The beta2 integrin CD11c distinguishes a subset of cytotoxic pulmonary T cells with potent antiviral effects in vitro and in vivo
© Beyer et al. 2005
Received: 14 September 2004
Accepted: 12 July 2005
Published: 12 July 2005
The integrin CD11c is known as a marker for dendritic cells and has recently been described on T cells following lymphotropic choriomeningitis virus infection, a systemic infection affecting a multitude of organs. Here, we characterise CD11c bearing T cells in a murine model of localised pulmonary infection with respiratory syncytial virus (RSV).
Mice were infected intranasally with RSV and expression of β2 integrins and T lymphocyte activation markers were monitored by flow cytometry. On day 8 post RSV infection CD11c+ CD8+ and CD11c- CD8+ T cells were assessed for cytokine production, cytotoxic activity and migration. Expression of CD11c mRNA in CD8+ T cells was assessed by quantitative PCR.
Following RSV infection CD11c+ CD8+ T cells were detectable in the lung from day 4 onwards and accounted for 45.9 ± 4.8% of CD8+ T cells on day 8 post infection, while only few such cells were present in mediastinal lymph nodes, spleen and blood. While CD11c was virtually absent from CD8+ T cells in the absence of RSV infection, its mRNA was expressed in CD8+ T cells of both naïve and RSV infected mice. CD11c+, but not CD11c-, CD8+ T cells showed signs of recent activation, including up-regulation of CD11a and expression of CD11b and CD69 and were recruited preferentially to the lung. In addition, CD11c+ CD8+ T cells were the major subset responsible for IFNγ production, induction of target cell apoptosis in vitro and reduction of viral titres in vivo.
CD11c is a useful marker for detection and isolation of pulmonary antiviral cytotoxic T cells following RSV infection. It identifies a subset of activated, virus-specific, cytotoxic T cells that exhibit potent antiviral effects in vivo.
Beta2 integrins, which are restricted to leukocytes, consist of a common β-chain (CD18) and the distinct α-chains CD11a (LFA-1), CD11b (Mac-1/ CR3) or CD11c (p150,95/ CR4) . While CD11a is expressed widely on leukocytes, expression of CD11b and CD11c was thought to be confined to cells of myeloid origin and these molecules have been used as markers to define certain cell populations, e.g. CD11b for macrophages  and CD11c for dendritic cells . β2 integrins are of critical importance for the development of functional immune responses; a mutation in the CD18 gene, resulting in decreased expression of β2 integrins and in defective migration of granulocytes, causes an immune defect termed leukocyte-adhesion deficiency . CD11a/LFA-1 has been shown to be involved in T cell activation , T cell recruitment  and target cell killing  by binding to its ligand intercellular adhesion molecule-1 (CD54) thus mediating adhesive cell-cell-interactions. Over the past ten years several groups have reported that CD11b is expressed on activated cytotoxic T cells [7, 8] and that it is also involved in T cell migration into inflamed tissues . CD11c expression on T cells has been detected initially on a population of intestinal intraepithelial lymphocytes  and more recently on CD8+ T cells following systemic viral infections . The studies regarding β2 integrins on T cells were mostly conducted in mouse-models of infection with LCMV, a virus that induces systemic immune responses involving many different organs . Here, we studied CD11c+ T cells during a localised infection of the lung with RSV, monitoring their distribution and comparing the pulmonary to the systemic immune response. We hypothesized that CD11c is a marker of antigen-specific cytotoxic T cells, which is expressed following activation, rather than being transferred from APC during cell-cell interactions. Such a transfer from APC to T cells has been described for the co-stimulatory molecule CD80 . In addition, we sought to define a function of CD11c when expressed on T cells following RSV infection.
Materials and methods
Virus and animals
Human RSV, type A2 from ATCC (Rockville, MD) free of mycoplasma contamination was used. The virus was cultured on HEp-2 cells from ATCC in Dulbecco's modified Eagle Medium (Invitrogen, Paisley, UK) containing 5% heat inactivated fetal calf serum and 1 % Penicillin / Streptomycin both from Sigma (Gillingham, UK).
