (Perbio Science, Stockholm, Sweden). NZW rabbits, presensitized against bacille Calmette–Guérin (BCG), were immunized with the resulting conjugate (c. 50–100 µg/rabbit per immunization) emulsified in incomplete Freund's adjuvant at >3-week intervals. All rabbits were terminally bled after five immunizations. The antibodies were affinity-purified using immobilized peptide columns (Sulpholink; Perbio). The affinity-isolated immunoglobulins were stored in phosphate-buffered saline (PBS) containing 0·2% crystalline bovine serum albumin (BSA).

Plasmids

The full-length murine CCR7 receptor cDNA (GenBank L31580) was cloned, using polymerase chain reaction (PCR) amplification, from mouse thymus mRNA using nested oligo nucleotide sense primers 5′-GCAGGATCAGATGAACTGCATG-3′ and 5′-GAGCACCATGGACCCAGGG-3′; and antisense primers 5′-CTGTGCATGTGTGCACCAC-3′ and 5′-CCTTGTAGTCCGGGCAGGG-3′. The PCR product was subcloned into the mammalian expression vector, pCDNA3.1 (Invitrogen, Paisley, UK), in the correct orientation, to generate the plasmid designated pCDNA3.1/CCR7.

pVAC1¹⁶ is a eukaryotic expression vector optimized for DNA vaccination, and is based on the plasmid pCI (Promega, Southampton, UK). pVAC1/OVA contains the gene encoding OVA, and pVAC1/OVAcyt contains a gene for a deletion variant of OVA downstream of the cytomegalovirus (CMV) promoter in pVAC1. This variant has a deletion between amino acids 20 and 145 of the coding sequence (GenBank V00383), which removes the secretion signal, resulting in an OVA gene product largely confined to the cytoplasm.

Transfection of COS-1 cells with murine CCR7

COS-1 cells were cultured in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mm l-glutamine, 200 mm HEPES, 100 U/ml penicillin and 100 µg/ml streptomycin, at 37°, in an atmosphere of 5% CO₂. Transient transfection was performed using lipofectamine™ (Invitrogen), according to the manufacturer's instructions. In brief, the day before transfection, 1 × 10⁶ COS-1 cells were seeded in a 25-cm² flask and allowed to adhere overnight. On the day of transfection, 5 µg of pCDNA3.1/CCR7 or mock-transfected cells, were mixed with transfection medium (DMEM, HEPES and l-glutamine; no FCS, penicillin or streptomycin) and 20 µl of lipofectamine, and incubated at room temperature for 30 min to allow DNA–lipid complexes to form. The cells were washed in transfection medium and the DNA–lipid mixture was applied to the cells (the total transfection-mix volume was 2 ml). The cells were incubated for 5–6 hr, after which the transfection mix was aspirated and 5 ml of complete culture medium was added. The cells were analysed by fluorescence-activated cell sorter (FACS) analysis 24 hr after transfection.

Cartridge preparation for particle-mediated immunotherapeutic delivery (PMID)

DNA was precipitated onto 2-µm diameter gold particles (DeGussa Metals Group, South Plainfield, NJ) in the presence of 0·05 m spermidine (Sigma, Dorset, UK) and 1 m calcium chloride (Fujisawa Inc., Melrose Park, IL), as previously described.^17,18 The DNA-coated gold particles were washed three times in absolute ethanol and resuspended in absolute ethanol containing 0·05 m polyvinylpyrrolidone (Sigma), then adsorbed onto the inner surface of Tefzel tubing (TFX Medical Inc., Jaffney, NH) by centrifugal force using a tube turner device (Barnant Co., Barrington, IL). The Tefzel tubing was subsequently cut into cartridges of 1·27 cm in length and stored desiccated at 4° until use. A cartridge contained a theoretical weight of 0·5 mg of gold microparticles and typically 0·6–0·8 µg of plasmid DNA, as determined by spectrophotometric analysis of DNA eluted from selected cartridges (Genequant II; Pharmacia Biotech., Cambridge, UK).

Short-term polyclonal activation

Fresh peripheral blood mononuclear cells (PBMC), spleen or lymph node cells (2 × 10⁶ cells/ml) from untreated C57BL/6 mice were plated in a six-well plate. Anti-mouse CD3 (0·5 µg/ml; clone 145-2C11; BD PharMingen, San Diego, CA) and anti-mouse CD28 (1 µg/ml; clone 37.51; BD PharMingen) antibodies or isotype-matched antibodies, were added to the wells. Cells were incubated in a humidified CO₂ incubator at 37° for 2 hr. Brefeldin A (Sigma) was added at a final concentration of 10 µg/ml and cells were incubated for an additional 4 hr before being stained for surface markers and intracellular cytokines (see below).

