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In inflamed lymph nodes, Ag-specific CD4+ and CD8+ T cells encounter Ag-bearing DCs and, together, this complex enhances the release of CCL3 and CCL4 that facilitate additional interaction with naïve CD8+ T cells. While blocking CCL3 and CCL4 has no effect on primary CD8+ T cell responses, it dramatically impairs the development of memory CD8+ T cell upon Ag re-challenge. Despite the absence of detectable surface CCR5 expression on circulating native CD8+ T cells, these data imply that naïve CD8+ T cells are capable of expressing surface CCR5 prior to cognate antigen-induced TCR signaling in inflamed lymph nodes; however, the molecular mechanisms have not been characterized to date. Here, we show that CCR5, the receptor for CCL3 and CCL4, can be transiently upregulated on a subset of naïve CD8+ T cells and this upregulation is dependent on direct contact with the HEV in inflamed lymph node (LN)3. Binding of CD62L and CD11a on T cells to their ligands CD34 and CD54 on the HEV can be enhanced during inflammation. This enhanced binding and subsequent signaling promote the translocation of CCR5 molecules from intracellular vesicles to the surface of the CD8+ T cell. The upregulation of CCR5 on the surface of the CD8+ T cells increases the number of contacts with antigen-bearing DCs, which ultimately results in increased CD8+ T cell response to Ag re-challenge.
The key to a successful adaptive immune response requires the physical interaction between rare APCs bearing cognate Ag and rare Ag-specific T cells (1 in 104 –106) within the secondary lymphoid organs including the lymph nodes (LN) (1). This interaction not only promotes the initial expansion of Ag-specific T cells but also creates a residual memory T cell population after the primary immune response has subsided. Development of these lymphocytes depends on helper activity provided by other immune cell types and soluble mediators within the inflammatory LN microenvironment. Although help from CD4+ T cell is not an absolute requirement to generate primary CD8+ T cell response, the presence of CD4+ helper T cells enhances the magnitude of CD8+ memory T cell generation (2). We and others have previously shown that the initial surveillance by naïve polyclonal CD8+ T cells of cognate antigens presented by dendritic cells (DCs) is facilitated by the local accumulation of CCL3 (MIP-1α) and CCL4 (MIP-1β), which are released by the complex between activated DCs and other antigen-specific CD4+ and CD8+ T cells (3, 4). This CCL3/CCL4-CCR5 chemokine interaction enhances the recruitment of non-antigen specific CD8+ T cells to the site of activated DCs in the LN, and increases potential antigen recognition by additional CD8+ T cells on DCs. Importantly, neutralizing the effects of CCL3/CCL4 during the early immune priming stage reduces the efficiency of polyclonal CD8+ T cell surveillance in a CCR5-dependent manner, and abrogates the helper-T cell enhanced long-term CD8+ memory T cell generation in vivo (3). The exact molecular mechanisms contributing to the efficacy of CCL3/CCL4-CCR5 interaction on naïve CD8+ T cells with regard to memory T cell generation remains unknown.
The LN is positioned at a location where naïve T cells and Ag-loaded DC encounter each other. Circulating naïve T cells first tether to the LN endothelium through the interaction of CD62L on T cells with Peripheral Node Addressin (PNAd), a shared motif expressed on several proteins including CD34 and Glycam-1 of the high endothelial venule (HEV) (5–10). These tethered T cells then roll on the endothelium, engaging surface CCR7 with CCL21 that is bound to heparan sulfate and collage-IV on the luminal surface of the HEV (11–13). Engagement of both CD62L and CCR7 strengthens T cell adhesion to the HEV. It also results in a conformational change of CD11a on the T cell (14). This conformational change from low- to high-affinity CD11a/CD18 facilitates stronger adhesion through CD54 located on the HEV, thereby promoting trans-endothelial migration of T cells through the HEV (5). Upon entry into the inflamed LN, a subset of naïve CD8+ T cells begin to navigate the complex LN microenvironment, guided by functional CCR5 molecule on the surface, for efficient cell-cell contact with activated DCs. Normally, only a minute number of naïve CD8+ T cells express detectable levels of CCR5 on the cell surface in the blood and LN (3, 4). However, previous published data implicated the importance of CCL3/CCL4-CCR5 chemokine signaling axis during vaccine-induced immune priming in the draining LN (DLN), suggesting that mechanisms exist for the expression and utilization of CCR5 by some naive CD8+ T cells in inflamed LNs that help to guide these cells to sites of activated T cell-DC complexes where high local concentrations of CCL3 and CCL4 exist.
In our present study, we find that a subset of circulating naïve, CCR5− CD8+ T cells up-regulate surface CCR5 protein expression early after entry into the inflamed LN in an antigen non-specific manner. While engagement of increased ligands for CD62L and CD11a on the inflamed HEV promotes adhesion and entry of naïve CD8+ T cells into LN, the same molecular interactions rapidly promote a subset of naïve CD8+ T cells to mobilize pre-formed intracellular CCR5 proteins from intracellular compartments to the cell surface. Furthermore, we found that naïve CCR5+ CD8+ T cell subset developed more robust memory response upon Ag-rechallenge and that this enhancement is associated with increased contact with Ag-bearing DCs in the inflamed LN.
