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. Author manuscript; available in PMC: 2014 Sep 15.
Published in final edited form as: J Immunol. 2013 Aug 9;191(6):3119–3127. doi: 10.4049/jimmunol.1200938

CCR7 Plays No Appreciable Role in Trafficking of Central Memory CD4 T Cells to Lymph Nodes

Bryan Vander Lugt *,#, Noah J Tubo *,**, Suzanne T Nizza *,**, Marianne Boes *, Bernard Malissen ††, Robert C Fuhlbrigge *,‡‡, Thomas S Kupper *,‡‡, James J Campbell *,‡‡
PMCID: PMC3784989  NIHMSID: NIHMS509474  PMID: 23935190

Abstract

CCR7−/− mice exhibit profound anomalies in LN and spleen architecture, which complicates the study of CCR7-mediated T cell trafficking in vivo. To circumvent this problem, we established in vivo models in which WT and CCR7−/− populations coexist within mice possessing normal lymphoid organs, and must compete for developmental niches within the tissues of these mice. Under the conditions we have created in vivo, we find the entry of memory CD4 T cells into LN from the blood to be independent of CCR7. Thus, the central memory CD4 T cells that traffic though LN, which are often defined by their expression of CCR7, do not appear to gain any competitive homing advantage by expressing this receptor. Furthermore, in contrast to cutaneous DC populations, we found that CCR7 deficiency had no appreciable effect on the exit of CD4 T cells from inflamed skin. Finally, we found that WT and CCR7−/− precursors were equally represented within the major thymic subpopulations, despite previous findings that CCR7 plays a role in seeding the thymus from bone marrow-derived T cell precursors.

INTRODUCTION

CCR7 is a homeostatic chemokine receptor believed to coordinate the correct positioning of lymphocytes and dendritic cells (DC) during immune cell development, routine immunosurveillance, and the generation of cognate immune responses. Proposed CCR7-mediated cell movements include the entry of bone marrow (BM)-derived T cell precursors into thymus (1–3), entry of naïve (4–6) and “central” memory (7, 8) T cells into lymph nodes (LN) from blood through high endothelial venules (HEV), positioning of T cells within T cell zones of secondary lymphoid organs (SLO) (6, 9, 10), and the exit of T cells (11, 12) and mature DC (9, 13) from non-lymphoid tissues.

CCR7 is expressed by all naïve lymphocyte subsets (14, 15), by “central” memory T cells (7, 8) and by mature migratory DC (13). Knockout mice lacking CCR7 or its ligands (CCL19 and CCL21) display numerous dramatic phenotypes, including paucities in several T cell subpopulations and severe diminution in T cell-mediated antigen responses (10). The SLO of these mice are small, lack distinct T and B zones and possess other histologically distinct irregularities (10). These diverse phenotypes have been construed as direct consequences of defective T cell trafficking (10, 16), but the potential indirect effects of T cell development within abnormal SLO microenvironments have not been fully explored.

We therefore created models in which CCR7-deficient T cell precursors could develop within relatively normal in vivo environments. In one such model, we reconstituted lethally irradiated hosts with mixed WT and CCR7-deficient BM. The presence of WT BM-derived cells preserved normal SLO architecture within recipients. This experimental design allowed us to directly compare trafficking behaviors between WT- and CCR7−/−-derived T cell subpopulations within each chimera.

We have found that many T cell phenotypes associated with CCR7 deficiency are most likely secondary effects of development within abnormal SLO microenvironments. Contrary to previous thought, our findings do not support the notion that CCR7 plays a discernable role in the trafficking of Ag-experienced CD4 T cells to the LN, either directly from the blood or from peripheral tissues such a skin.

MATERIALS AND METHODS

Mice

All experiments were performed with mice on the C57BL/6 background. Langerin-EGFP (LangEGFP) mice were provided by B. Malissen. CCR7−/− and LTα−/− mice were obtained from Jackson. C57BL/6, congenic CD45.1, and OT-II mice were obtained from Charles River. Animal housing and experimentation was in accordance with institutional guidelines.

Flow cytometry analysis and sorting

Directly conjugated antibodies were purchased from Ebioscience and Biolegend. E-selectin-Fc chimera was purchased from R&D and anti-human Fc-gamma was purchased from Jackson Laboratories. Single cell suspensions were stained on ice and analyzed on a BD FACSCaliber 6-color flow cytometer using FACSdiva software. Data analysis was done using FlowJo software. Naïve OT-II T cells were sorted using a core facility LSRII.

Bone marrow chimera generation and analysis

CCR7 competitive BMC - F1 CD45.1/CD45.2 mice were irradiated with 2 doses of 600 rads separated by 3 hours. Mice were immediately reconstituted with 5×106 red blood cell-depleted bone marrow cells comprised of 1:1 WT(CD45.1):CCR7−/−(CD45.2) bone marrow. 12 weeks after reconstitution, mice were used for experiments as indicated. WT and KO donor populations were distinguished by congenic markers, and ratios were calculated using absolute numbers. Langerhans cell BMC – CCR7+/− LangEGFP or CCR7−/− LangEGFP mice were irradiated and reconstituted with WT bone marrow as above. Ears were treated to remove hair (commercial Nair), split into dorsal and ventral halves, and floated on 1mg/ml Dispase II (Roche) in PBS for 30 min to separate epidermis from dermis. Epidermal sheets were directly analyzed by epifluorescent microscopy.