Adoptive transfer experiments

After lysis of red blood cells, splenocytes from D0.11.10 TCR RAG-2^−/− mice were washed in PBS and resuspended at 20 × 10⁶ cells/ml. Carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) was added to a final concentration of 5 µm and the cell suspension was incubated for 10 min at 37°. The cells were washed in ice-cold PBS containing 5% FCS. Labelled splenocytes (7 × 10⁶/200 µl per mouse) were injected intravenously into the tail vein of BALB/c recipients. One day later, groups of recipient mice were immunized once with pVAC1 or pVAC1/OVA. DNA-coated gold particles were delivered from cartridges at 500 p.s.i. at two sites onto the shaved abdomen (≈1·5-µg total DNA) by PMID using the Powderject™ XRI device (Powderject Vaccines Inc., Madison, WI).

Immunization experiments using pVAC1/OVAcyt

C57Bl/6 mice were DNA-immunized with pVAC1/OVAcyt, three times at 4-week intervals, by using PMID (Powderject, Inc.). Five days after the first and second boost, single-cell suspensions of spleen and lymph nodes were prepared and the cells were restimulated and subsequently stained for FACS analysis (see below).

In vitro restimulation

At the indicated time-points after immunization, blood, spleen and lymph nodes were collected, and single-cell suspensions of spleen and lymph node cells were prepared, followed by lysis of red blood cells. In the studies performed in C57Bl/6 mice, the cells were restimulated in vitro either with medium containing 300 nm of CD8-restricted OVA_257−264 peptide (SIINFEKL) plus 50 ng/ml of IL-2, or with 10 µg/ml of the CD4-restricted OVA peptide (TEWTSSNVMEERKIKV).¹⁹ Restimulation of spleen and lymph node cells from the adoptive transfer experiments (BALB/c mice) was performed by incubation of the cells with medium containing 10 µm of the CD4-restricted OVA_323−339 peptide (ISQAVHAAHAEINEAGR). The cells were incubated in a humidified CO₂ incubator at 37° for a total of 6 hr. Brefeldin A (10 µg/ml) was added after 2 hr and the cells were incubated for an additional 4 hr before being stained for surface markers and intracellular cytokines (see below).

Analysis by flow cytometry

Flow cytometric analysis was performed using standard settings on a FACScalibur flow cytometer (Becton-Dickinson, Oxford, UK). The CCR7- and mock-transfected COS-1 cells were stained, first with the CCR7 antibody (20 µg/ml; generated as described above) and then with phycoerythrin (PE)-labelled goat anti-rabbit immunoglobulin G (IgG) (2·5 µg/ml) (Sigma) 24-hr post-transfection.

For four-colour cell-surface staining, single-cell suspensions of spleen and lymph node cells were obtained by mechanical dissociation and the red blood cells lysed with ammonium chloride. Clumps were allowed to settle, and single cells were counted. A total of 1 × 10⁶ cells were used per test in 100 µl of buffer. A 100-µl volume of whole blood was used in each test. Anti-mouse CD4 or anti-mouse CD8, plus rabbit anti-mouse CCR7, anti-mouse CD62L–FITC and anti-mouse CD44–CyChrome, were added. Samples were incubated on ice for 30 min, then washed in cold buffer before removal of the supernatant. Streptavidin-ECD and anti-rabbit–PE were added and the cells were incubated on ice for 30 min and then washed with cold buffer. The whole-blood samples were lysed using the Whole Blood Lysing Reagents from Beckman Coulter (High Wycombe, UK). Samples were analysed using an EPICS XL (Beckman Coulter).