The mice used in this study were purchased from either Taconic, Inc. (Hudson, NY) or Jackson Laboratory (Bar Harbor, ME). We used 8–12 week-old male and female wild-type C57BL/6 mice (B6), C57BL/6 mice congenic for CD45 (CD45.1), OT-I TCR transgenic mice (15) on the Rag2−/− background, OT-II TCR transgenic mice (16), OT-I x CCR5−/ −, MHC-II KO, and β2-microglobulin KO mice. All animals were housed and handled according to NIH institutional guidelines under an approved protocol by CWRU IACUC# 2012-0126.
T cells from donor mice were isolated from LNs and spleen by passing tissue through a 40-μm filter to obtain single cell suspension. This cell suspension was treated with ACK lysis buffer to deplete red blood cells (RBCs). OT-I x CCR5+/+ mice are on a Rag2−/− background, and T cells isolated from these mice were 75–80% CD8+ without additional enrichment steps. CD8+ T cells from OT-I x CCR5−/− and C57BL/6 mice were enriched using negative selection with B220 and CD4 Dynabeads (Life Technologies, Grand Island, NY). OT-II T cells were enriched using negative selection with B220 and CD8 Dynabeads.
Inflamed LN were generated by injecting mice in the right footpad with either 10 μg LPS from Pseudomonas aeruginosa (Sigma-Aldrich, St. Louis, MO) or 20 μg CpG ODN (Klineman #1466: TCAACGTTGA; Klineman #1555: GCTAGACGTTAGGT; Life Technologies) admixed with Alum. PBS or PBS/Alum was injected into contralateral footpad as controls for LPS/Alum or CpG/Alum, respectively. For in vivo activation, 1x107 polyclonal T cells from C57BL/6 or monoclonal T cells from OTI x Rag2−/− mice were injected intravenously into recipient mice 24 or 48 h after footpad injections of LPS/Alum, CpG/Alum, or PBS/Alum. At different times both right (DLN) and left (non-draining LN; NDLN) popliteal LNs were harvested, single cell suspensions were stained with antibodies to CD8, CD45.2, and CCR5. For in vitro activation, both DLN and NDLN were removed 24 or 48 h after footpad injections and placed into 96-well V-bottom plates with 100 μl of RPMI-0.5% FBS to cover samples. For some experiments axial LN were also collected as additional NDLN. T cells were harvested from congenic mice as described above. One million T cells were added directly to the LN, which had been teased open with tweezers. To examine soluble effects of inflamed LN, the T cells (1x106 T cells) were added to the top well of a trans-well plate with 0.4μm diameter pores (Corning; Tewksbury, MA). In addition, T cells (1x106 T cells) were added to a 96 well plate with conditioned media that was collected from inflamed LN after 24 h in culture with serum-free media. For Ab-coated beads, 50 μl of sheep-anti-Rat IgG Dynabeads were suspended in 450 μl of FACS buffer and 1 μg of Ab (eBioscience Rat IgG; CD62L: clone Mel-14; CD11a: clone 2D7) for 30–90 min at 4°C. Five μg of Rat IgG was added for an additional 30 min at 4°C. Beads were re-suspended to final volume of 500 μl FACS buffer and aliquots (10, 30, or 100 μl) were added to 2 x 106 OTI T cells re-suspended in 500 μl FACS buffer, mixed at 4°C and transferred to 37°C for different times. For re-stimulation experiments, 2 x 106 cells were incubated with 100 μl anti-CD11a coated beads for 20 min at 37°C and then placed on magnets to remove non-adherent cells. After washing, cells were flushed off beads and incubated with biotinylated anti-CCR5 antibody with anti-biotin microbeads (Miltenyi) to remove CCR5+ cells. 2 x 106 CCR5− cells were then incubated again with 100 μl anti-CD11a coated-beads for 20 min at 37°C, and then examined for CCR5 expression by flow cytometry. To determine role of protein synthesis or transport in CCR5 expression, OT-I T cells were cultured with specific inhibitors of protein synthesis and transport for 2 h and 100 μl beads were added for 30 min at 4°C, transferred to 37°C for 20 min, washed and stained for expression of CCR5. To ensure inhibitors were capable of blocking protein synthesis or transport, OT-I T cells were cultured with inhibitors for 2 h and then placed in anti-CD3/CD28 coated wells for 24 h and examined for surface CD69 expression by flow cytometry. To measure memory responses, OTI T cells were cultured with inflamed LN for 5 h, homogenized and then sorted into CD45.2+CD8+CCR5+ or CD45.2+CD8+CCR5− populations using FACSAria (BD). One hundred thousand sorted T cells were then injected into naïve CD45.1+ mice along with 3 x 106 OTII T cells. The following day, mice were immunized with alum mixed with OVA257–264 (1 μg), and CpG (20 μg) in the presence or absence of OVA323–339 (10 μg). After 7 or 28 days, mice were sacrificed and spleen and DLN collected and restimulated with OVA257–264 (1 μg) for 4 h at 37°C in the presence of Brefeldin A. The total number of OTI T cells and the number of IFNγ producing cells from both spleen and LN were then enumerated based on FACS analysis.