Short-term Homing Assays

Blood homing assays – 5×107 LN and splenic lympyocytes from CD45.1 CCR7+/+ and CD45.2 CCR7−/− mixed 1:1 were injected retro-orbitally into recipient CD45.1/CD45.2 F1 mice. Two or eight hours after transfer, spleen and sdLNs were collected and analyzed by flow cytometry. Footpad homing assays – 5×107 mixed splenocytes were injected into the footpads of recipient mice. Popliteal LNs were collected 18 hours after transfer for analysis by flow cytometry.

DNFB Contact Hypersensitivity Response

50µl of 0.5% DNFB in 4:1 acetone:oil was painted onto shaved abdomen skin. 7 days after sensitization, mice were challenged with 5µl 0.5% DNFB solution applied directly to ear skin. 1 day after challenge, mice were treated with 25µg FTY720 (Cayman) i.p. Ears and sdLNs were collected 2 days after FTY720 treatment.

Isolation of Skin-infiltrating T cells

Ears were separated into dorsal and ventral halves and finely minced. Minced tissue was placed into 20ml isolation medium (HBSS supplemented with 10mM HEPES and 5mM EDTA) at 4°C with agitation by stir bar for 4–6 hours. Supernatant containing released lymphocytes was then passed through a 40µm filter and directly analyzed by flow cytometry.

Antigen-specific Responses

Immunization – Mice were immunized epicutaneously as previously described (17). Briefly, scotch tape was used to gently remove the cornified layer of ear skin, then skin was treated with acetone and cholera toxin adjuvant before administration of chicken ovalbumin323–339 peptide. For most OT-II experiments, 5×106 OT-II splenocytes were transferred retro-orbitally into recipient mice 24 hours prior to immunization. For memory OT-II experiments, 500 purified naïve OT-II T cells were transferred.

RESULTS

Generating Competitive Bone Marrow Chimeras

We created competitive WT/CCR7−/− mixed bone marrow chimeras (CCR7-BMC) similar to those we used previously to study CCR4 and CCR9 function in vivo (18–20). We reconstituted lethally irradiated WT hosts with 1:1 mixtures of BM from WT and CCR7−/− donors. We used congenic CD45 variants to distinguish host (CD45.1/CD45.2 double positive) from WT (CD45.1) and CCR7−/− (CD45.2) donors. [Note: all DC subsets required for presenting antigen to T cells are available in these chimeras from the host and WT BM donor, despite the additional presence of CCR7−/− DC populations].

After ≥12wk, we evaluated the relative contribution of each BM donor to individual cell populations within each host. A 1:1 ratio of WT-to-CCR7−/− cells (i.e., equivalent to the input population) would indicate that CCR7 expression provided no competitive advantage for WT over CCR7−/− cells. An n:1 ratio would indicate that CCR7 expression provided an n-fold advantage for WT over CCR7−/− cells. To correct for random differences in BM engraftment among recipients, we normalized the ratio of each cell type to a reference population not influenced by CCR7 function for each recipient. We used peripheral blood monocytes and neutrophils for this purpose. These two populations did no differ significantly from each other in WT:CCR7−/− ratio for any mouse used in these studies. Based on these two circulating leukocyte populations, the actual reconstitution efficiency ranged between 0.50 and 1.53 among individual recipients, in contrast to the ideal value of one (18–20)).

Identifying Naïve and Antigen-experienced T Cell Subsets

We used CD44 with either CD45RB (for CD4 T cells) or CD122 (for CD8 T cells) to distinguish between naïve and Ag-experienced populations [note: in both cases, CD44lo defines naïve cells, but CD45RB and CD122 help to establish the cutoff point between CD44lo and CD44hi cells]. An example of our phenotypic characterization is shown in Fig 1A, left panels. We confirmed that Ag-experienced CD4 and CD8 T cells defined by these criteria were over-represented in the CCR7−/− SLO, supporting our confidence in these immunophenotyping criteria (Supplemental Fig S1 and ref (10)).

Figure 1. Influence of CCR7-Deficiency on Accumulation of Naïve and Ag-Experienced T Cell Subsets within Lymphoid Organs.