For intracellular cytokine analyses, 1–2 × 10⁶ cells/sample were initially stained for surface markers, as described above. Cells were washed in FACS buffer, resuspended in 100 µl of reagent 1 from Immunotech's IntraPrep™ Permeabilization Reagent (Immunotech, Prague, Czech Republic) and incubated overnight in the dark at 4°. Cells were washed twice in FACS buffer and resuspended in 100 µl of reagent 2, to which anti-cytokine monoclonal antibodies (mAbs) had been added. The antibodies used were as follows: anti-mouse CD4Cy-chrome (clone H129.19); anti-mouse CD8Cy-chrome (clone 53-6.7); anti-mouse CD62L–PE (clone MEL-14); anti-mouse CD44–PE (clone IM7); anti-mouse IFN-γ–PE (clone XMG1.2); anti-mouse IL-2–PE (clone JES6-5H4), all from PharMingen (San Diego, CA); KJI-26 APC, specific for the TCR transgene (Caltag, Burlingame, CA); anti-mouse IL-4 (clone BVD6-24G2; Immunotech); goat anti-rabbit IgG–F (F-6005) or goat anti-rabbit IgG–PE (P-9537) (Sigma).

Results

Generation and analysis of specificity of the anti-mouse CCR7

The polyclonal rabbit anti-mouse CCR7 was generated by peptide immunization of six rabbits using a 24-mer corresponding to the predicted extreme N-terminus of the murine CCR7 protein (QDEVTDDYIGENTTVDYTLYESVC) (see the Materials and methods). The specific antibody response was determined after each immunization by indirect enzyme-linked immunosorbent assay (ELISA), using free synthetic peptide as antigen. After three immunizations, the serum titres were all >150 000 (data not shown). The affinity-isolated immunoglobulins were stored in PBS containing 0·2% crystalline BSA and further analysed by FACS. Antibodies useful for FACS analysis were isolated from the sera of two of the six rabbits immunized.

In order to determine whether the CCR7 antibody specifically bound to mouse CCR7, COS-1 cells were transiently transfected with mouse CCR7 and analysed by FACS. Twenty-four hours after transfection, the cells were incubated with the CCR7 Ab followed by PE-labelled anti-rabbit IgG. The expression of CCR7 was detected 24 hr after transfection with pCDNA3.1/CCR7 (Fig. 1a), whereas the antibody did not bind the mock-transfected COS-1 cells (Fig. 1b), thus confirming the specificity of the antibody.

Figure 1.

COS cells were transiently transfected with CC-chemokine receptor 7 (CCR7) and analysed by flow cytometry 24 hr later. The rabbit anti-mouse CCR7 binds to COS-1 cells transfected with pCDNA3.1/CCR7 (a) but not to mock-transfected COS-1 cells (b). Data are representative of one experiment out of two performed.

Distribution of CCR7 expression on T cells in different lymphoid compartments

The results in Fig. 2 clearly show that the vast majority of CD4 and CD8 T cells in lymph nodes are CCR7^high, whereas in the spleen and the blood there are clearly both CCR7 ^high and CCR7^1ow populations.

Figure 2.

Analysis of the phenotype of T cells in blood, spleen and lymph nodes. Cells from C57Bl/6 mice were prepared from blood, spleen and lymph nodes and, using four-colour immunofluorescence staining, were labelled with anti-CD4 (or anti-CD8)-biotin, streptavidin–ECD, anti-CD62L–fluorescein isothiocyanate (FITC), anti-CD44–CyChrome, rabbit anti-mouse CCR7 and anti-rabbit–phycoerythrin (PE). Samples were analysed on CD4 or CD8 cells, on an EPICS XL. Data are representative of one experiment out of two performed.

To compare the CCR7 expression with that of CD44 and CD62L on subpopulations of T cells from different lymphoid tissues, single-cell suspensions of lymph node, spleen and blood from untreated C57Bl/6 mice were prepared by four-colour staining, and FACS analysis was performed directly ex vivo (Fig. 2; Table 1)

Table 1.

Cell-surface phenotype of CD4 and CD8 T cells in lymph node, spleen and blood, collected from naive C57Bl/6 mice

	CCR7high	CD62Lhigh	CCR7low	CD62Llow
CD4 T cells
Tissue compartment	CD44low	CD44high	CD44low	CD44high
Lymph node	81·2%	18·8%	Very few cells	Very few cells
Spleen	70·9%	29·1%	9%	91·0%
Blood	74·9%	25·1%	6·2%	93·8%
CD8 T cells
Tissue compartment	CD44low	CD44high	CD44low	CD44high
Lymph node	84·8%	15·2%	Very few cells	Very few cells
Spleen	74·5%	25·7%	17·8%	82·47%
Blood	72·5%	27·5%	17·8%	82·2%

CD44 expression is indicated as a percentage within the CCR7^high CD62L^high, or the CCR7^low CD62L^low populations. CCR7, CC-chemokine receptor 7; CD62L, L-selectin.