The antibodies were purchased from eBioscience, BD Pharmingen, or BioLegend and are as follows: rat anti-mouse CD8α (clone 53.6.7), rat anti-mouse CD4 (clone GK1.5), biotinylated anti-mouse CCR5 (clone HM-CCR5), anti-mouse CD3e (clone 145.2C11), anti-mouse CD45.2 (clone A20), rat IgG2b, rat IgG2c, and Hamster IgG-biotin isotype control. Additional CCR5 antibody, MC-68, was a generous gift from Dr. Matthias Mack (17). Between 2 x 105 and 1x106 cells were washed in ice-cold FACS buffer (PBS/2.5mM EDTA/0.1% BSA), followed by incubation in blocking buffer (10% normal mouse serum in FACS buffer) for 15 min at 4°C. Antibodies were added 30 minutes on ice, except for anti-CCR5 antibody, which was incubated for 1 h on ice. The samples were then washed twice with ice-cold FACS buffer and the samples were run on Accuri C6 or FACScalibur flow cytometer. Fold increase of CCR5 expression was determined by using the following formula: [(%CCR5+ experiment – %CCR5+ isotype control)/(%CCR5+ control – %CCR5+ isotype control)]. To detect IFNγ-producing T cells, after stimulation with anti-CD3, T cells were first stained with anti-CD8 and CD45.2 prior to fixing with CytoFix/Perm (BD). Cells were then washed with Perm Wash buffer (BD) and then stained with anti-IFNγ Ab (clone XMG1.2). The data were then analyzed using FlowJo software (FlowJo, Inc).
To determine expression of CD54 and PNAd in LN, DLN or NDLN were isolated from mice 48 h after footpad injection of CpG/Alum or PBS/Alum, fixed in PLP buffer (periodate-lysine-paraformaldehyde) overnight, transferred to 30% sucrose for at least 6 h, frozen at −80°C in OCT, and then sectioned into 5 μm size slices and transferred to microscope slide. Slides were treated with Ag retrieval buffer, prior to being blocked with mouse serum, stained against PNAd (eBioscience Clone MECA-79) Ab and hamster anti-CD54 (Abcam ab171118; Cambridge, MA) using Pelco BioWave Pro (Redding, CA), washed and stained with secondary FITC Mouse anti-Rat IgG Ab (eBioscience) and AF-594 goat anti-hamster IgG (Life Technologies). Slides were examined on Leica SP5 confocal microscope (Leica Microsystems Inc, Buffalo Grove IL), and images were analyzed on LAS software using same settings (Leica Microsystems Inc). Regions of interest (ROIs) were drawn around PNAd+ regions and the relative intensities were determined for both the PNAd+ and CD54+ channels. Background intensities were determined by selecting ROIs in unstained areas and subtracting these values from both the PNAd and CD54 channels. The relative expression of CD54 in DLN and NDLN was determined using the formula: (Average intensity CD54 – background intensity) / Average intensity PNAd – background average intensity). For examining CCR5 localization, freshly isolated OTI T cells or cells isolated from DLN and NDLN were added on top of poly-lysine coated coverslips, spun down at low speed (600 rpm) for 30 sec, washed, and then fixed in 2% paraformaldehyde. Cells were then incubated with FACS buffer containing 0.05% Tween 20 for 20 min at room temperature to permeabilize cells. Cells were stained with anti-CD8-FITC, CD45.2-PerCp-Cy5.5, and anti-CCR5-biotin for 30 min at room temperature; washed 3X, and then incubated with SA-AF594 (Invitrogen) for an additional 30 min at room temperature. Slides were washed and air dried for 10 min before adding DAPI Fluoromount-G (Southern Biotech, Birmingham AL) followed by placement of a coverslip and sealing with nail polish. Images were collected on a Leica SP5 confocal microscope and CD45.2+ cells were selected and examined for expression of CD8 and CCR5. The amount of CCR5 that co-localized with CD8 was measured using Imaris software (Bitplane, Inc.). To measure DC-T cell interaction frequency, 1 x 106 BM-derived DCs were stained with Cell-tracker blue (10 uM), pulsed with 1 μg/ml OVA257–264 and 10 μg/ml LPS for 1 h at 37°C, washed, and then injected into the right footpad of C57BL/6 mice. Twenty-four hours later equal number of 5–10 x 106 SNARF-labeled OT-I+ T cells and CFSE-labeled CCR5 KO OT-I+ T cells were co-injected via tail vein. 5 h later draining and non-draining LN were removed, fixed in formalin O/N and then incubated in 30% sucrose O/N. LNs where then sliced into 5 μm sections, and examined via two-photon microscopy. The number of DC-T cell contacts per slide was determined using IMARIS software and averaged over multiple imaging fields in multiple LN samples.
Total RNA was isolated using TRIzol reagent per manufacturer’s protocol (GibcoBRL, Carlsbad, CA), and purified using RNeasy mini-kit (Qiagen). RNA quality was assessed by spectrophotometer absorption at 260/280 nm. RNA was converted to cDNA using 200 U of Moloney-murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Grand Island, NY) for 60 min at 37°C in the presence of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCL2, 10 mM DTT, 100 ng of Oligo dT 12–18, 0.5 mM dNTPs, and 40 U of recombinant RNase inhibitor. cDNA was amplified in the presence of FAM-labeled gene-specific primers and TaqMan universal master mix (AB Applied Biosystems, Foster City, CA) in a 96-well microtiter plate format on an ABI PRISM 7300 Sequence Detection System (AB Applied Biosystems). Each PCR reaction were performed in triplicate using the following conditions: 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Relative levels of RNA were determined using cycle threshold (Ct) (18). Ct values from target gene were subtracted by Ct value of GAPDH to determine relative expression (ΔCt = Ct target – Ct GAPDH). The concentration of gene-specific mRNA in samples were compared to untreated LN and was calculated by subtracting normalized values from treated groups from untreated LN (ΔΔCt = ΔCt test – ΔCt control) then determining relative concentration (2−ΔΔCt). For Micro Array analysis, naïve OT1 T cells were exposed to CpG-inflamed CD45.1 LNs for 6 h at 37°C, stained for surface expression of CCR5 and sorted into CCR5+ and CCR5− populations. Total RNA was isolated from both populations and subjected to Affymetrix GeneChip Mouse Gene 1.0 ST Array analysis to identify differentially expressed gene transcripts as described (Affymetrix Santa Clara, CA). Heat maps were generated and examined using Robust Multichip Analysis (RMA) Algorithm to determine the criteria of significance (FC<=−1.5 or >=1.5). All microarray data have been deposited in the GEO database (www.ncbi.nlm.nih.gov/geo) with accession number, GSE76820.