Figure 1

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Lethally irradiated mice (CD45.1/CD45.2) were reconstituted with a 1:1 mixture of bone marrow from WT (CD45.1) and CCR7−/− (CD45.2) mice. Twelve weeks after reconstitution, naïve and Ag-experienced CD4 and CD8 T cell populations from skin-draining LNs (sdLNs), spleen and thymus were analyzed for the relative contribution of cells from each donor. (A) Plots show gating criteria for each population examined (left panels), and the representation of each cells from each donor within the indicated population (right panels). Data are shown from a single representative CCR7-BMC mouse. [Please note that CD45.1/CD45.2 double positive cells excluded from the gates in the right panels represent host-derived cells that survived irradiation]. (B) WT:KO donor ratios of populations gated as in (A). Ratios were normalized to correct for engraftment efficiency of each donor BM type for each CCR7-BMC mouse as described in text. Left panel: Data analysis of 6 “resting” mice with each data point representing an individual mouse. Bars show mean ± s.d. Similar data were obtained in at least 6 independent analyses of 4–7 mice per analysis. Right panel: Data analysis of mice 3 days after cutaneous ear challenge of sensitized mice with DNFB. Each data point represents a single mouse from a 3-mouse experiment. Similar data were obtained in 3 independent analyses of 3–5 mice per experiment. Calculation of p-values was performed with a 1 sample 2-tailed t test against a hypothetical value of 1, n.s. indicates no significant advantage detected for WT cells over CCR7−/− cells. (C) Thymocyte populations from competitive CCR7-BMCs. Left panel shows gating criteria for each population analyzed. Right panel shows data from thymocyte populations analyzed as in (B). Each data point represents a single mouse from a 4-mouse experiment, representative of 2 experiments.

The presence of CCR7 on Ag-experienced T cells is a characteristic commonly used to identify the central memory subset, but this is not possible in a study involving CCR7−/− mice. Instead, for the purposes of this study, we used an operational definition for central memory T cells, i.e. their ability to enter LN directly from the blood via HEV (7, 8).

T cells that enter non-lymphoid tissues are often classified as effector memory (7, 8). However, many T cells that express markers required for entering non-lymphoid tissues (i.e. CLA and α4β7-integrin) also express CCR7, the purported central memory marker (15). Thus, for the purposes of this study, we consider T cells found within non-lymphoid tissues to be enriched but not pure effector memory populations.

CCR7 Does Not Influence Accumulation of Ag-Experienced T cells in Lymph Nodes

We found WT naïve cells from skin-draining LN (sdLN) to hold a nearly 20-fold competitive advantage over CCR7−/− naïve cells (Fig 1A& B). In contrast, WT Ag-experienced T cells had no appreciable advantage over their CCR7−/− counterparts. Data shown are from the cervical LN, but results were very similar for mesenteric LN (see supplemental Fig S2). In the spleen, we observed a small but significant skewing in favor of the WT donor in naive but not Ag-experienced populations (Fig 1B).

Similar trends were seen whether the tissue drainage area was inflamed (Fig 1B, right panel) or resting (Fig 1B, left panel). However, there was a very small but significant advantage for CCR7 expression by Ag-experienced CD4 but not CD8 populations within the inflamed LN.

CCR7 and Thymic Seeding

Several studies suggest that CCR7 plays a role in T cell development through its involvement in seeding the thymus with BM-derived T cell precursors (21, 22). However, our competitive assays reveal that thymic development proceeds with normal kinetics in the absence of CCR7. WT and CCR7−/− donor-derived cells were equally represented within the immature CD4/CD8 double positive population, as well as each of the single positive populations (Fig 1C).

Differential Requirements for Entry of CD4 versus CD8 Central Memory T Cells into LN from the Blood

Naïve T cells are thought to possess only a single route through which they may enter LN: from blood via HEV (23) (not including intranodal migration after entry to a connected LN through HEV (9)). However, Ag-experienced T cells can enter through the HEV or from peripheral tissues via the afferent lymph (24). As discussed above, those that enter through peripheral tissues are enriched in “effector” cells while those that enter through HEV are operationally defined as “central” memory cells (7, 8). The CCR7-BMC experiments shown in Fig 1 provide a “snapshot” of T cell trafficking that represents the combined contributions of both processes.

We wished to directly examine HEV-specific homing from blood to LN, and therefore designed a shorter-term model in which homing from the blood would reach near completion, but the vast majority of cells entering non-lymphoid tissues would not have sufficient time to enter the LN (25, 26). In this “short term” competitive assay, we transferred 1:1 mixtures of splenocytes from mature WT and CCR7−/− donors into non-irradiated recipients, and distinguished host-derived from donor-derived populations using the CD45 congenic markers described in Fig 1. Lymphoid organs from the recipients were harvested for flow cytometry either 2 hr or 8 hr after transfer. The ratio of WT to CCR7−/− cells within donor-derived populations was calculated and normalized to the input population (Fig 2).

Figure 2. Central Memory CD4 and CD8 T Cells Differ In Their CCR7 Requirements for Homing to LN Directly from Blood.