Female BALB/c AnNCrl mice, 8 to 12 weeks of age, free of specific pathogens, were obtained from Charles River Laboratories (Margate, UK) and kept under specific pathogen free conditions. All experimental animals used in this study were under a protocol approved by the Home Office, London, UK. Mice were infected under light anesthesia with isoflurane by intranasal inoculation of RSV (5 × 105 PFU in 70 μl). Controls were untreated or mock infected with RSV, inactivated by irradiation with UV-light. RSV infection was confirmed by plaque assay as described previously . Infection could be demonstrated in all infected animals tested but not in controls.
Mice were infected with RSV. On days 0 (naive controls), 4, 6, 8, 10, and 21 post infection mice were sacrificed and lungs, spleens, mediastinal lymph nodes (MLN) and blood cells were harvested. For secondary RSV infection mice were infected 28 days after primary infection. Allergic airway inflammation was induced by intraperitoneal injection of OVA in Alum on days 0 and 14. On days 21 to 23 mice were challenged intranasally with 75 μl 1% OVA in PBS. Mice were sacrificed 24 hours after the last OVA challenge. Organs were disrupted using a steel mesh, and red blood cells were lysed with ACK lysis buffer. For isolation of CD8+ cells lungs were minced, incubated in collagenase (Sigma) solution and mononuclear cells were prepared using a Ficoll (Biochrom) gradient (density = 1.09 g/l). Cell numbers were assessed by counting in a Neubauer chamber.
For intracellular cytokine staining, lung cell suspensions were cultured at a concentration of 106/ml medium in 24 well plates for 4 h in the presence of PMA (50 ng/ml), ionomycin (500 ng/ml) (both Sigma, Gillingham, UK) and Golgi Plug (1 μl/ml) (Becton Dickinson). Following fixation and permeabilization cells were stained with anti-IFN γ (XMG1.2) or isotype control.
Isolation of CD8+ cells
Mononuclear cells from lungs were pooled and incubated with anti-CD8-coated magnetic beads from Miltenyi (Bergisch-Gladbach, Germany) according to manufacturer's instructions. Cells were sorted by Auto-MACS (Miltenyi). Purity of CD8+ cells assessed by FACS was at least 90%. For sorting of CD8+ cells into CD11c+ and CD11c- populations, cells were labeled with anti-CD11c-Fitc and anti CD8-PE-antibodies and sorted on a FACSDiva (Becton Dickinson).
In vitro Cytotoxicity assay
The assay was performed as described . Briefly, P815 cells (ATCC) were used as target cells and labeled with 2 μM RSV-M2-peptide (ABC Synthesis, London, UK), washed extensively and cultured in 96-well U-bottom plates (104 cells/well in 100 μl). Sorted populations of CD8+CD11c+ or CD8+CD11c- were labeled with CFSE (1 μM, Invitrogen, UK) and added to the target cells in the concentrations indicated, resulting in a final volume of 200 μl/well. In some experiments effector cells were pre-incubated with 5 μg/ml anti-CD11c-mAb (HL3; BD) or isotype control-mAb (20 minutes, 4°C) prior to co-culture with target cells. Cells were incubated for 4 hr (37°C, 95% CO2), washed, and incubated with PBS/BSA/0.1% sodium-azide containing 7-Aminoactinomycin D (7-AAD) (20 μg/ml, Sigma, UK). Afterwards cells were washed and fixed in 1% PFA containing Actinomycin D (10 μg/ml, Sigma, UK) and analyzed directly on a FACScalibur (BD). Staining of CFSE-negative cells for 7-AAD was analyzed and specific lysis calculated with the formula:
%specific lysis = 100 × (% 7-AAD+ sample - % 7-AAD+ basal)/(100 - %7-AAD+ basal).
In vivo Cytotoxicity assay
CD11c+CD8+ or CD11c-CD8+ cells isolated by FACSDiva (Becton Dickinson) from lungs harvested on dpi 8, were adoptively transferred (2.5 × 105 cells per recipient) by intra-tracheal application 1 hour after RSV infection of recipient mice. On dpi 4 RSV titers in the lungs were determined by plaque assay as described .