All error bars represent standard deviation from the mean and the statistical analysis was performed using either student-t test or one-way ANOVA with InStat Software (Graph Pad).
While expression of CCR5 is classically associated with post-TCR activated T cells and considered absent on the surface of naïve T cells under steady state (19), available data implicate an important functional role for CCR5 on a small subset of naïve T cells during the initiation of inflammation-mediated primary immune responses in inflamed LNs (3, 4). To examine if the observed functional role for CCR5 is the result of a small number of previously activated T cells or an upregulation of surface CCR5 molecule on recent T cell immigrants in LN, naïve polyclonal T cells (CD44loCD62LhiCCR5−) from C57BL/6 (CD45.2+) mice were injected via tail vein into congenic (CD45.1+) recipients that were induced to undergo regional tissue and draining popliteal LN (DLN) inflammation upon injection of CpG/Alum in the right rear footpad 48 h prior to T cell inoculation. As controls, the contralateral footpad received either PBS/Alum injection or sham injection, thereby creating NDLN in the contralateral popliteal LN. At various times after adoptive T cell transfers, both DLN and NDLN were removed and isolated cells were stained for CD45.2, CD8, and CCR5. While there was some modest increase of surface CCR5 expression on transferred naïve T cells isolated from NDLN 4 hours after adaptive transfer, we observed >10-fold increase in CCR5 expression on the same transferred T cells in DLN (Figure 1A). Multiple experiments using either polyclonal C57BL/6 CD8+ T cells or monoclonal OTI CD8+ T cells showed similar results, although there was a wide range (~5–45 fold increase) in the absolute magnitude of CCR5 induction (Figure 1B). Similar results of CCR5+ T cell accumulation among transferred naïve CD8+ T cells in DLN were also seen using either LPS/Alum (Supplementary Figure 1) or LPS-treated BM-derived DCs (data not shown). Although to a lesser magnitude, there is also an enhanced accumulation of endogenous CCR5+ CD45.1+ CD8+ T cells in DLN (Supplementary Figure 2). Interestingly, we found that the surface expression of CCR5 was transitory, as it peaked between 2–4 h in DLN, began to decrease by 6 h (Figure 1C), and returned to basal expression level by 24 h (data not shown). These data suggest an accumulation of naive CCR5+ CD8+ T cells in the DLN; however, it does not discriminate whether this observation was the result of preferential in vivo recruitment and retention of contaminating CCR5+ donor memory T cells from the circulation or the induction of CCR5+ T cells from the CCR5− naïve donor T cell pool. The former scenario is unlikely due to several reasons. First, the total number of CCR5+CD45.2+CD8+ T cells in DLN far exceeds the total number of CCR5+ cells in the initial pool of transferred T cells. Second, the transfer of OT-I T cells isolated from Rag2−/− mice, which lacks memory T cells (20), resulted in the comparable appearance of CCR5+CD8+ T cell accumulation in DLN (Figure 1B). To provide further evidence for the direct conversion of CCR5−CD8+ T cells into CCR5+CD8+ T cells in the DLN, as well as to determine if such conversion is dependent on either direct cell-cell contact or mediated by some soluble factor from inflamed LN, DLN and NDLNs were removed from CD45.1 congenic mice 48 h after injection of CpG/Alum and teased apart in a 96-well plate. Naïve CCR5−CD45.2+CD8+ T cells were added either directly to the LNs ex vivo, or placed on top of a trans-well with small pore (0.4 μm) to prevent direct contact with LN but allow for soluble factors to pass through. This ex vivo experimental set up also prevented the potential ongoing recruitment of circulating CCR5+CD8+ memory T cells as a cause of the observed accumulation in vivo. Four hours following incubation, CD45.2+CD8+ T cells were stained for surface CCR5 expression. Interestingly, only direct contact between the DLN tissue and T cell resulted in greatest upregulation of surface CCR5 expression within CD45.2+CD8+ T cell population (Figure 1D), while the same T cells failed to upregulate CCR5 upon direct contact with NDLN ex vivo or following co-incubation with DLN through a trans-well. To interrogate whether this direct DLN contact-mediated CCR5 upregulation was dependent on MHC expression, naïve CCR5−CD45.1+CD8+ T cells were injected into C57BL/6 mice, MHC-II−/−, or β2-microglobulin−/− mice. We found that expression of either MHC-I or MHC-II was not necessary for the rapid induction of surface CCR5 on CD8+ T cells (Figure 1E).