Figure 2

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A 1:1 mixture of WT (CD45.1) and CCR7−/− (CD45.2) splenocytes was transferred intravenously into WT (CD45.1/CD45.2) recipient hosts. Two or eight hours after transfer, the sdLN and spleen of recipient mice were analyzed for the presence of donor cells. (A) Left cytometry plot shows the WT and KO components of the naïve CD4 T cell compartment within the mixed donor input population prior to transfer. Right cytometry plots show the WT and KO components of the naïve CD4 T cell compartments recovered from sdLN and spleen of recipient mice after transfer. Data in these panels are from a single representative recipient mouse. [Please note that CD45.1/CD45.2 double positive cells excluded from the gates in the FACs plots are comprised entirely of host cells]. (B) WT:KO ratios (normalized to that of the input population) calculated for donor T cell populations isolated two hours (left panel) or eight hours (right panel) after transfer. Two hour data are derived from two separate experiments of 3 recipients each. Eight hour data are derived from 8 recipient mice from a single experiment. Each data point represents an individual recipient mouse. Bars show mean ± s.d. Calculation of p-values was performed using a 1 sample 2-tailed t test against a hypothetical value of 1, n.s. indicates no significant advantage detected for WT cells over CCR7−/− cells.

CCR7 conferred a marked competitive advantage to both CD4 and CD8 naïve T cells for access to LN but not spleen (Fig 2). Also, as seen in the BM chimeras, WT and CCR7−/− Ag-experienced CD4 T cells were represented equally within LN and spleen. Thus, the central memory CD4 T cells that traffic though LN, which are often defined by their expression of CCR7 (7, 8), do not appear to gain any competitive homing advantage by expressing this receptor.

Interestingly, CCR7 did convey a significant advantage to Ag-experienced CD8 T cells in the LN but not spleen. Thus, unlike CD4 T cells, central memory CD8 T cells (or at least a subset of them) do indeed appear to utilize CCR7 for entry into LN from blood.

Differential Representation of Central Versus Effector Memory T cells

In our “short term” homing assay above, we further observed that the Ag-experienced-to-naïve ratios within the recovered CD4 populations from both LN and spleen were greatly reduced with respect to those of the input population at the 8 hr time point (Fig 3, left panel). This was not the case for the CD8 populations, where the ratios within the input and recovered populations were not significantly different (Fig 3, right panel). [Please note: data from CCR7−/− donor-derived cells were not considered in Fig 3].

Figure 3. Ag-Experienced CD4 and CD8 T Cell Populations Home Differentially from Blood to Peripheral tissues versus Lymphoid organs.

Figure 3

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The proportion of T cells within the WT CD4 and CD8 populations that displayed the Ag-experienced immunophenotype (as described in text) was calculated for input and recovered cells populations from the experiments reported in Fig 2. Bar graphs show mean ± s.d. Calculation of p-values was performed using the Mann-Whitney rank order test, n.s. indicates no significant difference from the input population.

Thus, a given Ag-experienced CD8 T cell in the circulation is most likely to enter a lymphoid tissue (just as likely as a naïve CD8 T cell), but a given Ag-experienced CD4 T cell is more likely to enter a non-lymphoid tissue. The Ag-experienced CD8 T cell population within blood and lymphoid organs is therefore comprised primarily of central memory cells. In contrast, Ag-experienced CD4 T cells in the LN at any given time most likely migrated from non-lymphoid tissue, and thus meet the operational definition of effector memory (7, 8).

WT and CCR7−/− T Cells Accumulate Equally Well within Inflamed Skin

Two recent studies propose that CCR7 is required for migration of T cells from peripheral tissues to the draining LN (11, 12). This is difficult to reconcile with our finding that Ag-experienced WT and CCR7−/− cells are equally represented within LN of our competitive chimeras (Fig 2). If CCR7 were indeed necessary for emigration from peripheral tissue, one would expect WT Ag-experienced CD4 T cells to greatly outnumber their CCR7−/− counterparts, because the majority of Ag-experienced CD4 T cells within LN are likely to have arrived there from peripheral tissue (Fig 3). Thus, our findings suggest that CCR7 is either unnecessary for this migration step, or that some other mechanism compensates for the proposed deficiency.

Nonetheless, if CCR7-deficient CD4 T cells were able to enter peripheral tissues but unable to exit efficiently, one would expect CCR7−/− cells to accumulate disproportionately within the peripheral tissue. To test this notion, we used skin as a representative peripheral tissue and returned to the CCR7 competitive bone marrow chimera model. We directed Ag-experienced T cells into the ear skin by inducing an anamnestic response through repeated topical immunization with ovalbumin + adjuvant (18). Interestingly, we found that WT and CCR7−/− cells accumulated equally within inflamed skin (Fig 4A). Thus, in the case of skin, Ag-experienced WT and CCR7-deficient T cells were equally represented within both the peripheral tissue (Fig 4) and the LN that drains that tissue (Fig 1).

Figure 4. CCR7 Does Not Affect the Accumulation of CD4 T cells within Inflamed Skin, nor Does it Influence Their Egress from Skin.

