Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential

CD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c+ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8+ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c+ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c- T cells. Furthermore, circulating CD11c+ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential.


Introduction
The ability to assess ongoing mucosal immune responses is critical for understanding hostpathogen responses and would assist mucosal vaccine development. In the case of the female genital tract (GT) immune responses, assays to determine the magnitude and quality of the immune response in mucosal tissues largely rely on sampling of peripheral blood. Measuring antigen-specific T cell responses directly is not always technically or economically feasible, therefore methods that provide an indirect measure of the immune response are essential.
We previously detected an increase in the expression of α chain integrin (αX, CD11c molecule) when analyzing blood samples from mice with lower GT Chlamydia infection and women with symptomatic bacterial vaginosis (J. Qualai et al., submitted for publication).
Integrins are widely expressed cell surface molecules, composed of non-covalently linked α and β subunits that allow cell-extracellular matrix and cell-cell interactions [1]. Among these integrins, CD11c/CD18 is one of the four members of the β2 leukocyte integrin family [1]. CD11c is also referred to as complement receptor 4, since it can mediate phagocytosis of inactivated complement C3b-opsonized particles, demonstrating a wider role in host defense than cell-cell adhesion [2]. There are a wide variety of ligands described for CD11c, including other adhesion molecules, bacterial cell wall components (including lipopolysaccharide), complement proteins, and matrix proteins [2]. The CD11c/CD18 complex is also reported to bind denatured proteins, perhaps acting as a danger signal in the context of innate immune defense [3].
CD11c is typically considered to be a marker of conventional dendritic cells (DC) [4], and is not often considered in the context of T cell responses [5]. Yet, CD11c can be expressed on NK cells and populations of activated T and B cells [6]. Indeed, studies in several different mouse models have associated CD11c expression with effector memory and regulatory T cell subsets (T EM /T regs ) [7][8][9][10][11][12], small gut intra-epithelial lymphocytes [13,14], and the development of experimental autoimmune encephalomyelitis [15]. The exact role of CD11c is unclear but was found to correlate with increased cytotoxicity, effector, or regulatory function (reviewed in [16]). CD11c expression has also been associated with activated antigen-specific T cells expanded in response to infection or vaccination, and has been correlated with the potential of these cells to secrete IFN-γ [9,12]. Recent reports have also described a unique cellular subset in mice and humans that contains key features of both T cells and DCs [17]. Interestingly, these unusual cells are characterized by the expression of T cell receptor (TCR), major histocompatibility complex (MHC) II, and CD11c [17].
Our hypothesis is that increased CD11c expression in circulating T cells during bacterial infection of the GT indicates the activation and migration of innate-like T cells. Here, we have examined the phenotype of T cells expressing CD11c in the blood and GT of mice and women. We find that in addition to CD8 + T cells, γδ T cells represent an unconventional T cell subset that expresses CD11c under physiological conditions. These innate populations should be considered when evaluating mucosal immune responses to infection.

Ethics statement
All animal procedures were approved and supervised by the Animal Care Committee of the Germans Trias i Pujol Health Science Research Institute (IGTP) and by the Department of Environment of the Catalan Government (approval number # 7066).
Informed written consent was obtained from all participants and the study protocols and questionnaires were approved by the University Hospital Germans Trias i Pujol (HUGTP, Badalona, Spain) Clinical Research Ethics Committee (reference numbers # EO-11-074 and # PI-14-070). The study was undertaken in accordance with the Declaration of Helsinki and the requirements of Good Clinical Practice.

Samples for differential gene expression analysis
Six to eight week-old female C57BL/6 mice were bred in house. Mice were kept in ventilated cages with sterile food and water ad libitum, under specific-pathogen-free conditions at the animal facility for experimental models of the IGTP, Spain. A group of six females were treated subcutaneously with 3mg of Depo-Provera six days prior to euthanizing in order to homogenize the endocrine effects on immunity by synchronizing the menstrual cycle. All mice were euthanized with isoflurane (inhalation excess) and blood was immediately obtained by cardiac puncture.