The analyses show that the majority of blood, spleen and lymph node T cells express both CCR7 and CD62L (Fig. 2a, 2d, 2g, 2j, 2m, 2p) and that these cells are predominantly CD44^low (Fig. 2c, 2f, 2i, 2l, 2o, 2r; Table 1), which is consistent with a naive T-cell phenotype. The frequency of CCR7^low CD44^high and CD62L^low CD44^high cells was considerably higher in blood (Fig. 2c, 2f, 2b, 2e) and spleen (Fig. 2i, 2l, 2h, 2k) than in lymph nodes (Fig. 2o, 2r, 2n, 2q), which is consistent with a more differentiated effector memory population. Furthermore, in lymph node, spleen and blood, some cells were found to be CD62L^high CD44^high in the CCR7^high T-cell populations (Table 1), suggestive of a central memory phenotype. There were also CCR7^high, cells which were CD62L^low (Fig. 2a, 2d, 2g, 2j, 2m, 2p), indicating further complexity in the co-regulation of these markers.

Cytokine production of CCR7^high and CCR7^low T cells

Cytokine profiles of murine CCR7^high and CCR7^low T cells, derived from blood, spleen and lymph node, were investigated. Cells from untreated C57Bl/6 mice were polyclonally stimulated (αCD3 and αCD28) for 6 hr and analysed by FACS. Naïve T cells have slower kinetics than memory T cells and the short-term stimulation was chosen to preferentially study memory T cells (data not shown). IL-2-producing CD4⁺ T cells were predominantly detected in the CCR7^high CD4 T-cell subpopulations, as shown in Fig. 3(a). In contrast, IFN-γ was mainly produced by blood and spleen CCR7^low CD4 T cells. No detectable levels of IFN-γ were detected in lymph node CD4 T cells. Within CD8 T cells, IL-2 was predominantly produced by CCR7^high CD8 T cells (Fig. 3a). However, in contrast to CD4 T cells, and to that previously reported, both CCR7^low and CCR7^high CD8 T cells from blood and spleen produced IFN-γ. Lower levels of IFN-γ were detected in lymph node compared with blood and spleen, and then only in the CCR7^high CD8 lymph node T cells (Fig. 3a). Unstimulated T cells showed very low or undetectable levels of cytokine production in spleen (Fig. 3b), lymph node and blood (results not shown).

Figure 3.

Cytokine production by CC-chemokine receptor 7 (CCR7)^high- and CCR7^low-expressing CD4 and CD8 T cells from blood, spleen and lymph node following short-term polyclonal stimulation using αCD3 and αCD28 monoclonal antibodies (mAbs) (a) or from spleen with no polyclonal stimulation (b). Data are representative of one experiment out of three performed.

In order to analyse the correlation between CCR7 expression and cytokine profile on T cells during the effector phase of a secondary immune response, C57Bl/6 mice were immunized with pVAC1/OVAcyt (or pVAC1 as control) using PMID. The mice were immunized three times at 4-week intervals, spleens and lymph nodes were collected 5 days after the first and second boost, and the cells were peptide-restimulated and further analysed by FACS. Figure 4 shows the result obtained 5 days after the third immunization (almost identical results were seen after the second immunization, data not shown). Antigen-specific IFN-γ-producing CD8 T cells were predominantly detected within the CCR7^low T-cell population, as shown in Fig. 4(a), 4(c). Interestingly, IL-2 was also produced predominantly by CCR7^low CD8 T cells (Fig. 4e, 4f, 4g), whereas very low frequencies of IL-2 producers were detected within the CCR7^high subpopulation. The same pattern of IFN-γ and IL-2 production was seen in CD4 T cells, although the CD4 T-cell response was much weaker (Figs 4b, 4d, 4f, 4h).

Figure 4.

Most interleukin (IL)-2 and interferon-γ (IFN-γ)-producing T cells show low expression of CC-chemokine receptor 7 (CCR7) during the effector stage of an in vivo recall immune response. Mice were ovalbumin (OVA)-immunized three times (at 4-week intervals). Spleen and lymph node cells were restimulated in vitro with OVA peptide for 6 hr followed by fluorescence-activated cell sorter (FACS) analysis. Cells restimulated with CD8-restricted OVA peptide were gated on CD8 and analysed for the correlation between CCR7 versus IL-2 and IFN-γ (a, c, e and g). Cells restimulated with CD4-restricted OVA peptide were gated on CD4 and analysed for the correlation between CCR7 versus IL-2 and IFN-γ (b, d, f and h). All plots are from a single C57BL/6 mouse. Data are representative of one experiment out of four performed. Background levels of cytokine-producing cells from mock-immunized mice were always ≤0·1%.