Figure 4

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CCR7-BMC mice were immunized epicutaneously with ovalbumin and adjuvant every 10 days, and ear skin was harvested for analysis 2 days after the third challenge. (A) Gating schemes for WT and CCR7−/− CD4 T cell populations pooled from the inflamed skin of 2 mice. (B) Egress of CD4 T lymphocytes from inflamed skin. The ears of CCR7-BMC mice were immunized as in A. After challenge, the immunized chimeras were split into three groups. The first group was sacrificed on day 2, and inflamed skin obtained for T cell isolation. The second group was treated with FTY720 on day 2: this compound effectively sequesters T lymphocytes within secondary lymphoid organs, removing them from the blood and thus eliminating the circulating pool of skin-homing cells and preventing further infiltration T cell infiltration into the skin. The third group was not further manipulated until day 4, when both the second and third groups were harvested for T cell isolation from skin. Each dot represents the CD45.1:CD45.2 ratio for CD3+CD4+ T cells isolated from both ears of a single CCR7-BMC mouse.Calculation of p-values was performed using an unpaired 2-tailed t test, n.s. indicates that no significant difference was detected between the WT and CCR7−/− populations.

Efficient Exit of CCR7−/− CD4 T Cells from Skin

To further evaluate the role of CCR7 in T cell emigration from peripheral tissues, we sought to isolate the tissue-emigration step from the other elements contributing to accumulation within LN. [Note: this experiment focuses specifically on Ag-experienced cells because naïve cells do not home to non-lymphoid tissues (24, 27)]. Using our CCR7-BMC model, we allowed T cells to accumulate within inflamed ear skin for 2 days, at which time we inhibited further skin recruitment by treating the chimeras with FTY720, thus sequestering circulating T cells within lymphoid organs (28). We then assessed the relative efficiency with which WT vs. CCR7−/− cells exited the skin by determining the ratio of WT to CCR7−/− T cells of skin-infiltrating cells immediately prior to FTY720-treatment and then 2 days later on day 4 (Fig 4B). We also assessed this ratio for mice that were not treated with FTY-720 but harvested on the same day as the treated mice

The ear skin of FTY-720-treated mice possessed approximately 30% of the cells present in the ears than untreated mice on day 4 after challenge, showing that the CD4 population in skin is reduced over this time period when new T cells are prevented from entering. However, the CD45.1:CD45.2 ratio remained near 1:1 under all conditions tested (Fig 4B). If CCR7 were required for T cell emigration from skin, one would have expected CCR7−/− cells to remain trapped in the ear skin while the WT cells exited efficiently, resulting in a decreased WT:CCR7−/− ratio, which was not the case.

CCR7 and Migration of DC to LN from Peripheral Tissues

As our findings regarding CCR7 and the exit of CD4 T cells from skin did not agree with the conclusions of previous studies, we next used the CCR7-BMC model to observe the accumulation of skin-derived dendritic cells within sdLN, for which CCR7 function has also been proposed as a requirement for exit from skin. Our model confirms that CCR7 expression confers a strong competitive advantage for accumulation of MHCIIhi migratory DCs in sdLN (Fig 5A). CCR7 also confers a small but significant advantage to MHCIIlo populations within LN, especially when compared to the same populations from spleen (where CCR7 had no effect).

Figure 5. Subpopulations of DC Possess Differential CCR7 Requirements for Accumulation within sdLN.

Figure 5

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(A) DC populations from sdLN and spleen of CCR7-BMCs. FACs plots (left panels) show gating scheme used to analyze WT and CCR7−/− donor contribution for the indicated populations. DC subpopulations were divided into CD11cint/MHCIIhi (not found in spleen), CD11chi/MHCIIloCD8hi, and CD11chi/MHCIIloCD11bhi as indicated. WT:KO donor ratios were calculated for the indicated populations of 4 mice from a single experiment. Similar data were obtained in 4 independent experiments of 3–6 mice each. Calculation of p-values was performed using a 1 sample 2-tailed t test against a hypothetical value of 1, n.s. indicates no significant advantage detected for WT cells over CCR7−/− populations. (B) WT or CCR7−/− mice expressing EGFP under the Langerin promoter (LangEGFP) were lethally irradiated and reconstituted with WT bone marrow, so that the only host-derived DC surviving radiation were expected to be EGFP+ Langerhans cells. After 12 weeks to allow full reconstitution of the chimeric mice, epidermal sheets were analyzed by epifluorescent microscopy to identify radio-resistant GFP+ Langerhans cells (left panels). FACs panels show MHCIIhi DCs from the sdLNs of chimeric mice analyzed for migrating skin-derived GFP+ LCs (right panels).

It was not possible to assess migration of Langerhans cells (LC) to sdLN using the model in Fig 5A, because LC precursors in skin are highly radioresistant (29): the LC population in the CCR7-BMC model is comprised solely of host-derived cells, which are systematically excluded from analysis (30). We therefore modified our BM adoptive transfer approach to directly assess the importance of CCR7 in LC migration.