To further interrogate the cellular mechanisms responsible for this transient surface CCR5 expression in vivo, we injected increasing number of OT-I T cells in vivo to see whether the CCR5− → CCR5+ conversion is a saturable and limiting event. When LNs were analyzed 4 h after adoptive transfer of CCR5− OT-I T cells, we found that the percentage of CCR5+ T cells decreased dramatically in the DLN with increasing number of T cell inoculum (Figure 1F), further suggesting that CCR5 expression was not caused by selective retention of contaminating CCR5+ donor T cells since we would expect the percentage of CCR5+ T cells to either remain the same or increase with increasing number of injected T cells. Instead, we observed that equivalent total numbers of CCR5+ donor T cells were found in DLN regardless of the sizes of the T cells inocula (Figure 1G). These data suggest that there were limited sites within the inflamed LN that were capable of inducing CCR5 expression in newly arrived T cells within the 4 hours following the adoptive T cell transfer.
To identify factor(s) that may influence surface CCR5 expression in newly arrived naïve CD8+ T cells in inflamed LN, we performed qPCR analysis of selected target proteins that are important for initial T cell binding to the HEV within the inflamed DLN. Due to the huge influx of immune cells into LN during inflammation, we compared the gene expressions of our targeted genes with the expression of CD31 mRNA as a surrogate marker for endothelial cells. We examined CD34 and Glycam1, two competing molecules that possess the same PNAd motif important in binding to CD62L on incoming naïve T cells. We found that while DLN exhibited a slight reduction in CD34 mRNA 48h after CpG/Alum stimulation, the level of mRNA for Glycam1 was significantly reduced in DLN relative to steady state LN as compare to NDLN (100-fold reduction versus 2–3 fold reduction; Figure 2A). As Glycam1 exists as a soluble molecule that may inhibit T cell interaction with the HEV, a reduction in Glycam1 may help facilitate binding of circulating T cells to HEV (8–10). On the other hand, the mRNA for CD54, which binds to activated CD11a/CD18 complexes on T cells and provides the final step for T cell binding to the HEV, was significantly increased in the DLN 48 h after CpG/Alum injection (Figure 2A). Together, the molecular changes in the HEV during inflammation favors homing of circulating T cells to the DLN.
Next, we examined tissue sections of steady state LN as well as both DLN and NDLN for the expression of PNAd and CD54. While we observed a reduction in mRNA for Glycam-1 and slight reduction in CD34 mRNA expression (Figure 2A), we did not see a change in the overall PNAd expression in DLN HEV (Figure 2B). The PNAd expression within the LN is reflective of CD34 expression, as Glycam1 exist as a soluble form of PNAd and decreases during inflammation (21). We also observed an increase in CD54 staining along the HEV, in agreement with the increased mRNA abundance (Figure 2A). We found that majority of CD54 found in the DLN was associated with PNAd+ regions (Figure 2B). When we analyzed regions of interests (ROI) around PNAd+ areas, we observed a two-fold increase in CD54 protein relative to PNAd in DLN as compared to that in NDLN (Figure 2C).
Next, we examined whether these HEV-associated ligands may be responsible for the rapid surface CCR5 expression on incoming naïve CCR5− CD8+ T cells. To test if interactions with CD62L or CD11a on T cells can promote CCR5 expression, we cultured OT-I T cells with Ab-coated beads to cross-link either CD62L or CD11a to induce surface CCR5 expression as determined by flow cytometry. We found that both anti-CD62L and anti–CD11a antibody-coated beads could induce CCR5 expression within the first 10 minutes after cross-linking (Figure 2D). As ligation of CD62L promotes conformational change of CD11a/CD18 which allows for binding to and signaling through CD54, either direct activation through CD11a or cross-linking of CD62L is sufficient to induce surface CCR5 expression on naïve CCR5−CD8+ T cells in vitro. This induction occurred rapidly and was dependent on the degree of stimulation (Figure 2E). Not all naïve CCR5− were capable of expressing CCR5, as only a subset of cells that failed to express CCR5 after primary exposure to anti-CD11a coated beads could upregulate CCR5 after a secondary exposure to anti-CD11a coated beads (Figure 2F).
CCR5 protein has been shown to exist in pre-formed intracellular vesicles in human T cells (22, 23). Therefore, we examined naïve OT-I T cells for the presence of intracellular CCR5 protein using confocal microscopy. We injected naïve surface CCR5−OT-I T cells into mice that received footpad injection of CpG/Alum 48 h prior, and isolated NDLN and DLN 3 h later to detect CCR5 protein expression. Immunofluorescence showed that CCR5 could be found in vesicles below the surface of a subset of unstimulated naïve OT-I T cells (Figure 3A), with a baseline of ~15% CCR5 signal co-localized with CD8 expression (Figure 3B). Similarly, newly arrived transferred OT-I cells in NDLN also exhibited ~10% co-localization of CD8 and CCR5 proteins. The extent of CCR5 and CD8 co-localization was significantly increased to 30% in OT-I cells isolated from DLN (Figures 3A, B). By fixing and permeabilizing OT-I T cells, we were able to detect intracellular CCR5 protein expression in a small number of T cells as determined by flow cytometry (Figure 3C). To interrogate whether increased surface CCR5 expression was caused by the mobilization of pre-existing protein pool or through de novo synthesis, OT-I cells were incubate in the presence of inhibitors that block synthesis or transport of protein prior to incubation with anti-CD11a coated beads (Figures 3D, E). We found that surface CCR5 was still induced by CD11a cross-linking in the presence of either actinomycin D or cyclohexamide, albeit at reduced levels. The addition of Brefeldin A or monensin, inhibitors of transport within the trans-Golgi network, similarly caused only a slight reduction in CCR5 expression after CD11a stimulation. We found that all treatments failed to completely eliminate the appearance of surface CCR5, supporting the notion that CCR5 was being transported to surface, at least in part, via pre-existing vesicles and does not require de novo synthesis. This is in contrast to CD8+ T cells stimulated with anti-CD3/CD28, where these same inhibitors significantly reduced expression of the activation marker CD69 (supplemental Figure 3).