Only a proportion of primary effector CD4 T cells that have undergone multiple cell divisions show down-regulation of CCR7 and CD62L

To analyse the changes in CCR7, CD62L, CD44 and CD69 on antigen-specific cells during the transition from naïve to effector cells in vivo, the primary response of OVA-specific CD4 T cells was followed after a single immunization with pVAC1/OVA. Naïve splenocytes from DO11.10 TCR RAG-2^−/− mice were CFSE-labelled and adoptively transferred into BALB/c mice. One day later, the recipients were immunized with pVAC1/OVA or pVAC1 empty vector control, using PMID technology. Before adoptive transfer, all OVA-specific CD4⁺ T cells (recognized by the monoclonal antibody, KJI-26) were naïve, as characterized by a CCR7^high CD62L^high CD44^{low/intermediate} CD69^low phenotype (data not shown). Analysis of lymph nodes and splenocytes 4 days postimmunization with pVAC1.OVA revealed that CCR7 remained highly expressed on the KJI⁺ CD4 T cells during the first cell divisions (Fig. 5). However, after multiple divisions (≥5), down-regulation of CCR7 was evident on a proportion of the cells. Similarly, CD62L was highly expressed on all cells during the first cell divisions, but a subpopulation of CD62L^1ow cells was evident after five or six cell divisions (Fig. 5). As expected, CD44 was up-regulated early during cell divisions but remained low on cells that had not divided (Fig. 5), suggesting that that the majority of cells in the lymph nodes had been specifically activated in vivo. Some cells also showed high expression of the CD69 activation marker, as has been observed previously in this model,²⁰ although this was down-regulated in cells that had undergone cell divisions (Fig. 5).

Figure 5.

During a primary immune response, in vivo down-regulation of CC-chemokine receptor 7 (CCR7) and L-selectin (CD62L) occurs after multiple cell divisions and coincides with the expression of effector cytokines. Naïve splenocytes from DO11.10 T-cell receptor (TCR) recombination-activating gene-2 (RAG-2)^−/− mice were carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled and adoptively transferred into BALB/c mice. One day later, the recipients were immunized with pVAC1/ovalbumin (OVA). Four days, later cells from draining lymph nodes (inguinal and periaortic) were restimulated in vitro (OVA-peptide +αCD28) for 6 hr and analysed further by flow cytometry. The cells were gated on CD4 and KJI-26 and analysed for the expression of the surface markers CCR7, CD62L, CD44, and CD69 versus CFSE, or for intracellular cytokine production [interleukin (IL)-2, IL-4 or interferon-γ (IFN-γ)] versus CFSE. All plots are from a single mouse. Analysis of five other mice produced very similar results. Cytokine production in the absence of OVA peptide for in vitro restimulation was < 0·05%.

Interestingly, the down-regulation of CCR7 and CD62L was coincident with the expression of the effector cytokines IL-4 and IFN-γ, whereas IL-2 was produced from the first cell division (Fig. 5). Cytokine production was not observed in the absence of in vitro restimulation with peptide (results not shown), indicating that the ex vivo lymph node T cells were not producing cytokines constitutively.

For mice immunized with the pVAC1 empty vector, very few cells (<0·5%) had undergone clonal expansion in vivo; the expression of CD69 was similar to pVAC1.OVA-immunized mice, but no cytokines were detected following in vitro restimulation with peptide, which is consistent with previously published data for this model.²⁰

Discussion

It has previously been shown that the expression of the homing and chemokine receptor, CCR7, may divide human memory T cells into two functionally distinct subsets. It has been suggested that CCR7^high CD62L^high central memory T cells, without immediate effector ability, traffic through secondary lymphoid tissue, whereas CCR7^low CD62L^1ow effector memory T cells preferentially circulate outside the lymphoid tissue.⁹ Recently, further support has been provided for the existence of distinct memory T-cell subsets in terms of function and migratory pattern, in that murine memory CD8⁺ T cells (induced by a viral infection) isolated from non-lymphoid tissues show high levels of lytic activity directly ex vivo, whereas memory cells isolated from lymphoid tissue do not, after short-term in vitro restimulation.^21–24 Moreover, unprimed murine T cells display a typically naïve phenotype, as characterized by low/intermediate expression of CD44, but high expression of the lymph node homing receptor, CD62L. Upon activation, CD44 is up-regulated and remains high following the transition from effector to memory T cells. In contrast, the expression of CD62L on effector and memory T cells has been shown to be more variable and remains high on certain T cells, but is down-regulated on others. This divergence has been suggested to correlate with distinct characteristics and patterns of migration in the same way as chemokine receptors show polarized patterns of expression.^9,11