We used the Langerin-EGFP mouse strain that allows observation of LCs by virtue of EGFP expression under the Langerin promoter (29). We bred this strain with the CCR7−/− strain to obtain CCR7+/−/EGFP+ or CCR7−/−/EGFP+ littermates, each serving as hosts for WT bone marrow after lethal irradiation. Through this design, all EGFP+ DC in the epidermis and sdLN are LC of host origin, and were thus CCR7+/− or CCR7−/− depending on the host. All radiosensitive DC were of donor (WT) origin. Eight weeks after reconstitution, we found comparable densities of EGFP+ cells within the epidermis of both chimera types (Fig 5B, left panels). However, only CCR7+/− LC accumulated within the sdLN (Fig 5B, right panels).

Tissue-Selective Homing Capabilities of CCR7−/− T Cells

The entry of CD4 T lymphocytes into skin is thought to require an “imprinting” step, whereby naïve cells acquire skin-selective homing molecules after recognizing skin-derived antigen within the sdLN (24, 27, 31). As CCR7−/− naïve T cells have great difficulty entering LN (Fig 1&2), one would expect them to have more restricted access to the imprinting apparatus than WT naïve cells. It is therefore noteworthy that we found any CCR7−/− cells within skin at all (Fig 4).

One of the most important molecules directly involved in skin-selective T cell homing is the carbohydrate ligand for E-selectin (E-lig, known as CLA in humans) (32–34). CD103, the ligand of E-cadherin is considered important for extended retention of T cells in skin (35).

We isolated CD4 T cells from the sdLN draining the inflamed ear skin of our CCR7-BMC mice to determine E-lig and CD103 expression (Fig 6A). We found no significant difference between the WT and CCR7−/− populations regarding expression of either molecule. Furthermore, when gating specifically on the CD4+/E-lig+ population from CCR7-BMC mice, we observed a 1:1 ratio for WT and CCR7−/− cells (Fig 6B).

Figure 6. CCR7-deficient T cells Efficiently Contribute to Long-term Skin-homing Pool.

Figure 6

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CCR7-BMC mice were immunized epicutaneously with ovalbumin and adjuvant every 10 days, and ear skin was harvested for analysis 4 days after the third challenge. (A) FACs plots (left panels) showing sdLN CD4+/CD44hi populations from WT and KO donors analyzed for expression of E-selectin ligand (E-lig) and CD103. Percentages for each quadrant are shown for one representative CCR7-BMC mouse. Right Panel: Bar graphs (mean ± s.d.) summarize donor data (mean ± s.d.) for each quadrant of an experiment of 7 mice. Calculation of p-values was performed using an unpaired 2-tailed t test, no significant differences were found between the WT and KO populations. (B) From the same experiments presented in (A), total CD4+/CD44hi/E-lig+ (i.e. skin-selective T cells) from sdLN were analyzed for WT and KO donor contribution. WT:KO donor ratios (right panels) were calculated for skin-selective populations and compared to naïve and total Ag-experienced populations as described in Fig 1B. As shown in previous figures, naïve cells were the only population to show a significant effect from CCR7 deficiency.

Imprinting of Ag-specific T Cells with E-lig during the Primary Immune Response

The data shown in Fig 6A derive from mice immunized multiple times with ovalbumin + adjuvant prior to analysis. As we have shown Ag-experienced CCR7−/− T cells to have normal access to skin (Fig 4), it is possible that the observed skin-specific imprinting of CCR7−/− T cells occurred in the Ag-experienced population rather than “conventionally” in the naïve population.

We therefore wished to directly observe the relative efficiency with which WT vs CCR7−/− cells became imprinted during a primary immune response. It was not feasible to track Ag-responsive endogenous naïve T cells after a primary immune response due to the expected small numbers of responding cells. We instead used a TCR-transgenic adoptive-transfer model. CD4 splenocytes from OT-II mice (which express a transgenic TCR for ovalbumin) were transferred into host mice (17, 19). The OT-II donors were bred onto either the WT or the CCR7−/− background.

Tissue-specific imprinting is believed to occur within the nodes that immediately drain the site of inflammation (24, 27). If our experiments were to show that CCR7−/− naïve OT-II cells could be imprinted normally, it would raise the possibility that sdLN are unnecessary for skin-selective imprinting. We therefore included LTα−/− recipients as a control: LTα−/− mice have no detectable peripheral LN, and thus provide a baseline for skin-specific homing without an sdLN contribution.

We topically immunized the ears once with ovalbumin + adjuvant (17, 19). Skin-infiltrating cells were isolated from the inflamed skin five days later, and OT-II cells were enumerated (Fig 7A). As shown previously in this model, accumulation of WT OT-II cells within normal recipients was relatively large [note: only negligible numbers of OT-II cells are found within skin inflamed with adjuvant alone (17, 19)]. Strikingly, very few CCR7−/− cells accumulated within normal ear skin, and very few WT OT-II cells accumulated within LTα−/− ear skin. The functional spleen possessed by LTα−/− mice is therefore not sufficient to replace sdLN for imprinting naïve T cells.

Figure 7. CCR7-mediated Access to sdLNs is Required for Efficient Primary Skin-specific Response.