We have previously identified that the chemokines CCL3 and CCL4, which were produced in high concentrations locally at sites of activated DCs - CD4+ T cell interactions in the draining lymph node, were essential for the enhancement for the magnitude of helper T cell-dependent memory CD8+ T cell responses (3). To test if the ability of naïve T cells to rapidly express surface CCR5 in DLN can modulate the magnitude of antigen-specific memory CD8+ T cell response, naïve CCR5− OT-I T cells were added directly to exposed CpG/Alum inflamed CD45.1+ LN in vitro for 4 h prior to sorting the T cells into CD45.2+CD8+CCR5+ or CD45.2+CD8+CCR5− fractions (Figure 4A). 1 x 105 OT-I T cells from each cohort were injected along with 3 x 106 naïve OT-II T cells into separate recipients (Figure 4A). Twenty-four hours later mice were immunized with OVA257–264 and CpG/Alum in the right footpad with or without OVA323–339 to provide CD4+ T cell help. At 7 and 28 days post immunization, mice were sacrificed, spleens and LN were removed and the cells were re-stimulated with OVA257–264 for 4 h in the presence of Brefeldin A. The total number of OT-I T cells and the number of IFNγ–producing OT-I cells in both the spleen and LN were enumerated. While we did not find significant differences in the total number of OT-I cells (Figure 4B) or in the number of IFNγ-producing OT-I cells in the LN on day 7 (Figure 4C), we observed significantly elevated total numbers of OT-I T cells on day 28 in the spleen and LN of mice that received CCR5+ sorted OT-I T cells when compared to those that received CCR5− sorted OT-I T cells (Figure 4D). Importantly, more IFNγ-producing OT-I T cells were found in mice that received the naïve CCR5+ OT-I cohort (Figure 4E). The level of enhancement in memory OT-I cell number and functional recall in the CCR5+ sorted OT-I cohort was the same or slightly higher than bulk, un-sorted naïve OT-I transfer. Importantly, the magnitude of the total number of OT-I T cells and IFNγ-producing OT-I T cells isolated from mice that received CCR5− sorted OT-I T cells was similar to that found in mice receiving naïve OT-I cells with homozygous germ-line CCR5KO. This supports our earlier work that showed CCL3 and CCL4 signaling through naïve CCR5+ CD8+ T cells during the very early phase of a primary immune response is critical in augmenting the magnitude of the late CD8+ memory T cell recall response (3, 4). Naïve CCR5+ OT-I T cell fraction did not exhibit increased CD69 or CD44 expression following in vitro exposure to inflamed LN prior to sorting, indicating that OT-I T cells in the CCR5+ sorted population was not otherwise activated prior to transfer (Supplemental Figure 4A, 4B). In further support of this, we used gene array analysis to examine expression of genes associated with development of memory phenotype in freshly isolated cells prior to adoptive transfer in vivo. We found that there was no significant difference in expression of genes in the sorted CCR5− or CCR5+ T cells (Supplemental Figure 4C, 4D), again confirming that CCR5 expression alone does not confer a memory phenotype and that our CCR5+ fraction did not contain contaminating endogenous memory T cells.
To gain additional insight into how early CCR5 expression on naïve CD8+ T cells affect access to potential antigenic stimulation by LN DCs, we transferred differentially fluorescent labeled, naive WT and CCR5 KO OT-I T cells into mice that were foot-pad injected 24 hours prior with fluorescent labeled, LPS and OVA257–264 pulsed bone marrow-derived DCs. Seven hours later the DLNs were harvested and examined by static two-photon microscopy (Figure 4F). The numbers of CD8+ T cell-DC contacts were determined for multiple LN samples (Figure 4G). As expected, significantly more WT OT-I T cells were able to gain access and form contacts with antigen-bearing DCs as compared with co-transferred naïve CCR5 KO OT-I T cells, suggesting a distinct advantage for the CCR5+ fraction of the naïve T cells to have early and ready access for priming by newly arrived antigen-bearing DCs from the periphery.
Under steady state, CD4+ T cells and CD8+ T cells exhibit distinct LN surveillance behavior, with CD8+ T cells traversing the LN almost twice as long as that of CD4+ T cells (~18–21 hrs versus ~12 hrs, respectively) (24). During their LN transit, CD8+ T cells were estimated to interact with an average of ~313 APCs compared to ~160–200 APCs contacts made by CD4+ T cells (24). In generating a primary immune response, therefore, the adaptive immune system must rely on additional biochemical or physical cues to facilitate the necessary physical contacts between the rare antigen-bearing APC and the even rarer antigen-specific T cells in order to combat the offending agent in a timely manner. We have previously identified that inflammatory chemokines, CCL3 and CCL4, are central in enhancing this antigen surveillance by naïve polyclonal CD8+ T cells to sites of productive CD4+ T cell - DC interactions during the early LN priming phase (3). The ability of these naïve T cells to utilize the CCL3/CCL4 - CCR5 chemokine axis correlates with the magnitude of helper CD4+ T cell-enhanced memory CD8+ T cell generation. Hugues and colleagues subsequently confirmed this critical chemokine signaling axis by demonstrating that productive antigen-specific CD8+ T cell – DC interaction also enhances polyclonal CD8+ T cell surveillance of the antigen-bearing DCs, again in a CCL3/CCL4-CCR5 dependent manner (4). These data imply that, although not expressed on the surface of naïve T cells in circulation or under non-inflamed states, CCR5 is functional in at least a fraction of naïve CD8+ T cells in the inflamed LN (13). Here, we presented molecular mechanisms that shed new insights into how naïve CD8+ T cells utilize CCR5 to accomplish this feat in vivo and in vitro.