It has been difficult to generate antibodies against murine CCR7 because of strong sequence conservation. This has not permitted direct comparison of results obtained in humans with those in murine systems. Using the approach outlined above we have generated a rabbit polyclonal antisera that stains CCR7-transfected but not mock-transfected COS-1 cells. Together with the pattern of the antisera staining on CD4 and CD8 T cells in different lymphoid compartments, these results provide confidence that the antisera is specific for the CCR7 molecule. The results of CCR7 staining in combination with other memory markers, CD62L and CD44, and functionally for IL-2 and IFN-γ production, are consistent with those observed in other human and murine studies.^9,13 Our results support the existence of phenotypic and functionally distinct memory T-cell subsets, but further indicate additional complexity in the system. In agreement with previous reports, we found that most CCR7^high CD4 and CD8 T cells co-expressed CD62L. Moreover, the frequency of CCR7^high T cells within the CD62L^1ow subpopulation was higher in lymph node T cells compared with peripheral blood T cells, further indicating an essential role for CCR7 in promoting immigration into lymph nodes. This indicates differential regulation between CCR7 and CD62L, and adds to the complexity of different memory phenotypes observed in different tissues. Notably, although the vast majority of T cells in lymph nodes were clearly CCR7^high, a small population of CCR7^low cells may be present, predominantly within the CD4 T-cell subpopulation, suggesting that CCR7^low T cells also gain access to lymph nodes. These cells should not enter the lymph node via high endothelial venules but may have gained access to lymph nodes through afferent lymph vessels.²⁵ Alternatively, CCR7 may have been down-regulated within the lymph node by binding to one of its ligands, EB11 ligand chemokine (ELC), which is constitutively expressed in lymph nodes and has been shown to reversibly down-regulate CCR7.²⁶

Analysis of cytokine production after short-term (6-hr) polyclonal stimulation showed that splenic and peripheral blood CCR7^high CD4 T cells produced IL-2, whereas CCR7^low CD4 T cells produced both IL-2 and IFN-γ. These results are in agreement with previous reports from the human system, where it was shown that polyclonal stimulation of CCR7^high T_CM cells resulted in IL-2 production, whereas stimulation of the more differentiated CCR7^low T_EM cells resulted in production of effector cytokines IFN-γ and IL-4, as well as IL-2.⁹ Although we cannot separate naïve T cells from T_CM cells on the basis of CCR7 expression, we believe that the cytokine-producing cells detected after 6 hr of restimulation are of memory type owing to the slower cytokine response of naïve CD4 and CD8 T cells compared with memory CD4 T cells.^7,24 Moreover, the fact that no/very low numbers of IL-2- or IFN-γ-producing cells were detected in CCR7^low CD4 lymph node cells after short-term polyclonal stimulation may indicate functional differences between CCR7^low CD4 T cells derived from lymph nodes compared with CCR7^low CD4 T cells derived from spleen and blood.

Within the CD8 T cells, IL-2 was predominantly detected in the CCR7^high subpopulation. However, in marked contrast with earlier findings in humans, a substantial proportion of IFN-γ-producing cells was found within the CCR7^high CD8 T-cell subpopulation in addition to that detected within the CCR7^low CD8 T-cell subpopulation. These contrasting results may be explained by the fact that T cells from two different species, human and murine, were analysed. Nonetheless, this may suggest that murine CCR7^high CD8 T_CM cells have the capacity to produce IFN-γ in addition to IL-2 or, alternatively, that CD8 T_EM cells may be able to re-express CCR7 while functionally still acting as T_EM cells.