Differential gene expression and gene set enrichment analysis
Microarray gene expression profiling with Mouse Genome 430 PM Strip arrays was performed following manufacturer recommendations (Affymetrix Inc, Santa Clara, CA) with specific adaptations based on the picoprofiling method [18]. Image intensities were extracted with Affymetrix GeneAtlas System software, normalized and summarized by RMA and analyzed for differential gene expression by Limma [19] with false discovery rate multiple testing significance correction. NK-related γδTCR, iNKT CD4 + and iNKT CD4cells specific gene sets [20] were tested by Gene Set Enrichment Analysis (GSEA) [21] for significant enrichment in CD11c + T cells.

Animal model
Chlamydia muridarum elementary bodies were purified by discontinuous density gradient centrifugation and the number of inclusion-forming units were determined as previously described [22,23]. Eight week-old C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained in accordance with University of California Davis Research Animal Resource guidelines, USA. Estrus was synchronized and 7 days after, 1x10 5 C. muridarum in 5μL sucrose/phosphate/glutamate buffer were deposited directly into the vaginal vaults with a blunted pipet tip [23].

Tissue processing
Blood was collected by retro-orbital bleeding and erythrocytes were immediately removed by ACK lysing buffer (Life Technologies). Leukocytes were washed with FACS buffer (PBS with 2% FBS) and stored on ice until use. Mouse GT were removed and leukocytes isolated as described [24]. Briefly, vagina, cervix, uterine horns and oviducts were minced into small pieces, digested in 500mg/L collagenase IV (Sigma) for 1 hour at 37˚C with constant stirring. Leukocytes were purified by percoll density gradient centrifugation (GE Healthcare), washed with FACS buffer and stored on ice until use.

Phenotyping of human blood
Healthy normal donors (ND) were recruited from the clinical trials unit of the HUGTP. For unconventional T cell subset phenotyping (n = 10, 27±4 years old), 1ml of blood was lysed by ammonium Lysis Buffer (BD Pharm Lyse, BD Biosciences) and processed for staining. Leftover blood (*9ml) was used to isolate peripheral blood mononuclear cells (PBMC) by density gradient centrifugation over

Statistical Analysis
Data are reported as the mean and the standard deviation (SD) for each group using Prism 4.0 software (GraphPad Software). For animal samples, statistical analyses were performed by Student's t test or by Paired T test when comparing the positive and negative fraction of the same sample. For all the other analyses in human samples, which did not pass normality test (p<0.05), non-parametric tests were employed. A p value of <0.05 was considered significant.

Accession codes
Microarray data presented in this article are deposited into the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE68934.

Murine activated CD11c + T EM cells are enriched for NK gene signatures
In order to characterize circulating CD11c + T cells, we initially analyzed gene expression in activated CD3 + CD62L -CD44 + CD11c + and CD3 + CD62L -CD44 + CD11c -T EM cells recovered from naïve female mice. Multiple related genes were expressed two to eight fold higher in CD11c + than CD11c -T cells ( Table 1). Many of these genes are expressed in NK and non-conventional memory T cell subsets but are not highly expressed in conventional αβ T cells [20]. Gene Set Enrichment Analysis revealed significant enrichment for γδT, NK and CD4 -iNKT cells when looking at the entire dataset (S1 Fig). Thus, CD11c expressing circulating T cells display a gene signature similar to γδ T and CD4 -iNKT cells.