We found that a fraction of naïve cells among both polyclonal and TCR transgenic CD8+ T cells could be induced rapidly to express CCR5 on the cell surface after entering an inflamed LN that is induced by TLR agonists (CpG and LPS; Figure 1 and Supplementary Figure 1) or TLR-activated DC vaccines (data not shown). This expression was transient and reached a maximum density between 2–4 h upon T cell arrival in the inflamed LN before returning to basal levels by 24 h (Figure 1C and data not shown). This rapid induction of surface CCR5 expression does not depend on TCR engagement with either cognate / MHC or self-peptide / MHC complexes, as similar magnitude of CCR5 up-regulation was observed in MHC class I-deficient host as compared with wild type or MHC class II-deficient hosts (Figure 1E). Neither does this process require nascent CCR5 gene transcription or protein synthesis, as cyclophosphamide and actinomycin treatments failed to completely inhibit CCR5 proteins expression on naïve CD8+ T cell surface (Figure 3D). These observations agree well with previously published intravital imaging reports in which the chemotactic behavior of naïve CD8+ T cell towards the CCL3/CCL4 gradient was evident in a small fraction (<5%) of recently arrived naïve CD8+ T cells in LNs that were undergoing productive cognate antigen-mediated DC-CD4+ T cell or DC-CD8+ T cell interactions (3, 4). As the duration of surface CCR5 expression lasts only for a few hours on naïve CD8+ T cells (Figure 1C), and as ligand-mediated receptor downregulation further reduces CCR5 expression in the absence of cognate peptide / MHC – TCR engagement (25), the relative proportion of naïve CD8+ T cells that are capable of responding to local CCL3/CCL4 gradient would be expected to diminish over time. Furthermore, during inflammation, HEVs undergoes structural changes that favor increased recruitment of T cells from the circulation (Figure 2B). As CD8+ T cells have prolonged intranodal transit time (~18–21 hrs) as compared with that of CD4+ T cells (~12 hrs), these data suggest that, within the available CD8+ T cell pool, the fraction of naïve CD8+ T cells capable of responding to CCR5 ligands would decrease as LN inflammation ensues over time. These data also predict that the highest impact of CCL3/CCL4-CCR5 signaling on the intranodal naïve CD8+ T cell fate resides in the early phases of an inflammation-induced primary LN response.
We found similar maximum numbers of donor CD44loCD62hiCCR5+ CD8+ T cells that were acutely converted from a CCR5− state in the DLN irrespective of the sizes of transferred T cell inocula (Figure 1G), suggesting that CCR5 expression is not intrinsically uniform in every naïve CD8+ T cell arriving in the LN from the general circulation, but is rather promoted on specific subset of CD8+ T cells that have access to limited interaction sites within the LN. Our in vitro studies show a requirement for direct contact with the internal contents of an inflamed LN, further supporting the idea that CCR5 is being induced in CCR5− naïve CD8+ T cells in anatomically distinct sites within the LN (Figure 1D). These intriguing data, therefore, begs the question: What is the unique feature of the inflamed LN environment that could promote CCR5 expression in naïve CD8+ T cells in the absence of cognate antigen/MHC – TCR engagement?
Three sequential steps are necessary for T cell entry into LN through the HEV. The first step involves the rolling and tethering of the T cell via CD62L interaction with a group of sialomucins on the HEV that contains the PNAd motif. The PNAd motif is required for binding of L-selectin and is expressed in a few proteins including Glycam-1, CD34, Sgp200 and on a subset of MAdCAM-1 (10). The second step is ligation of chemokine receptor CCR7 on T cells with luminal CCL21 on HEV. While both CCL19 and CCL21 bind to CCR7, only CCL21 contains a hydrophobic region that forms complexes with heparan sulfate, gp38 and collagen IV on the surface of the HEV to enhance rolling of T cells (11, 13). Binding of both CD62L and CCR7 on T cells is important, since inhibition of either pathway greatly reduces T cell adhesion and trans-endothelial migration through the HEV (26–28). While interaction between CCR7 and CCL21 results in a strong interaction between T cell and HEV, signals from both CD62L and CCR7 are capable of promoting a conformation change of CD11a/CD18 on the T cell through inside-out signaling (29). This conformational change of CD11a/CD18 allows for the third step to take place, which is to allow a strong binding of T cells to CD54 on the HEV. This final step results in outside-in signaling in T cells, allowing for transmigration of T cells into the LN through the HEV (30, 31).