Figure 7

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Splenocytes from OT-II mice on the WT or CCR7-deficient background (as indicated) were separately transferred into CD45 congenic recipient hosts of either WT or LTα-deficient background. One day after transfer, recipients were immunized epicutaneously with ovalbumin and adjuvant. Five days after immunization, ear skin (A) and sdLN (B) were harvested for analysis of CD4 T cells. (A) Absolute number of skin-infiltrating OT-II cells per ear was calculated as in Fig 4, in which cells from 3 mice were pooled per condition. Bar graph shows mean ± range for two experiments of 3 mice each. (B) Total CD4+/CD44hi/E-lig+ T cell populations from sdLN were analyzed for content of OT-II donor cells. Bar graph shows data from 4 mice in a single experiment as mean ± s.d. (in this case CCR7−/− OT-II cells compared to CCR7+/− littermates). Similar data were obtained from 3 independent experiments using 4 mice each. [Note: no sdLN data available for LTα−/− mice, as these mice do not possess LN].

In similar experiments, we examined OT-II cells within the sdLN of recipient mice (Fig 7B). We found CCR7−/− OT-II cells to be severely impaired in their ability to become imprinted for skin-selective homing. This was most likely due to the paucity of CCR7−/− naïve cells within the sdLN, implying dramatically lower numbers of CCR7−/− OT-II cells available to undergo the imprinting process. [Note: it is not possible to present sdLN data from LTα−/− mice, as they do not possess any LN].

DISCUSSION

We have for the first time assessed the contribution of CCR7 to various T lymphocyte and dendritic cell homing processes in vivo without the confounding variable of T cell development within grossly aberrant lymphoid organs. We find that CCR7 is not involved in the migration of “central” memory CD4 T cells to LN through the HEV, nor is it involved in the exit of lymphocytes from peripheral tissue (i.e. skin).

Thymic Development

Our CCR7-BMC system does not show that developing thymocytes gain any advantage by expressing CCR7. Both WT and CCR7−/− thymocytes were equally represented in the DP and SP populations (Fig. 1C). Although other evidence supports a role for CCR7 (along with CCR9) in seeding the thymus with BM-derived T cell precursors (1, 2, 36, 37), this role appears to be diluted in later developmental stages, perhaps due to prodigious expansion during the DP stage (38). However, the effect of CCR7 deficiency is dramatically different from that of CCR9 deficiency, where similar competitive BMC models showed CCR9−/− cells to be >10-fold less abundant than WT cells at most stages of thymic development (20, 39).

A second role proposed for CCR7 in thymus is the migration of positively selected SP cells from the cortex to the medulla (40, 41). Our findings in the CCR7-BMC are not inconsistent with such a role: CCR7 had a small but significant effect on naïve CD4 and CD8 numbers within the spleen. As homing from blood to spleen does not involve HEV (and thus is not thought to require CCR7 (24, 42)), it is possible that this finding reflects a slightly lower output of CCR7−/− naïve T cells from the thymus.

Homing of Naïve T Cells to LN From Blood

Naïve WT cells had a very strong advantage over naïve CCR7−/− cells in both the CD4 and CD8 LN populations, in some cases reflecting a greater than 20-fold difference (Figs 1&2). Regardless of the reason for slightly fewer CCR7−/− naïve T cells in the spleen, the dramatically larger influence of CCR7 on accumulation of naïve T cells in LN compared to spleen confirms the crucial role of this receptor in homing of naïve T cells to from the blood to LN via the HEV.

Homing of Central Memory Cells to LN from Blood

For the purposes of this study, we operationally defined “central” memory T cells as those capable of entering the LN directly from blood. Although expression of CCR7 was the original marker proposed for central memory T cells (7, 8), we find this receptor to have no demonstrable role in the entry of central memory CD4 T cells into LN from the blood (Fig 2). Our assays require direct competition between WT and CCR7−/− populations for accumulation within specific niches of a given tissue or organ, and thus would allow detection of even a redundant role for CCR7, but this was not seen. Although it has been previously reported that some Ag-experienced T subsets possess alternative, less efficient CCR7-independent mechanisms for homing to LN (43–45), it was unexpected that CCR7 would convey no detectable competitive advantage whatsoever. Thus it appears that CCR7 plays no role in the defining function of central memory T cells.

CD8 central memory T cells, in contrast, were characterized by a significant CCR7 requirement for LN entry from blood (Fig 2). This dependence was somewhat less than that of naïve CD8 T cells, consistent with the notion that CD8 central memory T cells do indeed possess alternative (but less efficient) CCR7-independent mechanisms for LN entry from blood (43–45). It was also noteworthy that Ag-experienced CD8 T cells were more likely to enter LN from the blood than Ag-experienced CD4 T cells (Fig 3). This suggests that the circulating Ag-experienced CD8 T cell population is comprised mostly of central memory cells, whereas the corresponding CD4 population is comprised mostly of effector memory cells. This result fits well with previous work suggesting that effector memory CD8 T cells are localized primarily to non-lymphoid tissue rather than the circulating blood (46).