Murine CD11c + T cells express high levels of NK1.1 in blood after vaginal infection
Next, we performed flow cytometry analyses of blood and GT obtained from C57BL/6 mice vaginally infected with Chlamydia muridarum. Seven days after infection, the frequency of T cells expressing CD11c + in peripheral blood increased from 1.53% to 6.04% as reported (J. Qualai et al., submitted for publication). Within this fraction we analyzed the expression of several NK-associated molecules (NK1.1, DX5, NKG2A, NKp46), specific T cell phenotypes (CD8α, CD1d-restricted iNKT cells, γδTCR) and adhesion molecules (CD103, CCR10). Part of the gating strategy is shown in S2 Fig. The frequency of CD11c + T cells expressing CD8α in blood was similar before and after infection, while NK1.1 expression increased markedly following infection (Fig 1A and 1B). Before infection, a significant fraction of CD3 + CD11c + T cells also expressed DX5 and NKG2A (Fig 2), but these markers either decreased or remained the same after infection (Fig 2). The frequency of CD3 + CD11c + T cells within the GT increased from 0.5±0.2% to 4.1±1.6% seven days after infection (p = 0.0001). In marked contrast to blood, the expression of CD8 by CD11c + T cells strikingly increased in these tissues after Chlamydia infection (Fig 1A). NK1.1 was expressed at high levels by GT CD11c + T cells before infection and significantly decreased after infection (Fig 1B). The expression of DX5 and NKG2A was less frequent at baseline in GT CD11c + T cells compared to blood, while the activating receptor NKp46 was expressed by approximately 40% of cells (Fig 2). After infection, the frequency of NKG2A expression by CD11c + T cells significantly increased (p = 0.006), while NKp46 decreased (p = 0.0009) (Fig 2). Thus, CD3 + CD11c + T cells increase in both blood and GT after vaginal infection and express different patterns of surface markers within these compartments.

Murine CD11c + T cells include high frequencies of γδT cells and iNKT cells
The total frequency of iNKT or γδ T cells in blood did not change after vaginal infection; however, it did increase in the GT of the infected mice (p<0.003; data not shown). When comparing CD11c + and CD11csubsets, there was a clear enrichment of these unconventional T cells within the CD11c + fraction in both tissues (Fig 1C and 1D). Thus, under physiological conditions, CD11c + T cells included almost exclusively all iNKT cells and high proportions of γδ T cells. Of CD3 + CD11c + T cells in the blood, 27-39% of them were iNKT and 50-60% were γδ T cells and this frequency did not change after vaginal infection (Fig 1C and 1D). Nevertheless the frequency of CD1d-tetramer + cells in the CD11c + T cell fraction decreased in the GT after infection (Fig 1C). Actually, when performing the analysis in combination with CD8 and NK1.1 expression, it was clear that while NK1.1 + γδ T cells characterized the major subset expanding in blood, a single CD8 + phenotype was the major contributor to the increment of CD11c + T cells observed in the GT (Fig 3).

CD103 expression is exclusively associated to CD11c + T cells in mice
Previous work in a mouse model of colitis determined that CD11c + CD8 + T regs are CD8α + CD103 + NK1.1in the small intestine, but CD8α -CD103 -NK1.1 + in the colon [10]. We analyzed the expression of these molecules and included CCR10, which could indicate migration of CD11c + T cells into the GT. The ligand for this chemokine, CC-chemokine ligand 28, is expressed in the genital tract, and B cells homing there express CCR10 [27]. Co-expression of CD103 and CD11c was high in the blood and GT of uninfected mice (Fig 1E). Additionally, among circulating T cells, CCR10 expression was found almost exclusively associated with the CD11c + subset (Fig 1F). This chemokine receptor represented a small fraction of CD11c + T cells, but expression was clearly enriched in CD11c + cells after infection (Fig 1F).
To summarize, it appears that the majority of CD11c + T cells in blood under homeostatic conditions are CCR10 -CD8α -CD103 + NK1.1 -, but this phenotype is less frequent in the GT (Fig 4). After vaginal infection, CD103 + NK1.1 + CD8αcells expand among circulating CD11c + T cells and CD8α + CD103 -NK1.1are elevated in the GT (Fig 4). Thus, none of these phenotypes illustrated the T regs described in the colitis model [10].