While CD34 mRNA expression did not change significantly in the inflamed LN, Glycam-1 dramatically decreased by over 100 fold at 48h after activation of inflammation. Both CD34 and Glycam-1 contain the PNAd motif; however, Glycam1 is secreted and has been postulated to inhibit LN homing of T cell by competing with CD34 for binding to CD62L in the HEV (8–10). Elevated plasma levels of Glycam-1 have been observed shortly after inflammation induction, and the Glycam-1 level decreases after 12 h (21). This suggests that the loss of soluble Glycam-1 production during inflammation enhances effective CD62L-mediated homing of circulating naïve T cells through binding to CD34 on the HEV. Concurrently, CD54 protein expression dramatically increased in PNAd+ regions of the HEV 48 h after stimulation, which further enhances homing of circulating naïve CD8+ T cells to the inflamed LN. LN inflammation induced by either CpG ODN or LPS can promote surface CCR5 expression in naïve CD8+ T cells. These PAMPs are known to promote the release of inflammatory cytokines such as TNFα and IL6, and could upregulate CD40 expression in DCs (32). IL-6 has also been shown increase CD54 expression in PNAd+ areas in the HEV (33, 34). Together, the molecular changes within the PNAd+ regions of the HEV result in preferential T cell recruitment to the inflamed LN and enhance CCR5 expression on T cells that arrive in such a LN.
Engagement of TCR and CD11a/CD18 results in the binding of T cells to target cells as well as the polarization and release of lytic granules and cytokine-containing granules (35–38). In this context, CD11a/CD18 plays an important role in directing the granules towards the target cell (39). While wild type OT-I T cells transferred into CD54−/− mice exhibit normal activation, proliferation and effector function acquisition upon primary Ag stimulation, they are unable to respond to Ag re-challenge (40). Using bone marrow chimera approaches, it has been shown that memory CD8+ T cells had reduced ability to respond to secondary Ag challenge in mice lacking CD54 in non-hematopoietic cells (41). These data further support our current observation that CD54 expression in the HEV contributes to memory CD8+ T cell development.
Chemokine receptors are membrane-bound molecules composed of 7 trans-membrane spanning domains, which are coupled to G-proteins (12). These receptors bind to specific chemokines and promote cellular migration by chemotaxis. While G-protein-coupled receptors (GPCRs) are mostly exported directly to the plasma membrane, their intracellular trafficking pathway may be modulated by specific proteins (42). In particular, CCR5 has been shown to reside in intracellular vesicles associated with the CD4 molecule in primary human T lymphocytes (22, 23). Ligation of CD62L either by antibody cross-linking or by using the CD62L ligands (fucoidan or sulfatide) can promote translocation of intracellular CXCR4 to the cell membrane surface in human T cells presumably from intracellular stores (43). In our current study, we observed surface CCR5 expression in mouse CD8+ T cells in as little as 10 min after cross-linking with antibody against either CD62L or CD11a. The exact molecular signaling responsible for translocation of intracellular CCR5 to the cell membrane remains to be studied.
While some naïve polyclonal CD8+ T cells express surface CCR5 in inflamed LN, not all naïve T cells do. This holds true for naïve CD8+ T cells from either wild type animals or TCR transgenic animals on a Rag2−/− background. Most strikingly, exposure of this naïve CCR5+ T cell cohort to cognate Ag was able to recapitulate the same magnitude of helper T-cell enhanced memory CD8+ T cell generation, whereas exposure of the CCR5− T cell cohort to the same cognate Ag produced long term memory CD8+ T cells to the same extent as observed in germ line CCR5-deficient CD8+ T cells (Figure 4). An insight into this phenomenon came from the observation that the CCR5− T cell cohort has a reduced ability to be induced into CCR5+ CD8+ T cells upon additional inflammatory exposure (Figure 2F). These data argue for differential epigenetic or metabolic regulation of thymic-derived CD8+ T cells in deciding which T cell subpopulations are better endowed to be precursor memory T cells. It also supports the notion that the presence of CCR5 alone is not associated with constitutive expression of genes required for the development of memory CD8+ T cells (Supplementary Figure 4). In this context, CCR5 serves as a target for cell entry by Human Immunodeficiency Virus (HIV). Based on our current report, we speculate that the early CCR5+ converters among naïve CD8+ T cells (hence the robust memory forming subpopulation) in inflamed LN may be targeted by the virus for elimination during early HIV infection, leading to the observed clinical development of robust anti-HIV effector T cells with subsequent collapse of memory T cell pool (44–47). More studies are needed to understand the genetic and epigenetic regulation of the early CCR5+ converters among the naïve T cell pool in order to fully understand the intriguing observation in the current study.
The authors wish to thank Dr. Matthias Mack for providing the anti-CCR5 antibody, MC-68.
1This work was supported by the following funding sources: NIH CA154656 (A.Y.H.), NIH EB007509 (D.S.B.), NIH GM007250 (R.D.D.), the Wolstein Research Scholarship (R.D.D.), the Steven G. AYA Cancer Research Foundation (D.A.), the Hyundai “Hope-on-Wheels” Program (A.Y.H.), the Samuel Szabo Foundation (A.Y.H.), the Marc Joseph Fund (A.Y.H.), the Pediatric Cancer Research Foundation (A.Y.H.) and the Theresia G. and Stuart F. Family Foundation (A.Y.H.).
3Abbreviations used in this article: PNAd, Peripheral Node Addressin; HEV, high endothelial venule; LN, Lymph Node; DLN, draining LN; NDLN, non-draining LN; ROI, region of interest;