Lungs of RSV infected mice were harvested on dpi 8, CD8+ cells were isolated and labeled with 1 μM CFSE. Cells were incubated with 20 μg/ml of anti-CD11c antibodies (HL3, BD or N418, Serotec) or isotype controls for 30 minutes at 4°C and washed afterwards. Subsequently, cells were adoptively transferred by intravenous injection into recipient mice, which were either naïve or RSV infected (dpi4). After 3 or 24 hours lungs, perfused with PBS via the right ventricle, and spleens were harvested, cell suspensions were generated and analyzed by FACS.
RNA was isolated from purified cells using RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, including a step for on column DNA digestion. The isolated RNA was transcribed into cDNA using Omniscript kit (Qiagen). Primers and probes for CD11c (Applied Biosystems, Warrington, UK) and murine GAPDH (Qiagen) were used according to manufacturers' instructions. Samples were done as duplicates and analyzed on an ABI Prism 7000 light cycler PCR machine (Applied Biosystems).
Data are expressed as mean ± standard deviation unless indicated otherwise. Groups were compared by students t-test. P values for significance were set at < 0.05.
RSV infection results in increases in numbers of CD11c+ CD8+ T cells in the lung
To analyze if CD11c is also expressed on T cells in other organs than the lung during RSV infection, CD8+ T cells were monitored in MLN, spleen and blood. In all of these organs small increases in CD11c+ CD8+ T cells were detected from dpi 4 onwards but their percentage returned to baseline rapidly. Only in MLN, numbers of CD8+ T cells expressing CD11c remained significantly elevated on dpi 10 (Figure 1c). However, numbers of CD8+ CD11c+ cells in the lungs were 8 – 12-fold higher than in any other organ at any time point assessed after RSV infection. Next, we asked if expression of CD11c on CD8+ T cells is unique to primary infection. Analysis of T cells from mice which were re-infected with RSV on dpi 28 showed that 31.9 ± 1.4% of pulmonary CD8+ T cells were positive for CD11c already 4 days after secondary infection. Again CD11c expression was much lower in MLN, spleen or blood and was not increased on CD3+CD8- cells (data not shown). Since both primary and secondary RSV infection resulted in appearance of CD8+ CD11c+ T cells we asked if CD11c expression on T cells is a general phenomenon in lung inflammation irrespective of etiology. To this end, allergic airway inflammation was induced in mice using a protocol, which leads to airway eosinophilia, as well as antigen-specific Th2 cytokine production by CD4+ lung cells (data not shown). In this setting, only 3.8 ± 1.0% of CD8+ T cells expressed CD11c and no changes were observed in the CD3+ CD8- population. To examine if other markers, expressed predominantly on cells of the myeloid lineage, are also increased on T cells during RSV infection we analyzed expression of CD11b and F4/80 on T cells from lungs, MLN, spleen and blood. Kinetics of CD11b expression on T cells paralleled CD11c expression, with lower percentages of CD11b+ CD8+ T cells in the lungs on dpi 8 and 10 (Figure 1c). Expression of the macrophage marker F4/80 was only detectable in significant amounts on splenic T cells on dpi 8 (Figure 1c).
Activation markers are expressed differentially on CD11c+ CD8+ and CD11c- CD8+ T cells
Since most activated CD8+ T cells expressed CD11c, a surface molecule of myeloid cells including APC, we explored the expression of molecules involved in APC T cell-interaction on pulmonary CD8+ T cells 8 days after RSV infection. While CD40 was detectable neither on CD11c+ nor on CD11c- CD8+ T cells, expression of CD80 was confined mostly to CD11c+ T cells (Figure 2a). In addition, we measured co-expression of the β2 integrins CD11c and CD11a or CD11b on CD8+ T cells following RSV infection. Compared to CD11c- CD8+ T cells, CD11a was up regulated on CD11c+ CD8+ cells and CD11b was co-expressed on a subpopulation of CD11c+ cells (Figure 2a).