Circulating T cells expressing CD11c are associated with γδ T cells but not iNKT cells in women
Next, we examined CD11c expression in CD3 + CD4 + /and CCR7 + / -T cells from healthy young women (normal donors, ND), and found an enrichment of this marker in the CCR7 -CD4fraction (Fig 5). We then analyzed subsets studied in the mouse model and examined Vα7.2 expression to determine mucosal associated invariant T (MAIT) cells, which are characterized by high CD161 expression (NK1.1 human homolog) [28]. We also adressed iNKT populations via expression of the invariant TCR α chain (Vα24-Jα18). We analyzed each phenotype in circulating CD3 + CCR7 -T cells, and compared their expression in CD11c + (1.9±0.7%) vs. CD11c -(35.4±10.0%) cells using a non-parametric paired T test (Fig 6A and S3 Fig left). The gating strategy used for these analyses excluded CD19 + and CD14 + cells, which marginally contaminated CD11c + T cells. The only subsets significantly enriched in the positive fraction were CD8 + and γδTCR + (Fig 6A). In contrast, MAIT cells (Vα7.2 + CD161 h ) and iNKT cells were lower in the positive than in the negative fraction (Fig 6A). Of note, γδ T cells represented 5.7±2.7% of the total CD3 + T cells in blood, and the frequency of CCR7 -CD11c + in this subset was of~10% (S4A Fig), from which ~78% were CD161 + and~18% were CD8 + . In fact, CD161 was similarly high in γδ T cells regardless of CD11c expression (S4B Fig), but CD8 expression was doubled in the CCR7 -CD11c + fraction of γδT cells compared to total CD3 + γδTCR + T cells (S4C Fig). Regarding CD8 + T cells, In six of these donors, we analyzed the expression of these markers after obtaining PBMC to determine if this process affected any of the T cell phenotypes determined. The overall differences between CD11c + and CD11c -T EM cells were mostly maintained, yet MAIT and iNKT significances were lost (Fig 6B and S3 Fig right). Thus, circulating CD11c + T EM cells represented at least two different populations in healthy women: a subset of γδ T cells with their constitutive high expression of CD161 and enriched CD8 expression, and a subset of CD8 + T cells with enhanced CD161, yet not belonging to the MAIT or iNKT lineages.

Human cervical tissue contains γδ T cells expressing CD11c
The same subsets were determined by flow cytometry in cervical samples from healthy women. First, we evaluated the impact of tissue digestion on the detection of these phenotypes in PBMC, since it has been reported that other markers are affected by collagenase treatment [29]. While we confirmed the loss of CD56 expression, we also detected a dramatic reduction on CD19, CD20 and CCR7 protein expression. However, expression of CD3/CD8/ CD14/CD16/CD161/αβTCR/γδTCR/Vα7.2/Vα24/CD11c/HLA-DR was not modified. Thus, we analyzed the same subsets without considering CCR7 expression, since most CD3 + T cells in these tissues were CCR7 -(>95%). Following the gating strategy shown in Fig 7A, CD11c expression on CD3 + CD14 -T cells represented an average of 3.3±1.5% for ectocervix and 3.7 ±2.5% for endocervix. Since there were no differences between any of the subsets analyzed in the ecto and endocervix (Fig 7B), we pooled both tissues in order to strengthen the statistical power of the analysis. This way, the proportion of γδTCR was of >9% in CD11c + vs.~1% in CD11cand the proportion of CD8 was of 59% in CD11c + vs. 46% in CD11c -(p<0.006 for both). In three of these samples, we also analyzed the activation marker CD69, which expression was of 86.0±9.5% in CD11c + vs. 74.5±10.1% in CD11c -(S5A Fig).