Entry and Exit of CD4 T Cell Populations To and From Non-Lymphoid Peripheral Tissues

Our finding that WT and CCR7−/− CD4 T cells in our CCR7-BMC were equally represented within both peripheral tissue (skin) populations and Ag-experienced sdLN populations (Figs 1B and 4A) did not fit with the notion that CCR7 is required for T lymphocyte exit from skin (12). Nonetheless, two recent studies suggested that T cells are more abundant within the skin of CCR7−/− mice than WT mice (47, 48). However, these latter two studies do not have the advantage of allowing direct, side-by-side comparison of WT and CCR7−/− cells within the skin of an individual mouse.

Thus, we chose to ask whether WT and CCR7−/− T cells could exit the skin with equal efficiency over time using our competitive BMC system (Fig 4B). Indeed, WT and CCR7−/− cells exited the skin at equal rates. Our data therefore do not support the conclusion that CCR7 confers a competitive advantage in the process by which CD4 T cells emigrate from peripheral tissues. We attempted to repeat a previous study (12) that proposed a key role for CCR7 in this process (Supplemental Fig S3). We modified the short-term competitive homing approach used in Figs 2&3 by injecting the mixed WT and CCR7−/− splenocytes into footpads. This approach was identical to that of the previous study (12), except for our use of CD45 congenic markers (instead of intravital dyes) to identify the donor-derived cell types. Our results were somewhat consistent with those previously reported (12), albeit the difference between WT and CCR7−/− was less pronounced (Supplemental Fig S3). In repeating these experiments, we realized (as did the authors of the initial study) that the number of donor-derived cells recovered from the draining node was exceedingly small, ~3 orders of magnitude less than the input population. This complication yielded considerable variation between experiments. It is not clear to us that the selective process leading to the appearance of this diminutive fraction of the input population in draining nodes is truly representative of the physiological mechanism by which tissue-resident T cells reach sdLN. More recent experiments by Braun et al., in which T cells were directly injected into afferent lymph, demonstrate that CCR7 is not required for T cell entry into the LN parenchyma (9) [note: our assay involves T cells isolated from the LN parenchyma as a whole, and would not detect the likely function of CCR7 in bringing them more deeply into T cell zones (9)]. Taken together, it appears that CCR7 is required neither for exit of T cells from skin nor for their entry into the sdLN.

As our model did not yield data consistent with a role for CCR7 in CD4 T cell egress from skin, we wished to assure ourselves that the well-established role of CCR7 in the migration of dendritic cells (DC) from skin to sdLN (13) was consistent with our model. We found that MHCIIhi/CD11clo “migratory” DC (49, 50), including Langerhans cells, were indeed highly dependent on CCR7 for migration to sdLN (Fig 5).

Development of Tissue-Selective Homing Properties in CCR7−/− T Cell Populations

It was noteworthy that a CCR7-deficient T cell population was present within the skin of CCR7-BMC mice, especially considering competition for the same niches from the WT population. The entry of CD4 T lymphocytes into skin is thought to require an “imprinting” step, in which naïve cells acquire skin-selective homing molecules by encountering skin-derived cognate antigen within sdLN (24, 27). As CCR7−/− naïve T cells have great difficulty entering peripheral LN in this model (Figs 1&2), one would expect these cells to have inefficient access to the imprinting apparatus.

Nonetheless, we found both WT and CCR7−/− cells to express E-lig equally well during anamnestic responses (Fig 6). This finding could bring into question the notion that encountering antigen within sdLN is necessary for skin-selective imprinting. To address this issue, we used the TCR-transgenic OT-II system to examine primary responses to cognate antigen.

After a single topical immunization, relatively large numbers of WT OT-II cells but very few CCR7−/− OT-II cells were found within the inflamed ear skin (Fig 7A). Furthermore, no OT-II cells were detectable within the inflamed skin when normal OT-II cells were transferred into LTα−/− recipients (which lack peripheral LN but possess a functional spleen). Thus, both peripheral lymph nodes and functional CCR7 are required to develop a skin-specific primary immune response.

This requirement for CCR7 in skin-selective imprinting during primary but not recall immune responses raises the possibility of an alternative tissue-selective imprinting mechanism, which does not require the presence of naïve T cells in the sdLN. One possible mechanism would involve the CCR7−/− Ag-experienced T cells present in the sdLN of our CCR7-BMC during the resting state. Perhaps such Ag-experienced cells have the ability to gain skin-selective homing capabilities within this environment, much like normal naïve cells.

Conclusion

We find that multiple effects of CCR7-deficiency previously attributed to the functional role of CCR7 in T lymphocyte trafficking can be traced back to secondary effects from T cell development within the abnormal LN and splenic environments characteristic of mice lacking CCR7 or its ligands. We were able to uncover these new insights by comparing the behavior of WT vs. CCR7−/− cells within mice in which the normal lymphoid environment had been reconstituted.

Supplementary Material

1

ACKNOWLEDGEMENTS

Grant Support: This work was supported by NIH RO1AR052810 (to R.C.F & M.B.) and R21AI092388 & R21AI097468 (to J.J.C.).

We thank Christoph Schlapbach for critical reading of the manuscript.

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