CD11c mRNA is expressed by both activated and resting CD8+ T cells
Surface molecules such as CD80 can be transferred from APC to T cells by cell-cell contact . Since both CD11c and CD80 are expressed widely on murine APC we asked whether the presence of CD11c on CD8+ T cells is due to expression by T cells rather than to membrane transfer from APC during T cell priming. CD8+ cells were purified from lungs and spleens of RSV-infected or naïve mice, RNA was isolated and the expression of CD11c analyzed by quantitative PCR. CD11c+ pulmonary cells served as a positive control while LA-4 cells, a pulmonary epithelial cell line, and P815 cells, a mastocytoma cell line, were used as negative controls. In two separate experiments, expression levels of CD11c normalized to GAPDH were nearly equal in CD11c+ (relative expression = 1.05 ± 0.06, n = 4) and CD8+ (relative expression = 1.08 ± 0.03) cells from the lung following RSV infection. Surprisingly, CD11c mRNA could also be detected in comparable amounts in CD8+ cells isolated from spleens of naïve mice (relative expression = 1.12 ± 0.08). CD11c mRNA was neither detectable in P815 cells nor in LA-4 cells. These findings suggest that CD11c mRNA is constitutively expressed in CD8+ T cells.
CD11c+ CD8+ cells are IFNγ producing, virus-specific, potent cytotoxic T cells
Preferential recruitment of CD11c+ CD8+T cells to the lung
Expression of β2 integrins on T cells following viral infections has been described in lymphatic organs [7, 8, 11] but knowledge about integrin expression on T cells following respiratory infections is scarce. Previously, we have described a population of CD8+ T cells in the lung expressing the β2 integrin CD11c following RSV infection . To characterize these cells in more detail, we have performed a time course analysis of CD3+ cells displaying CD11c, thus excluding cells of myeloid origin. In mediastinal lymph nodes, where CD11c+ cell numbers increase during inflammation [17, 18], CD8α expressing dendritic cells [19, 20] could be mistaken for CD11c+ CD8+ T cells if analyzed based on CD11c and CD8α only. Following RSV infection, CD11c expression on lung CD8+ T cells increased with up to 50% of CD8 T cells positive for CD11c on dpi8, while, in contrast to LCMV infection, percentages of CD11c+ CTL in spleen and blood were low. CD11c up-regulation on mucosal T cells has previously only been described in the intestine where a subpopulation of intraepithelial T cells expresses CD11c constitutively . In the lung, expression of CD11c on T cells was restricted to CD8+ T cells and was paralleled by increases in CD11b. In contrast to CD11b and CD11c, expression of F4/80 was hardly detectable on pulmonary T cells following RSV infection. Expression of F4/80 may be dependent on both mouse strain and organ analyzed: F4/80+ T cells seem to be more prevalent in C57Bl/6 than in BALB/c mice  and were detectable in significant amounts only on splenic CD8+ T cells, as shown here and by Lin and colleagues . In contrast to viral infections, allergic airway inflammation elicited by sensitization to OVA, resulted only in a small population of CD11c+ CD8+ T cells in the lung, demonstrating that up-regulation of CD11c is not a consequence of T cell activation and migration per se or of an inflammatory environment, but that it is dependent on the underlying pathology. Interestingly, reactivation of antigen-specific CD8+ memory T cells by OVA results in CD11c up-regulation when cells are primed with an OVA-expressing virus . This suggests that MHC class I-restricted antigen presentation as well as co-stimulatory factors induced by viral infection, e.g. IFNα, favor expression of CD11c on CD8+ T cells. Several studies have demonstrated up-regulation of CD11a , expression of CD11b [7, 8, 23] or CD11c  on T cells following viral infections. Here, we demonstrate that following RSV infection a population of CTL co-expresses CD11ahi, CD11b and CD11c. Furthermore CD11c mRNA was detectable in resting as well as activated CD8+ T cells. Contamination by CD11c+ non-T cells cannot be excluded completely but is very unlikely to account for CD11c mRNA detection in CD8 T cells since no major difference in CD11c mRNA quantity was detected between CD8+ and CD11c+ cell populations. CD11c mRNA seems to be expressed constitutively in CD8+ T cells while expression of CD11c protein on the cell surface requires activation. This notion is supported further by the finding that activation markers were up regulated on CD11c+ T cells both in the lung and in the draining MLN. Interestingly, CD11b mRNA is also expressed in resting as well as activated human T cells but CD11b becomes detectable on the cell surface only following stimulation of T cells . Taken together these observations make it tempting to speculate that there may be a common mechanism of post-transcriptional regulation of β2 integrin expression following T cell activation.