Characterization of T Cell Subsets Expressing CD11c
Compared to blood, γδTCR + and CD161 + represented smaller populations of total CD3 + T cells in the cervix, while CD8 + T cells were clearly enriched in this tissue. The mean frequency of CD11c + in γδ T cells was of over 20% (S4E Fig), from which CD161 was again independent of CD11c expression (S4F Fig), while CD8 was significantly enriched compared to total CD3 + γδTCR + T cells (S4G Fig). Finally, CD11c + represented 5.8±2.9% of total CD8 + T cells (S4E Fig), in which CD161 was also significantly different from total CD3 + CD8 + T cells (S4H Fig). Regardless of differences in the percentage of CD8 and other subsets between blood and mucosa in women, similar findings were observed concerning the subsets enriched in CD11c + T cells. CD11c + T cells are highly enriched for adhesion molecules expression but not CD103 in women Additionally, we studied the association of CD11c expression with a variety of cellular adhesion molecules in thirteen ND from a previous homing study (J. Qualai et al., submitted for publication). We observed that many of these molecules were enriched for the CD11c-positive fraction of circulating CCR7 -CD4 -T cells: CCR2, CCR9, CCR10, CXCR6, α1β1, α4β7 (Fig 8A and  8C-8G), as well as CXCR3 (18.7±2.9% vs. 4.5±1.89) and α4β1 (37.4±5.2% vs. 4.5±1.2%) (p = 0.0002; data not shown). In contrast, expression of CCR5 was higher in the negative fraction compared to the positive (Fig 8B), while cutaneous lymphocyte antigen and CD103 were not different between these two subsets (data not shown). Additionally, activation markers HLA-DR + and/or CD38 + were also significantly increased in CD11c + compared to CD11c -T cells (all p0.0006; Fig 8H and data not shown). Thus, compared to the animal model, CD103 was not particularly expressed in CD11c + T cells. Still, and similarly to the mouse data, CCR10 was enriched in CD11c + T EM cells, as occurred for most of the other chemokine receptors and other adhesion molecules measured, except for CCR5. This suggests that subsets of T cells expressing CD11c + are activated T cells with high homing potential to a variety of tissues and mucosal compartments.

CD11c expression in γδ T cells is associated with higher IFNγ secretion after activation
CD11c expression in T cells has been associated with multiple effector functions, such as increased IFN-γ secretion [9,12]. In order to address if activated γδ T cells also differ in their capacity to secrete IFN-γ based on CD11c expression, we stimulated PBMC for 18 hours with HMBPP and then determined IFN-γ secretion and activation. HMBPP is an intermediate of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis used by many pathogens, and represents the most potent stimulant known of the major γδ T cell population in human peripheral blood [30]. No IFN-γ secretion was detected in conventional T cells or control samples, while 5.9±4.1% of the CD11c + and 3.4 ±2.1% of the CD11cγδ T cells secreted IFN-γ after stimulation (Fig 9A). When samples from the same individual were compared based on CD11c expression we detected significant differences in both, IFN-γ secretion and activation of γδ T cells based on this marker (Fig 9B and 9C). IFN-γ-secreting γδ T cells in PBMC from healthy women after HMBPP activation. (a) Comparison of the frequency of γδTCR + CD11c + /and γδTCR -CD11c + / -T cells that secrete IFNγ in PBMC from the same individual (n = 6) after 20 hours of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) activation. Gating strategy consisted on the following consecutive gates: lymphocytes, singlets, CD3 + T cells, γδTCR + / -, CD11c + /and IFNγ/CD69 + expression. Each bar represents the mean ± SD of control (squares) and HMBPPstimulated PBMC (circles) samples. Data were analyzed using the non-parametric Friedman test for repeated measures, with Dunn's multiple comparisons post-hoc test. Graphs below show the comparison in the frequency of (b) IFN-γ-secreting γδ T cells and (c) CD69 expression in γδ T cells based on CD11c expression in HMBPP-activated PBMC from the same individual (n = 6). Data were analyzed using Wilcoxon matchedpaired signed-ranked test. doi:10.1371/journal.pone.0154253.g009