Contradictory results have been reported regarding the expression of CCR7 on activated T cells. A number of studies have demonstrated an up-regulation of CCR7 protein expression and/or mRNA early after polyclonal in vitro activation of PBL/CD4 T-cell lines.^23,24 In contrast, others have shown a lack of CCR7 mRNA expression in effector CD8 T cells analysed 8 days postviral infection in vivo, whereas naïve Ag-specific CD8 T cells expressed high levels of CCR7.^27,28 The difference in CCR7 expression on activated cells observed in the different studies might be explained by the length of time after antigen stimulation (number of cell divisions) that the cells were analysed and/or by the different methods used for activation of the cells (i.e. polyclonal in vitro stimulation [phytohaemagglutinin (PHA)/anti-CD3 versus Ag-specific in vivo stimulation]. We found that after adoptive transfer of naïve CCR7^high KJI⁺ CD4 T cells followed by PMID using pVAC1/OVA, the expression of CCR7 remained high during the first five or six cell divisions, after which a proportion of the cells showed down-regulation of CCR7. Interestingly, the down-regulation of CCR7 coincided with the down-regulation of CD62L and with the induction of IL-4 and IFN-γ production. These results indicate that CCR7 is down-regulated on more differentiated effector cells and not on early activated effector cells. However, the fact that only a proportion of the cells that underwent multiple cell divisions down-regulated CCR7 (and CD62L), indicates that multiple cell divisions are necessary but not sufficient to induce the down-regulation. Other factors, such as the duration of TCR stimulation and the amount and quality of extrinsic factors, such as cytokines, have been proposed to play important roles in the differentiation process.^29–31

It has been reported that when restimulated in vitro, CCR7^high memory CD4 T cells lost their CCR7 expression and a proportion of the cells produced effector cytokines, indicating that stimulation of T_CM can gave rise to effector cells and possibly T_EM.⁹ However, the fate of T_CM and T_EM cells during a recall response in vivo has not been investigated. It has been proposed that T-cell differentiation from naïve to T_CM to T_EM cells is a linear and irreversible event.^29,32 However, others have proposed a non-linear model of T-cell memory differentiation in which some CCR7^low memory T cells have the capability to regain the expression of CCR7 and function as central memory T cells, possibly depending on the presence or absence of antigen.³¹ Analysis of the cytokine profile of effector T cells during the clonal expansion (5 days postboost) of a secondary immune response in vivo, showed that most IL-2 and IFN-γ producers were found within the CCR7^low subpopulation of CD8 and CD4 T cells. In contrast, very few CCR7^high cytokine-producing T cells could be detected. The fact that the vast majority of cytokine-producing effectors were CCR7^low may suggest that CCR7 is down-regulated on T_CM when reactivated during a secondary immune response. We believe that it is highly unlikely that all the OVA-responding cells were of T_EM type actually before the immunization, as the same pattern was seen in both spleen and lymph nodes and T_EM cells should have limited access to lymph nodes. Furthermore, this suggests that in vivo stimulation of murine CCR7^high memory T cells results in CCR7^low effector cells.

An important question that needs to be answered is whether boost immunizations result in a higher CCR7^low : CCR7^high ratio of memory T cells compared with a primary immunization. This can have important implications for vaccine development and protocols, where it may be desirable to predominantly generate T_CM but at the same time may be necessary to boost the effector immune response. The expression of CCR7 on the T_CM provides a ‘sign post’ into lymph nodes where interaction with professional APCs and clonal expansion occurs. It may be important that a substantial proportion of the memory T cells are CCR7^high T_CM cells with the capacity to produce IL-2 and to clonally expand on the next encounter with antigen to prevent exhaustion or dysfunctional memory responses.

The results reported here have validated an antibody against CCR7 in mice and enabled a comparison with similar studies in humans. The CCR7 antibody can be used to determine the balance between T_CM and T_EM populations following PMID. This may allow correlation of the proportion of T_CM versus T_EM with protective responses and possibly provide a rationale for vaccine design.

Abbreviations

Ab

antibody

Ag

antigen

APC

antigen-presenting cell

BCG

bacille Calmette–Guérin

BLAST

basic local alignment search tool

CCL

CC-chemokine ligand

CCR7

CC-chemokine receptor 7

CD62L

L-selectin

ELC

EB11 ligand chemokine

FACS

fluorescence-activated cell sorter

FCS

fetal calf serum

IFN-γ

interferon-γ

OVA

ovalbumin

PE

phycoerythrin

PMID

particle-mediated immunotherapeutic delivery

PNAd

peripheral-node addressin

PPD

purified protein derivative of tuberculin

RAG-2

recombination-activating gene-2

T_CM

central memory T cells

T_EM

effector memory cells

TCR

T-cell receptor