Assessing functional properties of CD11c+ CD8+ T cells, we found that these were more efficient than CD11c- CD8+ T cells in IFNγ production, target cell lysis in vitro and induction of viral clearance in vivo. This is likely due to the fact that the CD11c+ CTL population contains the majority of activated T cells specific for the RSV M282–90-peptide, the immuno-dominant CTL epitope of RSV in BALB/c mice. Thus, we show an association of adhesion molecule expression and high cytotoxic activity of pulmonary CD8+ T cells during a respiratory viral infection, parallel to findings in lymphoid organs in LCMV infection [8, 11]. A subset of CD11c- CD8+ T cells expressed high levels of the activation marker CD69 and low levels of CD62L. Cytotoxic effects induced by CD11c- CD8+ cells could be due to this subpopulation of activated CD11c- CTL. We cannot exclude this, since we did not assess cytotoxic effects of isolated subpopulations of CD11c- CD8+ T cells. We believe though, that contamination with CD11c+ CTL is a more likely explanation for cytotoxic activity observed when CD11c- cells were used as effectors. In contrast to a study implicating CD11c on human T cell lines in target cell binding  we could not detect an inhibitory effect of anti-CD11c-mAb on the cytotoxic activity of T cells. In addition to target cell binding, β2 integrins are involved in migration of leukocytes and a role for CD11b in recruitment of T cells into inflamed areas has been shown by antibody blocking experiments . In our model, the percentage of CD11c+ cells within the CD8+ T cell population differed between the organs assessed, being lowest in MLN and spleen, higher in the blood, where a short-lived increase of these cells was noted during the acute phase of infection, and highest in the lung. This indicates that CD11c marks effector T cells, which are recruited to the site of infection. An analogous pattern of tissue distribution has been described for VLA-4hi T cells following intra-cerebral LCMV infection . Interestingly, following adoptive transfer we observed efficient recruitment of CD11c+ CD8+ T cells to the lung in naïve mice, while only a small percentage of these cells were recruited to the spleen. This preferential recruitment of CD11c+ CD8+ T cells to the lung was even more pronounced in RSV infected recipients. Pre-treatment of transferred cells with anti-CD11c-mAb did not reveal a unique function of CD11c for homing of activated CTL to the lung. These observations suggest that CD11c is a marker for activated CTL, which are recruited preferentially to the lung, while the CD11c molecule itself may not be directly involved in the process of T cell recruitment to the lung. Functional redundancy of β2 integrins in target cell binding or migration into effector sites is a possible explanation of our results. CD11a has been shown to be important for target cell binding , recruitment of activated lymphocytes into the lung [5, 27] and generation of an effective cytotoxic T cell response during primary RSV infection .
We demonstrate that the β2 integrin, CD11c, identifies a subset of activated, virus-specific, cytotoxic CD8+ T cells. These cells are preferentially recruited to the lung in RSV infection and exhibit potent antiviral effects in vivo. CD11c is expressed by CD8+ T cells, but this molecule may not be directly involved in effector functions. Thus, following respiratory viral infections CD11c is a useful marker for detection and isolation of activated, cytotoxic, pulmonary T cells, provided distinction from myeloid cells is taken into account.
antigen presenting cells
- CFSE 5-(and-6)-carboxyfluorescein diacetate:
cytotoxic T lymphocytes
Glycerol aldehyde phosphate dehydrogenase
lymphotropic choriomeningitis virus
major histocompatibility complex
mediastinal lymph nodes
respiratory syncytial virus
This work was supported by Grants 067454 from the Wellcome Trust, 01GC9802 from Bundesministerium für Bildung und Forschung and Schw 597/2-1 from Deutsche Forschungsgemeinschaft. We thank Niga Nawrolny for technical assistance with FACS sorting.
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