Discussion
We have attempted to characterize the cellular subsets found in murine and human circulating and GT T cells marked by CD11c expression. While NK1.1 and CD103 expression was tightly associated with CD11c expression in T cells from female mice, these markers were not common in CD11c + T cells isolated from women. Furthermore, although both iNKT and γδ T cell populations were enriched in this fraction in the blood and GT of mice, only the second subset was consistently found in CD11c + T cells from women. These cells are highly activated, express high levels of adhesion molecules, and secrete higher levels of IFN-γ upon activation, when compared to CD11c -T cells. Thus, considering that CD11c + T cells increase after infection or vaccination in different mouse models [9,12], but also during symptomatic vaginosis (J. Qualai et al., submitted for publication), we need to reconsider CD11c + T cells in the context of mucosal immune responses.
In murine models of viral infection and graft-versus-host disease, TCR stimulation induces CD11c up-regulation in CD8 + T cells [14,16]. CD11c expression has been associated with gain of effector function, identification of antigen-specific T cells during infection or vaccination (9,12), and specific T regs [16]. Protection provided by these cells derives from high IFN-γ secretion and up-regulation of effector mechanisms (i.e. granzyme), which account for both effector and regulatory functions [9,16]. Gene expression analyses of activated CD11c + T EM cells revealed up-regulation of some of these effector mechanisms to be highly related to NK properties [20,31]. Our data show a clear association between CD11c and NK1.1 expression, which strikingly increases after vaginal infection in mice. Although expression of this molecule on T cells was first employed to exclusively define NKT cells [32], it is now clear that other subsets of nonconventional and activated T cells express NK1.1 or CD161 in mice and humans, respectively [28]. In mice, two different genes encoding proteins with opposite functions share the NK1.1 epitope, namely Klrb1b and Klrb1c [33]. According to the gene expression analyses we performed, only Klrb1b was significantly up-regulated in blood, which would suggest an inhibitory function, although this was not confirmed. However, γδ T cells expressing NK1.1 are the main IFNγ-producers among γδ T cell populations [12] and, as occurred for other NK markers addressed here, differences in the expression of activator (i.e. NKp46) or inhibitory (NKG2A) molecules between blood and tissue could also exist for this epitope. Similarly, in humans, CD161 + γδ T cells are efficient producers of IFN-γ but not of interleukin (IL)-17A [34]. In healthy women, we also detected an enrichment of CD161 expression in CD11c + CD8 + T cells from blood and cervix; while in γδ T cells, this marker was already high. Although the role of this molecule as inhibitory or co-stimulatory has not yet been fully defined, CD161 + T cells, including MAIT and CD161 + γδ T cells, have been shown to commonly respond in an innatelike manner to IL-12/IL-18 stimulation independent of TCR activation [28].
Several potential mechanisms could also contribute to the expression of CD11c on T cells. In particular, protein or RNA transfer from activated antigen presenting cells via trogocytosis or external vesicles/exosomes during infection have been described [35,36]. Although further research should clarify the role of these processes here, the fact that these differences exist during baseline physiological conditions suggest constitutive expression of CD11c by T cells, as others have observed [7]. From all the T cell-subsets analyzed, besides CD8 + T cells, the most consistent population expressing CD11c was γδTCR + . Indeed, one of the genes that differentiates γδ T cells from other non-conventional T cell subsets is Itgax, which encodes the αX integrin [20]. About 10 years ago, Brandes et al. described antigen presenting cell functions by γδ T cells in humans [37]. These cells have signs of pre-activation (CD69 + ) along with up-regulation of a variety of co-stimulatory and adhesion molecules (including HLA-DR and CD11c) [37]. Similarly we found that γδ T cells expressing CD11c are more activated, while CD11c + CD3 + T EM cells expressed increased levels of HLA-DR/CD38 and of most of the cellular adhesion molecules addressed compared to CD11c -T cells.
In human blood, γδ T cells and specifically Vγ2Vδ2 + T cells, quickly expand after infection in response to microbial metabolites [31,37]. Thus, we could expect an increase on these cells in the context of GT disorders caused by Chlamydia or Gardnerella spp. due to production of these metabolites [30]. Although we have not yet been able to confirm if the increment of CD11c + T cells during bacterial vaginosis is due to an increase on this subset, CD11c expression increased in circulating γδ T cells of Chlamydia-infected mice, which demonstrated expansion of CD11c + NK1.1 + γδ T cells in blood seven days after infection (Fig 3). Moreover, CD11c + and γδTCR + cells also increased in the GT after infection. The role of γδ T cells during C. trachomatis infection was examined in a knockout model of pneumonia by Rank´s group [38]. Although these cells were protective during the first 3-7 days post-infection, they were potentially deleterious at later stages [38]. As suggested, the diversity of their cytotoxic and regulatory effects may impact differently on the course of various inflammatory processes [38]. Additionally, based on the level of CD11c expression, a regulatory (CD11c high ) and an effector (CD11c low ) subset have been described for CD11c + CD8 + T cells in mice [8]. Future investigations should elucidate if all CD11c + T cell subsets, including γδ T cells, can be similarly distinguished, even in humans.
Further, there was a clear association between CD103 and CD11c expression in mice. CD103 is an αE integrin induced by transforming growth factor (TGF)-β necessary for tissue retention [31,39]. Interestingly, inducible TGF-β was up-regulated >3-5 times in CD11c + T cells ( Table 1). CD103 expression, together with the absence of CCR7 and expression of CD69, is one of the hallmarks of tissue-resident memory (T RM ) CD8 + T cells [39]. In naïve mice, only T cells expressing CD11c expressed CD103 in blood and GT, and this association was maintained after infection. Therefore, CD11c could potentially be another marker for T RM in mice. Although no differences were observed in the frequency of CD103 expression based on CD11c expression in the cervix of women, CD11c + T cells in this tissue may also contain CD8 + T RM cells, since not all T RM cells express CD103 + [40]. Undoubtedly, the relationship between these different phenotypes needs further clarification.
Intraepithelial CD103 + CD11c + T cells with an activated phenotype that mediate inflammatory gut pathology during infection have also been described in mice [13]. These cells, composed of~50% γδ T and αβ T cells, depend on CCR2 for recruitment and, actually, CD11c + T cells express higher levels of CCR2 than CD11c -T cells [13]. In other models, expression of adhesion molecules such as α4β1 on γδ T cells control trafficking into tissue [41]. We determined CCR2 and α4β1 expression in CCR7 -CD4 -T cells, among other adhesion molecules, and found that during physiological conditions CD11c + are almost exclusively associated with expression of most of these molecules. Likewise, CD161 ++ CD8 + T cells, including different unconventional phenotypes, have been proposed to possess critical functions in diverse tissues because of their specific tissue-homing expression patterns [42].
In summary, several animal models evidence that CD11c + CD8 + /CD11c + NK1.1 + T cells are functionally more potent than the ones not expressing CD11c [7,[9][10][11][12]. Following respiratory syncytial virus infection, only CD11c + CD8 + T cells show signs of recent activation, including up-regulation of CD11b/CD69, and are recruited preferentially to the lung [7]. The characterization of the major subsets included in CD11c + T cells during physiological and infection conditions performed here may recapitulate similar findings described in the literature for these related phenotypes. In humans, this may include the recent description of αβ T cells with DC properties [17], as reported before for γδ T cells [37], and possibly even CD8 + T RM [39,40]. Further research on the role of the different subsets included in this phenotype during mucosal infection is warranted.
Supporting Information S1 Fig. Significant gene set enrichment of NK biomarkers among differentially up-regulated genes in CD11c positive versus CD11c negative cells. A signal-to-noise ratio (SNR) statistic was computed by GSEA software for each gene in a gene set compared to the rank list of the genes assayed on the microarray ranked according to their correlation with CD11c + , this means positively correlated with log2 expression ratios. Significantly enriched gene sets cluster in the up-regulated end of the ranked list have positive enrichment scores of the gene set used for the comparison (red). The graph on the bottom of each panel represents the non-redundant list of genes ranked by differential gene expression between the CD11c + and CD11c -T cells. On each panel, the vertical black lines indicate the position of each of the genes of the studied in the gene set of interest within the rank ordered, non-redundant data set. The green curve corresponds to the ES (enrichment score) curve, which is the running sum of the weighted enrichment score generated by the GSEA software. Shown below are the normalized enrichment scores (NES) for each plot, which are equivalent to the value of the ES curve at the leading edge of the curve (where the statistic reaches its maximum value for a particular gene set). Results show that genes up-regulated in CD11c + cells are significantly enriched for all four gene sets, as judged by the density of hits (black vertical bars) localized at the tip of the blue region with p<0.05 and false discovery rate (FDR) <0.25, but show more significant enrichment in γδ T and iNKT CD4 + than in iNKT CD4cells.